Title of Invention

MODULATORS OF HUMAN G PROTEIN-COUPLED RECEPTORS FOR THE TREATMENT OF HYPERGLYCEMIA AND RELATED DISORDERS

Abstract The present invention relates to methods of identifying whether one or more candidate compounds is a modulator of a G protein-coupled receptor (GPCR) or a modulator of blood glucose concentration. In certain embodiments, the GPCR is human. The present invention also relates to methods of using a modulator of the GPCR. A preferred modulator is agonist. Agonists of the invention are useful as therapeutic agents for lowering blood glucose concentration, for preventing or treating certain metabolic disorders, such as insulin resistance, impaired glucose tolerance, and diabetes, and for preventing or treating a complication of an elevated blood glucose concentration, such as atherosclerosis, heart disease, stroke, hypertension and peripheral vascular disease.
Full Text MODULATORS OF HUMAN G PROTEIN-COUPLED RECEPTORS FOR THE TREATMENT OF HYPERGLYCEMIA AND RELATED DISORDERS
This application claims the benefit of priority from the following provisional application, filed via U.S Express Mail with the United States Patent and Trademark Office on the indicated date U.S Provisional Number 60/561,954,1 filed April 13, 2004 The disclosure of the foregoing provisional application is herein incorporated by reference in its entirety.
FIELD OF THE INVENTION
The present invention relates to methods of identifying whether one or more candidate compounds is a modulator of a G protein-coupled receptor (GPCR) or a modulator of blood glucose concentration. In certain embodiments, the GPCR' is human. The present invention also relates to methods of using a modulator of the GPCR. A preferred modulator is agonist Agonists of the invention are useful as therapeutic agents for lowering blood glucose concentration, for preventing or treating certain metabolic disorders, such as insulin resistance, unpaired glucose tolerance, and diabetes, and for preventing or treating a complication of an elevated blood glucose concentration, such as atherosclerosis, heart disease, stroke, hypertension and peripheral vascular disease.
BACKGROUND OF THE INVENTION
The following discussion is intended to facilitate the understanding of the invention, but is not intended nor admitted to be prior art to the invention.
A. Hyperglycemia
Blood glucose concentration typically is maintained within a narrow, range. An elevation in blood glucose concentration normally leads to an increased release of insulin, which then acts on target cells to increase glucose uptake. Dysregiilation of blood glucose homeostasis can lead to persistent elevated blood glucose concentration, or hyperglycemia. Some individuals with hyperglycemia may proceed to develop type 2 diabetes. Chronic exposure of tissues to hyperglycemia may result in diverse complications including microvascular problems of neuropathy, retinopathy and nephropathy and the macrovascular complications of stroke, coronary heart disease, and peripheral vascular disease. Hyperglycemia is a major and growing medical problem in need of better management options [Nesto, Reviews in Cardiovascular
Medicine (2003) 4:S11-S18; the disclosure of which is hereby incorporated by reference m its entirety]

B. G Protein-Coupled Receptors
Although a number of receptor classes exist in humans, by far the most abundant and therapeutically relevant is represented by, the G protein-coupled receptor (GPCR) class. It is estimated that there are some 30,000-40,000 genes within the human genome, and of these, approximately 2% are estimated to code for GPCRs.
GPCRs represent an important area for the development of pharmaceutical products: from approximately 20 of the 100 known GPCRs, approximately 60% of all prescription Pharmaceuticals have been developed. For example, in 1999, of the top 100 brand name prescription drugs, the following drugs
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interact with GPCRs (the primary diseases and/or disorders treated related to the drug is indicated in
parentheses):
Claritin® (allergies) Prozac® (depression) Vasotec® (hypertension)
Paxil® (depression) Zoloft® (depression) Zyprexa®(psychotic disorder)
Cozaar® (hypertension) I mitrex® (migraine) Zantac® (reflux)
Propulsid® (reflux disease) Risperdal® (schizophrenia) Serevent® (asthma)
Pcpcid® (reflux) Gaster® (ulcers) i Atrovent® (bronchospasm)
Effexor® (depression) Depakote® (epilepsy) Cardura®(prostatic hypertrophy)
Allegra® (allergies) Lupron® (prostate cancer) Zoladex® (prostate cancer)
Diprivan® (anesthesia) BuSpar® (anxiety Ventolin® (bronchospasm)
Hytrin® (hypertension) Wellbutrin® (depression) Zyrtec© (rhinitis)
i
Plavix® (Ml/stroke) ToproI-XL® (hypertension) Tenormin® (angina)
Xalatan® (glaucoma) Singulair® (asthma) Diovan® (hypertension)
Hamal® (prostatic hyperplasia) (Med Ad News 1999 Data).
GPCRs share a common structural motif, having seven sequences of between 22 to 24 hydrophobic amino acids that form seven alpha helices, each of which spans the membrane (each span is identified by number, i.e., transrnembrane-1 (TM-1), transmembrane-2 (TM-2), etc.). The transmembrane helices are joined by strands of amino acids between transmembrane-2 and transrnembrane-3, transmembrane-4 and transmembrane-5, and transirtembrane-6 and transmembranc-7 on the exterior, or "extracellular" side, of the cell membrane (these are referred to as "extracellular" regions 1, 2 and 3 (EC-1, EC-2 and EC-3), respectively). The transmembrane helices are also joined by strands of amino acids between transmembrane-1 and transmembrane-2, transmembrane-3 and transmembrane-4, and transmembrane-5 and transmembrane-6 on die interior, or "lntracellular" side, of the cell membrane (these are referred to as "ratracellular" regions 1,2 and 3 (IC-1, IC-2 and IC-3), respectively). The "carboxy" ("C") terminus of the receptor lies in the intracellular space within the cell, and the "amino" ("N") terminus of the receptor lies in the extracellular space outside of the cell.
1 Generally, when a ligand binds with the receptor (often referred to as "activation" of the receptor), there is a change in the conformation of the receptor that facilitates coupling between the intracellular region and an intracellular "G-protein." It has been reported that GPCRs are "promiscuous" with respect to G
pioteins, i.e, that a GPCR can interact with more than one G protein. See, Kenakin, T, 43 Life Sciences
i 1095 (1988). Although other G proteins exist, currently, Gq, Gs, Gi, Gz and Go are G proteins that have
been identified. Ligand-activated GPCR coupling with the G-protern initiates a signaling cascade process (referred to as "signal transduction"). Under normal conditions, signal transduction ultimately results in cellular activation or cellular inhibition, Althoughj not wishing to be bound to theory, it is thought that the IC-3 loop as well as the carboxy terminus of the receptor interact with the G protein.
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There are also promiscuous G proteins, which appear to couple several classes of GPCRs to the phospholipase C pathway, such as Gccl5 or Gtxl6 [Offermanns & Simon, J Biol Chem (1995) 270:15175-80], or chimeric G proteins designed to couple a large number of different GPCRs to the same pathway, e g phospholipase C [Milhgan & Rees, [Trends in Pharmaceutical Sciences (1999) 20:118-24]
Under physiological conditions, GPCRs exist in the cell membrane in equilibrium between two different conformations' an "inactive" state and an "active" state. A receptor in an inactive state is unable to link to the intracellular signaling transduction pathway to initiate signal transduction leading to a biological response Changing the receptor conformation to the active state allows linkage to the transduction pathway (via the G-protein) and produces a biological response.
A receptor may be stabilized ha an active state by a Iigand or a compound such as a drug. Recent
discoveries, including but not exclusively limited to modifications to the amino acid sequence of the
receptor, provide means other than! ligands or drugs to promote and stabilize the receptor in the active state
conformation. These means effectively stabilize the receptor in an active state by simulating the effect of a
Iigand binding to the receptor Stabilization by such ligand-independent means is termed "constitutive
receptor activation
RUP43

RUP43 (where it is understood that endogenous RUP43 may be GPR131, e.g. GenBank® Accession No. NM_J70699) has recently, been reported to act as a receptor for bile acid [European Patent
Application Number 02717114.9 published as EP1378749 on 07 January 2004; and Kawamata et aL, J Biol
i Chem (2003) 278:9435-9440; the disclosure of each of which is hereby incorporated by reference in its
entirety]. RUP43 expression within leukocytes was reported to be specific to monocytes, and bile acid acting at monocyte RTJP43 was reported to inhibit expression of tumor necrosis factor alpha (TNFa). Compounds disclosed in EP13787J49 may be used in methods of the subject invention.
SUMMARY OF THE INVENTION
Applicants have unexpectedly discovered that agonists of RUP43 increase glucose uptake in adipocytes and in skeletal musclelceUs. Applicants disclose that agonists of RUP43 have unexpected utility
for lowering blood glucose concentration in a mammal. Applicants ¦ further disclose novel compounds
i having agonist activity at RUP43 and uses therefor.
In a first aspect, the invention features a method of identifying one or more candidate compounds as a modulator of a RUP43 GPCR, said receptor comprising a GPR131 amino acid sequence, wherein the
receptor couples to a G protein; cpmpnsing the steps of.
i
(a) contacting the candidate compound with the receptor; and
i
(b) determining whether the receptor functionality is modulated;
wherein a change in receptor functionality is indicative of the candidate compound being a modulator of a RUP43 GPCR j
In certain embodiments, >the GPR131 amino acid sequence is selected from the group consisting of:
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(a) the amino acid sequence of SEQ ID NO:2;
(b) amino acids 2-330 of SEQ IB NO:2;
(c) amino acids 2-330 of SEQ ID NO:2i with the proviso that the RUP43 G protein-coupled
receptor does not comprise the methionine residue at amino acid position 1 of SEQ ID NO:2;
i
(d) the amino acid sequence of a G protein-coupled receptor encoded by a polynucleotide
comprising a nucleic acid sequence, said nucleic acid sequence being obtainable by a process comprising performing PCR on a human DNA sample using primers SEQ ID NO.3 and SEQ ID NO:4;
(e) the amino acid sequence of SEQ ID NO:6;
i
(f) the amino acid sequence of a G protein-coupled receptor encoded by a polynucleotide
comprising a nucleic acid sequence, said nucleic acid, sequence being obtainable by a process comprising
performing PCR on a human DNA sample using primers SEQ ID NO:7 and SEQ ID NO:8;
(g) the amino acid sequence of SEQ ID |NO:2 wherein the alanine at amino acid position 223
of SEQ JD NO:2 is substituted with lysine;
(h) ammo acids 2-330 of SEQ ID NO;2 wherein the alanine at amino acid position 223 of SEQ ID NO:2 is substituted with lysine;
(l) amino acids 2-330 of SEQ ID NO.2 ^herein the alanine at ammo acid position 223 of SEQ ED NO:2 is substituted with lysine, with the proviso! that the RUP43 G protein-coupled receptor does not comprise the methionine residue at amino acid position 1 of SEQ ID NO:2; and
Q) the amino acid sequence of a G protbin-coupled receptor encoded by a polynucleotide that hybridizes under stringent conditions to the complement of SEQ ID NO:1.
In certain embodiments, said RUP43 GPCR is recombinant In certain embodiments, said
contacting comprises contacting with a host cell or with membrane of a host cell that expresses the GPCR,
i
wherein said host cell comprises an expression vector! comprising a polynucleotide encoding the receptor.
In some embodiments, said contacting is carried out in the presence of a known ligand of the GPCR. In some embodiments, said contacting is earned out in the presence of a known modulator of the GPCR. In some embodiments, said contacting is carried out in the presence of a known agonist of the GPCR. In some embodiments, said known agonist of the GPCR is Compound 1, Compound 2, or Compound 3 In some embodiments, said known agonist of the GPCR is Compound 1. In some embodiments, said known agonist of the GPCR is Compound 2. In some embodiments, said known agonist of the GPCR is Compound 3. In some embodiments, said known agonist is present at about EC50 to about EC75 for the means of said determining
The invention also relates to a method of| identifying one or more candidate compounds as a modulator of blood glucose concentration in a mammal, comprising the steps of:
contacting the candidate compound with ajGPCR comprising a GPR131 amino acid sequence, wherein the receptor couples to a G protein; and
determining whether the receptor functionality is modulated;
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wherein a change in receptor functionality is indicative of the candidate compound being a modulator of blood glucose concentration in a mammal.
la certain embodiments, th'e GPR.131 amino acid sequence is selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:2;
(b) aniino acids 2-330 of SEQ ID NO:2;

(c) amino acids 2-330 of SEQ ID NO:2, with the proviso that the RUP43 G protein-coupled
receptor does not comprise the methionme residue at amino acid position 1 of SEQ ID NO.2;
(d) the aniino acid sequence of a G protein-coupled receptor encoded by a polynucleotide
comprising a nucleic acid sequence, said nucleic acid sequence being obtainable by a process comprising
performing PCR on a human DNA sample using primers SEQ ID NO:3 and SEQ ID NO:4;
(e) the'amino acid sequence of SEQ ID NO:6;,
(f) the amino acid sequence of a G protein-coupled receptor encoded by a polynucleotide
comprising a nucleic acid sequence, said nucleic acid sequence being obtainable by a process comprising
performing PCR on a human DNA sample using primers SEQ ID NO 7 and SEQ ID NO:S;
(g) the amino acid sequence of SEQ ID NO.2, wherein the alanine at amino acid position 223
of SEQ ID NO:2 is substituted with Iysine;
(h) amino acids 2-330 of SEQ ID NO.2 wherein the alanine at amino acid position 223 of SEQ ID NO:2 is substituted with lysme;
(i) amino acids 2-330 of SEQ ID NO:2 wherein the alanine at ammo acid position 223 of SEQ ID NO.2 is substituted with Iysine, with the proviso that the KUP43 G protein-coupled receptor does not comprise the methionine residue at amino acid position 1 of SEQ ID NO:2; and
0) the amino acid sequence of a G protein-coupled receptor encoded by a polynucleotide that hybridizes under stringent conditions to the complement of SEQ ID NO-1
In certain embodiments, an increase in receptor functionality is indicative of the candidate
i
compound being a compound that lowers blood glucose concentration in a mammal.
In certain embodiments, said GPCR is recombinant In certain embodiments, said contacting comprises contacting with a host cell or with membrane of a host cell that expresses the GPCR, wherein said host cell comprises an expression vector comprising a polynucleotide encoding the receptor.
In some embodiments, ;said contacting is carried out in the presence of a known ligand of the GPCR In some embodiments, said contacting is carried out in the presence of a known modulator of the GPCR. In some embodiments,! said contacting is carried out in the presence of a known agonist of the GPCR In some embodiments, said known agonist of the GPCR is Compound 1, Compound 2, or Compound 3 In some embodiments, said known agonist of the GPCR is Compound 1. In some embodiments, said known agonist of Hie GPCR is Compound 2. In some embodiments, said known agonist of the GPCR is Compound 3 In some embodiments, said known agonist is present at about EC50 to about EC75 for the means of said determining.
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In certain embodiments, said one or more candidate compounds is not an antibody or an antigen-binding derivative thereof
In certain embodiments, said one or more candidate compounds is not a peptide.
In certain embodiments, said one or more candidate compounds is not a bile acid.
In some embodiments, the GPR13I amino a'cid sequence is the amino acid sequence of SEQ ID NO:2. In some embodiments, theGPRBl amino acid sequence is a variant of the amino acid sequence of SEQ ID NO:2. In some embodiments, said variant of the amino acid sequence of SEQ ID NO:2 is an allelic variant or mammalian ortholog of said amino acid sequence. In some embodiments, said variant of the amino acid sequence of SEQ ID NO:2 is a non-endogenous, constitutively activated mutant of said amino acid sequence or of an allelic variant or mammalian ortholog of said amino acid sequence. In certain embodiments, said variant of the amino acid sequence of SEQ ID NO:2 is a biologically active fragment of
said amino acid sequence or of an allelic variant or mammalian ortholog of said amino acid sequence In
I certain embodiments, said biologically active fragment of the amino acid sequence of SEQ ID NO:2 or of
an allelic variant or mammalian ortholog of said amino acid sequence is the ammo acid sequence of SEQ ID NO:2 or of an aflelic variant or mammalian ortholog of said amino acid sequence absent the N-tenrunal methionine. In certain embodiments, said variant of jthe amino acid sequence of SEQ ID NO:2 is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at leastiabout 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, said variant of the amino acid sequence of SEQ ID NO:2 is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at leastjabout 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about]99% identical to the ammo acid sequence of SEQ ID NO:2.
In certain embodiments, said RUP43 GPCR bomprising a GPR131 amino acid sequence is a fusion protein further comprising one or more epitope tags. | In some embodiments, said fusion protein comprising one or more epitope tags is the amino acid sequence of SEQ ID NO:6.
In certain embodiments, said G protein leads to an increase m the level of intracellular cAMP In some preferred embodiments, said G protein is Gs.
In certain embodiments, said G protein is pertussis toxin sensitive. In certain embodiments, said G protein is Gi or Go In certain embodiments, said G protein is Gi. In certain embodiments, said G protein is Go.
In certain embodiments, said G protein is Gal 5 or Gal6. In certain embodiments, said G protein is Gal5. In certain embodiments, said G protein is Gal6.
In certain embodiments, said G protein is Gq.
In certain embodiments, said method furtiier|comprises the step of comparing the modulation of the receptor caused by the candidate compound to a second modulation of the receptor caused by contacting the receptor with a known modulator of the receptor. |In certain embodiments, said known modulator is an
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agonist. In certain embodiments, said agonist is Compound 1, Compound 2, or Compound 3. In certai embodiments, said agonist is Compound 1 In certain embodiments, said agonist is Compound 2. In certai embodiments, said agonist is Compound 3.
In some preferred embodiments, said determining or said comparing is through the measurement c GTPyS binding to membrane comprising said GPCR. In certain embodiments, said GTPyS is labeled wit
[35S]
In certain embodiments, said determining or said comparing is through the measurement of th level of a second messenger selected fiom the group consisting of cyclic AMP (cAMP), cyclic GM (cGMP), inositol triphosphate (Tp3), diacylglycerol (DAG), MAP kinase activity, and Ca2+. In certai preferred embodiments, said second messenger is cAMP. In certain preferred embodiments, the level c cAMP is increased. In certain embodiments, said measurement of cAMP is carried out using whole-ce adenylyl cyclase assay. In certain! embodiments, said measurement of cAMP is carried out with membran
comprising said GPCR In certain embodiments, said second messenger is MAP kinase activity. In certai
i embodiments, the level of MAP kinase activity is increased.
In some preferred embodiments, said determining or said comparing is through CRE-reporter assa; In certain embodiments, said reporter is luciferase. In some embodiments, said reporter is p-galactosidase.
!
In certain embodiments, said determining or said comparing is through measurement c intracellular IP3
In certain embodiments', said determining or said comparing is through measurement c intracellular Ca2+.
In certain embodiments,! said determining or said comparing is through measurement of glucos uptake by adipocytes obtained from a mammal
In certain embodiments,[said determining or said comparing is through measurement of glucos uptake by skeletal muscle cells obtained from a mammal.
In certain preferred embodiments, said determining or said comparing is through the use of Melanophore assay.
In a second aspect, the invention features a compound of Formula (II):

or a pharmaceutically acceptable salt thereof,
wherein1
R, is H or C^ alkyl;
R2 is a 2-methyl-4,5,6,7^tetrahydro-2H-indazol-3-yl group, or
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Ri and R2 together with the nitrogen to which they are bonded form a 3,4-dihydro-2H-quinoline-l-yl group, and
Rio and R] i are each independently H or halogen
In a third aspect, the invention features a modulator of a GPCR identified according to a method of the. first aspect In certain embodiments, the modulator is not an antibody or an antigen-binding derivative thereof. In certain embodiments, the modulator is not a peptide. In certain embodiments, the modulator is not a bile acid. In certain embodiments, the modulator is a compound that increases glucose uptake in adipocytes obtained from a mammal. In certain embodiments, the modulator is a compound that increases glucose uptake in skeletal muscle cells obtained from a mammal.
The invention also features a modulator of a GPCR identifiable according to a method of ^s first aspect, tn certain embodiments, the modulator is not an antibody or an antigen-binding derivative thereof. In certain embodiments, the modulator is not a peptide. In certain embodiments, the modulator is a compound that increases glucose uptake in adipocytes obtained from a mammal. In certain embodiments, the modulator is a compound that increases glucose uptake in skeletal muscle cells obtained from a mammal.
In certain embodiments, said modulator is [selected from the group consisting of agonist, partial
agonist, inverse agonist and antagonist. In certain embodiments, said modulator is an agonist In certain
i
embodiments, said modulator is a partial agonist. In certain embodiments, said modulator is an inverse agonist In certain embodiments, said modulator is an antagonist.
In certain embodiments, said modulator is [preferably an agonist. In certain embodiments, said agonist is a compound according to die second aspect
In some embodiments, said modulator is an agonist with an ECso of less than 10 uM, of less than 1
uM, of less than 100 nM, or of less than 10 nM. In some embodiments, said modulator is an agonist with an
i
EC50 of less than a value selected from the interval of 10 nM to 10 uM. In some embodiments, said modulator is an agonist with an EC50 of less than a jvalue selected from the interval of 10 nM to 1 uM. In some embodiments, said modulator is an agonist with an EC50 of less than a value selected from the interval
of 10 nM to 100 nM. In certain embodiments, said EC50 is determined using an assay selected from the
i group consisting of: whole cell cAMP assay learned using transfected HEK293 cells expressing
recombinant RUP43 GPCR polypeptide having the amino acid sequence of SEQ ID NO:2 or 6; and
1
melanophore assay carried out using transfected melanophores expressing recombinant RUP43 GPCR polypephde having the ammo acid sequence of SEQ ID NO:2 or 6. In some embodiments, said modulator is an agonist with an EC50 of less than 10 JIM, of less than 1 uM, of less than 100 nM, or of less than 10 nM in said assay. In some embodiments, said modulator is an agonist with an EC50 of less than 10 uM in said assay, of less than 9 uM in said assay, of less than ,8 uM in said assay, of less than 7 \xlA in said assay, of less than 6 uM in said assay, of less than 5 uM in said assay, of less than 4 uM in said assay, of less than 3 pM in said assay, of less than 2 uM in said assay, of less than 1 uM in said assay, of less than 900 nM in
said assay, of less than 800 nM in said assay, of less than 700 nM in said assay, of less than 600 nM in said
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assay, of less than 500 nM in said assay, of less than 400 nM in said assay, of less than 300 nM m said assay, of less than 200 nM in said assay, of less than 100 nM in said assay, of less than 90 nM in said assay, of less than 80 nM in said assay, of less than 70 nM in said assay, of less than 60 nM in said assay, of less than 50 nM in said assay, of less than 40 nM n said assay, of less than 30 nM in said assay, of less than 20 nM in said assay, or of less than 10 nM in said assay. In some embodiments, said modulator is an agonist with an EC50 in said assay of less than a value selected from the interval of 10 nM to 10 uM. In some embodiments, said modulator is art agonist with an EC50 in said assay of less than a value selected from the interval of 10 nM to 1 uM. In some embodiments, said modulator is an agonist with an EC50 in said assay of less than a value selected from the interval of 10 nM to 100 nM.
In some embodiments, said modulator is selective for the GPCR.
In some embodiments, said modulator is Compound 1 ("Cmpd#l, see Table 1), Compound 2
("Cmpd#2", see Table 1), or Compound 3 ("Cmpd#3", see Table 1). In some embodiments, said modulator
is Compound 1 In some embodiments, said modulator is Compound 2. In some embodiments, said
modulator is Compound 3. j
In some embodiments, said modulator is orally bioavailable In some embodiments, said oral bioavailabihty is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%3 or at least 45% relative to intraperitoneal administiation. In some embodiments, said oral bioavailablity is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative to intraperitoneal administration
In some embodiments, said orally bioavailable modulator is further able to cross the blood-brain
barrier. ¦
In a. fourth aspect, the invention features a method of preparing a pharmaceutical or physiologically acceptable composition comprising admixing a carrier and a modulator of a RUP43 GPCR, said receptor comprising a GPR131 amino acidisequence. hi certain embodiments, the modulator is not an antibody or an antigen-binding derivative thereof. In certain embodiments, the modulator is not a peptide. In certain embodiments, the modulator is a: compound that increases glucose uptake in adrpocytes obtamed from a mammal. In certain embodiments, the modulator is a compound that increases glucose uptake in skeletal muscle cells obtained from a mammal. In certain embodiments, the modulator is selected from the group consisting of agonist, partial agonist, inverse agonist, and antagonist. In certain embodiments, the modulator is an agonist. In certain embodiments, the modulator is a partial agonist. In certain embodiments, the modulator is an inverse agonist In certain embodiments, the modulator is an antagonist. In certain embodiments, the modulator is preferably an agonist In certain embodiments, said agonist is a compound according to the second aspect
The invention also features a method of preparing a pharmaceutical or physiologically acceptable composition which comprises identifying a modulator of a RUP43 GPCR, wherein said receptor comprises a GPR131 ammo acid sequence, identifiable by a method according to a method of fas first aspect In certain embodiments, the modulator is
1
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identified according to a method of the first aspect In certain embodiments, the modulator is not an
antibody or an antigen-binding derivative thereof. In certain embodiments, the modulator is not a pcptide

In certain embodiments, the modulator is preferably an agonist.! In certain embodiments, the modulator is a
compound that increases glucose uptake in adipocytes obtained from a mammal. In certain embodiments,
the modulator is a compound that increases glucose uptake m skeletal muscle cells obtained from a mammal In certain embodiments, the modulator is selected from the group consisting of agonist, partial agonist, inverse agonist, and antagonist In certain embodiments, the modulator is an agonist In certain embodiments, the modulator is a partial agonist In certain embodiments, the modulator is an inverse agonist. In certain embodiments, the modulator is an antagonist. In certain embodiments, the modulator is preferably an agonist In certain embodiments, said agonist is a compound according to the second aspect.
In certain embodiments, said composition .is pharmaceutical. In certain embodiments, said
composition is physiologically acceptable.
In some embodiments, said modulator is an agonist with an EC50 of less than 10 uM, of less than 1 (iM, of less than 100 nM, or of less than lOnM. In some embodiments, said modulator is an agonist with an ECso of less than a value selected from the interval}of 10 nM to 10 \iM. In some embodiments, said modulator is an agonist with an ECS0 of less than a value selected from the interval of 10 nM to 1 uM. In some embodiments, said modulator is an agonist with an EC50 of less than a value selected from the interval of 10 nM to 100 nM. In certain embodiments, said EC50 is determined using an assay selected from the group consisting of: whole cell cAMP assay earned using transfected HEK293 cells expressing recombinant RUP43 GPCR polypepude having the amino acid sequence of SEQ ID NO:2 or 6; and iuelanophore assay carried out using transfected melanophores expressing recombinant RUP43 GPCR polypeptide having the ammo acid sequence of SEQ ID NO:2 or 6 In some embodiments, said modulator is an agonist with an EC50 of less than 10 uM, of less than 1 uM, of less than 100 nM, or of less than 10 nM in said assay In some embodiments, said modulator is an agonist with an ECSo of less than 10 uM in said assay, of less than 9 u,M in said assay, of less than 8 M in said assay, of less than? uM in said assay, of less than 6 uM in said assay, of less than 5 uM in said' assay, of less than 4 uM in said assay, of less than 3 uM in said assay, of less than 2 uM in said assay, of less than I uM in said assay, of less than 900 nM in said assay, of less than 800 nM in said assay, of less than 700 nM in said assay, of less than 600 nM in said assay, of less than 500 nM in said assay, of less than 400 nM in said assay, of less than 300 nM in said assay, of less than 200 nM in said assay, of less than 100 nMinsaid assay, of less than 90 nM in said assay, of less than 80 nM in said assay, of less than 70 nM in said assay, of less than 60 nM in said assay, of less
than 50 nM in said assay, of less than 40 nM n said assay, of less than 30 nM in said assay, of less than 20
1
nM in said assay, or of less than 10 nM in said assay.] In some embodiments, said modulator is an agonist with an EC50 in said assay of less than a value selected from the interval of 10 nM to 10 uM. In some embodiments, said modulator is an agonist with an EGS0 in said assay of less than a value selected from the interval of 10 nM to 1 uM. In some embodiments, said modulator is an agonist with an EC50 in said assay of less than a value selected from the interval of 10 nM to 100 nM.
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In some embodiments, said modulator is selective for the GPCR.
In some embodiments, said modulator is Compoundi 1, Compound 2, or Compound 3. In some
embodiments, said modulator is Compound 1, In some embodiments, said modulator is Compound 2. In
some embodiments, said modulator is Compound 3.
In some embodiments, said modulator is orally biqavailable. In some embodiments, said oral
bioavailabdiry is at least 1%, at least] 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%,
at least 35%, at least 40%, or at least 45% relative to intraperitoneal administration. In some embodiments,
said oral bioavauablity is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45%
relative to lnirapentoneal administration,
In some embodiments, said orally bioavailable modulator is further able to cross the blood-brain
barrier
In a fifth aspect, the invenhpn features a method of modulating the activity of a RXJP43 GPCR, said receptor comprising a GPR131 amino acid sequence, comprising the step of contacting the receptor with a modulator of the receptor. In certain embodiments, the modulator is identifiable by a method according to a method of 1he first aspect. In certain embodiments, the modulator is identified according to a method of the
first aspect In certain embodiments, the modulator is not an antibody or an antigen-binding derivative
thereof. In certain embodiments, the modulator is not a peptide. hi certain embodiments, the modulator is
selected from the group consisting of agonist, partial agonist, inverse agonist, and antagonist. In certain embodiments, the modulator is aniagonist. In certain embodiments, the modulator is a partial agonist In certain embodiments, the modulator is an inverse agonist. In certain embodiments, the modulator is an antagonist In certain embodiment, the modulator is preferably an agonist. In certain embodiments, the modulator is a compound that increases glucose uptake in adipocytes obtained from a mammal. In certain embodiments, the modulator is a compound that increases glucose uptake in skeletal muscle cells obtained from a mammal. In certain embodiments, said agonist is a compound according to the second aspect.
The invention also features a method of modulating the activity of a RUP43 GPCR, said receptor
i comprising a GPR131 amino acid sequence, comprising the! step of contacting the receptor with a modulator
of Hie receptor, wherein the mbdulator is identifiable by a method of the first aspect In certain embodiments, the modulator is identified according to a method of the first aspect. In certain embodiments, the modulator is not an antibody or an antigen-binding derivative, thereof, hi certain embodiments, the modulator is not a peptide. In certain embodiments, the modulator is a compound that stimulates glucose uptake in adipocytes obtained from a mammal. In certain embodiments, the modulator is a compound that stimulates glucose uptake in skeletal muscle cells obtained from a mammal. In certain embodiments, the modulator is selected from, the group consisting of agonist, partial agonist, inverse agonist, and antagonist In certain embodiments, the modulator is an agonist In certain embodiments, the modulator is a partial agonist hi certain embodiments, the modulator is an,inverse agonist In certain embodiments, the modulator is an antagonist, m certain embodiments, the modulator is preferably an agonist In certain embodiments, said agonist is a compound according to the second aspect.
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In some embodiments, said modulator is an agonist with an EC50 of less than 10 uM, of less than 1 uM, of less tiian 100 nM, or of less than 10 nM. In some embodiments, said modulator is an agonist with an ECW of less than a value selected from the interval of 10 nM to 10 M. In some embodiments, said modulator is an agonist with an EC50 of less than a v,alue selected from the interval of 10 nM to 1 uM. In some embodiments, said modulator is an agonist with an EC50 of less than a value selected from the interval of 10 nM to 100 nM. In certain embodiments, said|EC50 is determined using an assay selected from the
group consisting of: whole cell cAMP assay tarried using transfected HEK293 cells expressing
i recombinant RUP43 GPCR polypeptide having the ammo acid sequence of SEQ ID NO:2 or 6; and
melanophore assay carried out using transfected melanophores expressing recombinant RUP43 GPCR polypeptide having the ammo acid sequence of SEQ ID NO:2 or 6. In some embodiments, said modulator is an agonist with an ECS0 of less than 10 uM, of less than 1 uM, of less than 100 nM, or of less than 10 nM in said assay. In some embodiments, said modulator is an agonist with an EC50 of less than 10 uM in said assay, of less than 9 uM in said assay, of less than S uM in said assay, of less than 7 uM in said assay, of [ess than 6 uM in said assay, of less than 5 uM in said assay, of less than 4 uM in said assay, of less than 3 uM in said assay, of less than 2 uM in said assay, of less than 1 uM in said assay, of less than 900 nM in said assay, of less than 800 nM in said assay, of less [than 700 nM in said assay, of less than 600 nM in said assay, of less than 500 nM in said assay, of less than 400 nM in said assay, of less than 300 nM in said assay, of less than 200 nM in said assay, of less than 100 nM in said assay, of less than 90 nM in said assay, of less than 80 nM in said assay, of less than 70 nMl in said assay, of less than 60 nM in said assay, of less than 50 nM in said assay, of less than 40 nM n said assay, of less than 30 nM in said assay, of less than 20 nM in said assay, or of less than 10 nM in said assay. In some embodiments, said modulator is an agonist
with an ECso in said assay of less than a value selected from the interval of 10 nM to 10 uM. In some
i embodiments, said modulator is an agonist with an EC50 in said assay of less than a value selected from the
interval of 10 nM to 1 uM. In some embodiments, said modulator is an agonist with an EC50 in said assay of less than a value selected from the interval of 10 nivl to 100 nM.
In some embodiments, said modulator is selective forthe GPCR.
In some embodiments, said modulator is Compound 1, Compound 2, or Compound 3. In some
embodiments, said modulator is Compound 1. In sbme embodiments, said modulator is Compound 2 In
some embodiments, said modulator is Compound 3.
In some embodiments, said modulator is 'orally bioavailable. In some embodiments, said oral bioavailability is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%,
at least 35%, at least 40%J or at least 45% relative to intraperitoneal administration. In some embodiments,
i
said oral bioavailablity is at least 20%, at least 25%,]at least 30%, at least 35%, at least 40%, or at least 45% relative to intraperitoneal administration.
In some embodiments, said orally bioavailable modulator is further able to cross the blood-brain
barrier.
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In certain embodiments, said contacting comprises administration of the modulator to a membrane comprising the receptor.
In certain embodiments, said contacting comprises administration of the modulator to a cell comprising the receptor
In certain embodiments, said contacting comprises administration of the modulator to a tissue comprising the receptor.
In certain embodiments, saijd contacting comprises administration of the modulator to an individual comprising the receptor. In certain embodiments, said administration of the modulator to an individual comprising the receptor is oral ¦ In certain embodiments, said individual is a mammal. In certain embodiments, said individual is a non-human mammal. In certain embodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit, rflouse, rat, non-human primate or human. In certain embodiments, said mammal is a mouse, rat, non-humap. primate, or human. Most preferred is human.
In a sixth aspect, the invention features a method of modulating the activity of a RUP43 GPCR, said receptor comprising a GPR131 amino acid sequence, wherein said modulation is for lowering blood glucose concentration in an individual in need of said modulation, comprising contacting said receptor with a therapeutically effective amount of a modulator of the receptor. In certain embodiments, Ihe modulator is an agonist
The invention also feature, s a method of modulating the activity of a R.UP43 GPCR, said receptor
i
comprising a GPR131 amino ac^id sequence, wherein said modulation is for preventing or treating a metabolic disorder in an individual in need of said modulation, comprising contacting said receptor with a therapeutically effective amount of a modulator of the receptor. In certain embodiments, the modulator is an agonist, m certain embodiments, the metabolic disorder is selected from the group consisting of:
(a) diabetes;
(b) impaired glucose; tolerance;
(c) insulin resistance; and
(d) hyperinsulinemia.
In some embodiments, diabetes is type 1 diabetes. In certain preferred embodiments, diabetes is
I 4
type 2 diabetes. In certain embodiments, the metabolic disorder is diabetes. In certain embodiments, the
i metabolic disorder is type 1 diabetes. In'certain embodiments, the metabolic disorder is type 2 diabetes. In
certain embodiments, the metabolic disorder is impairedi glucose tolerance. In certain embodiments, the metabolic disorder is insulin1 resistance. In certain embodiments, the metabolic disorder is hyperinsuhnemia. In certain embodiments, the metabolic disorder is related to an elevated blood glucose concentration in the individual.
The invention also features a method of modulating the activity of a RUP43 GPCR, said receptor comprising a GPR131 amino acid sequence, wherein said modulation is for preventing or treating a complication of an elevated blood glucose concentration in an individual in need of said modulation,
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comprising contacting said receptor with a therapeuUcally effective amount of a modulator of the receptor. In certain embodiments, the modulatoi is an agonist [n certain embodiments, the complication is selected from the group consisting of;
(a) Syndrome X;
(b) atherosclerosis;
(c) atheromatous disease;
(d) heart disease;
(e) hypertension;
(f) stroke;
(g) neuropathy;
(h) retinopathy;
(1) nephropathy; and
(j) peripheral vascular disease.
Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, coronary artery disease, and high blood pressure. In certain embodiments, the complication is Syndrome X. In certain embodiments, the complication is atherosclerosis. In certain embodiments, the complication is atheromatous disease. In certain embodiments, the complication is heart disease. In certain embodiments, the complication is cardiac insufficiency. In certain embodiments, the complication is coronary insufficiency. In certain embodiments, the complication is coronary artery disease. In certain embodiments, the complication is coronary artery disease. In certain embodiments, the complication is high blood pressure. La certain embodiments, the complication is hypertension. In certain embodiments, the complication is stroke. In certain embodiments, the complication is neuropathy. In certain embodiments, die complication is retinopathy. In certain embodiments, the complication is neuropathy. In certain embodiments, the complication is peripheral vascuiari disease. In certain embodiments, the complication is polycystic ovary syndrome. In certain embodiments, the complication is hyperlipidemia.
In certain embodiments, the modulator is identifiable by a method according to a method of ihe first aspect. In certain embodiments, the modulator is identified according to a method of ihe first aspect. In certain embodiments, the modulator is not an antiboc y or an antigen-binding derivative thereof In certain embodiments, the modulator is not a peptide. In certain embodiments, the modulator is a compound that stimulates glucose uptake in adipocytes obtained from a mammal. In certain embodiments, the modulator is a compound that stimulates glucose uptake in skeletal muscle cells obtained from a mammal. In certain embodiments, said modulator is selected from the group consisting of agonist, partial agonist, inverse agonist, and antagonist In certain preferred, embodiments, said modulator is an agonist. In certain embodiments, said agonist is a compound according t) the second aspect.
In certain embodiments, said modulator is selective for the GPCR
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In some embodiments, said modulator is Compound 1, Compound 2, or Compound 3. In some
embodiments, said modulator is Compound 1. In some embodiments, said modulator is Compound 2 In
i i
some embodiments, said modulator is Compound 3.
In certain embodiments, said modulator is orally bioavailable. In some embodiments, said oral bioavailability is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, al least 30%, at least 35%, at least 40%, or at least 45% relative to intraperitoneal administration.. In some embodiments, said oral bioavailablity is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative to intraperitoneal administration.
In certain embodiments, said orally bioavailable modulator is further able to cross the blood-brain
barrier. j l
i In some embodiments, said modulator is an agonist with an EC50 of less than 10 jiM, of less than 1
pM, of less than lOOnM, oroflessithan lOnM. In some embodiments, said modulator is an agonist with an EC50 of less than a value selected from the interval of 10 nM to 10 uM. iln some embodiments, said modulator is an agonist with an EC5D of less than a value selected from the interval of 10 nM to 1 pM. In some embodiments, said modulator is an agonist with an EC50 of less than a value selected from the interval
of 10 nM to 100 nM. In certain embodiments, said EC50 is determined using an assay selected from the
i
group consisting of: whole cell cAMP assay carried using transfected HEK293 cells expressing
recombinant RUP43 GPCR polypeptide having the amino acid sequence of SEQ ID NO:2 or 6; and
1 melanophore assay carried out using transfected melanophores expressing, recombinant RUP43 GPCR
1 polypeptide having the ammo acid sequence of SEQ ID NO:2 or 6. In some embodiments, said modulator
1 is an agonist with an EC50 of less than 10 uM, of less than 1 uM, of less than 100 nM, or of less than 10 nM
in said assay. In some embodiments, said modulator is an agonist with an EC50 of less than 10 uM in said assay, of less than 9 uM in said assay, of less than 8 uM in said assay, of less than 7 uM in said assay, of less than 6 pM in said assay,, of less fhan'5 uM in said assay, of less than 4 uM in said assay, of less than 3 uM in said assay, of less than 2 uM in said assay, of less than 1 uM in said assay, of less than 900 nM in said assay, of less than 800 nM in said assay, of less than 700 nM in said assay, of less than 600 nM in said assay, of less than 500 nM in said assay, of less than 400 nM in said assay, of less than 300 nM in said assay, of less than 200 nM in said assay, of less than 100 nM in said assay, of less than 90 nM in said assay, of less than 80 nM in said assay,1 of less than 70 nM in said assay, of less than 60 nM in said assay, of less than 50 nM in said assay, of less1 than 40 nM n said assay, of less than 30 nM in said assay, of less than 20 nM in said assay, or of less than 10 nM in said assay. In some embodiments, said modulator is an agonist with an EC50 in said assay of less than a value selected from the interval of 10 nM to 10 uM. In some embodiments, said modulator is an agonist with an EQo in said assay of less than a value selected from the interval of 10 nM to 1 uM. In some embodiments, said modulator is an agonist with an EC5D in said assay ofless than a value selected from the interval of 10 nM to 100 nM.
In certain embodiments, said contacting comprises oral administration of said modulator to said individual.
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' '* PCT/US2005/012447
In certain embodiments, said individual is a mammal. In certain embodiments, said individual is a non-human mammal. In certain embodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit, mouse, rat, non-human pnmate or human. In certain embodiments, said mammal is a mouse, rat, non-human primate, or human. Most preferred is human.
In a seventh aspect, the invention features a riethod of lowering blood glucose concentration in an individual in need of said lowering, comprising contacting a therapeutically effective amount of a modulator of a RUP43 GPCR with said receptor, said GPCR comprising a GPR131 amino acid sequence. In certain embodiments, the modulator is an agonist
The invention additionally features a method of lowering blood glucose concentration in a mammal comprising providing or administering to a mammal in need of said lowering a modulator of RUP43 GPCR, said GPCR comprising a GPR131 amino acid sequence. In certain embodiments, the modulator is an agonist. In certain embodiments, the agonist of RUP43 GPCR is an agonist of GPR131 GPCR, where it is understood that GPR131 GPCR is endogenous RUP43 GPCR.
The invention also features a method,of preventing or treating a metabolic disorder in an individual in need of said prevention! or treatment, comprising contacting a therapeutically effective amount of a modulator of a RUP43 GPGR with said receptor, said receptor comprising a GPR131 amino acid sequence.
In certain' embodiments, the modulator is an agonist. In certain embodiments, the metabolic disorder is
I selected from the group consisting of:
(a) diabetes; (
(b) impaired glucose tolerance;
(c) insulin resistance; and
(d) hyperinsulinemia.
The invention additionally features; a method of preventing or treating a metabolic disorder comprising administering to a mammal in need of ^aid prevention or treatment a modulator of RUP43 GPCR, said receptor comprising a GPR131 amino acid sequence. In certain embodiments, the modulator is an agonist. In certain embodiments, the agonist of RUP43 GPCR is an agonist of GPR131 GPCR, where it is understood (hat GPRl'31, GPCR is endogenous RUP43 GPCR. In certain embodiments, the metabolic disorder is selected from the group consisting of:
(a) ' " diabetes; (b) impaired glucose tolerance;
(c) insulin resistance; and
(d) hyperinsulinemia. j
In some embodiments, diabetes is type 1 diabetes, In certain preferred embodiments, diabetes is type 2 diabetes. In certaini embodiments, the metabolic disorder is diabetes. In certain embodiments, the metabolic disorder is type 1 diabetes. In certain embodiments, the metabolic disorder is type 2 diabetes. In certain embodiments, the metabolic disorder is unpaired glucose tolerance. In certain embodiments, the metabolic disorder is insulin resistance. In \ certain embodiments, the metabolic disorder is
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PCT/US2005/012447
i bypennsulinerrua. In certain embodiments, the metabolic disorder is related to an elevated blood glucose
concentration in the individual. |
i
The invention also features |a method of preventing or treating a complication of an elevated blood glucose concentration in an individual in need of said prevention or treatment, comprising contacting a therapeuticaHy effective amount of a modulator of a RUP43 GPCR with said receptor, said receptor comprising a GPR131 anaino acid sequence. In certain embodiments, the modulator is an agonist. In certain embodiments, the complication is selected from the group consisting of.
(a) Syndrome X, (b) atherosclerosis; I
(c) atheromatous disease;
(d) heart disease;
(e) hypertension,
(f) stroke;
(g) neuropathy;
(h) rennopathy; j
(i) nephropathy; and
(j) peripheral vascular disease.
Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, coronary artery disease, and high blood pressure. In certain embodiments, the complication is Syndrome X. In certain embodiments, the complication is atherosclerosis. In certain embodiments, the complication is atheromatous disease, hi certain embodiments, the complication is heart disease. In certain embodiments, the complication is cardiac insufficiency. In certain embodiments, the complication is coronary insufficiency. In certain embodiments, the complication is coronary artery disease. In certain embodiments, the complication is high blood pressure. In certain embodiments, the complication is hypertension. In certain embodiments, the complication is stroke. In certain embodiments, the complication is neuropathy. In certain embodiments, the complication is retinopathy.l In certain embodiments, the complication is neuropathy. In certain embodiments,' the complication is peripheral vascular disease. In certain embodiments, the complication is polycystic ovary syndrome. In certain embodiments, the complication is hyperlipidemia.
The invention additionally features a method of preventing or treating a complication of an elevated blood glucose concentration comprising providing or administering to a mammal in need of said prevention or treatment a modulator of RUP43 GPCR, said receptor comprising a GPR131 amino acid sequence. In certain embodiments, the modulator is an agonist In certain embodiments, ,the agonist of RUP43 GPCR is an agonist of GPR131 GPCR, where it is understood that GPR131 GPCR is endogenous RUP43 GPCR. In certain embodiments, the complication is selected from the group consisting of:
(a) Syndrome X; ,
(b) atherosclerosis,
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(c) atheromatous disease;
(d) heart disease;
(e) hypertension;
(f) stroke,
(g) neuropathy;
(h) retinopathy;
(1) nephropathy; and
0) peripheral vascular disease
Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, coronary artery disease, and high blood pressure. In certain embodiments, the complication is Syndrome X In certain embodiments, the complication is atherosclerosis. In certain embodiments, the complication is atheromatous disease. In certain embodiments, the complication is heart disease. In certain embodiments, the complication is cardiac insufficiency, hi certain embodiments, the complication is coronary insufficiency, in certain embodiments, the complication is coronary artery disease. In certain embodiments, the complication is high blood pressure. In certain embodiments, the complication is hypertension. In certain embodiments, the complication is stroke. In certain embodiments, the complication is neuropathy. hi certain embodiments, the complication is retinopathy. hi certain embodiments, the complication is neuropathy. In certain embodiments, the complication is peripheral vascular disease. In certain embodiments, the complication is polycystic ovary syndrome. In certain embodiments, the complication is hyperlipidemia.
In certain embodiments, the modulator is identifiable by a method according to a method of ih&first aspect. In certain embodiments, the modulator is identified according to a method of ihe first aspect In certain embodiments, the modulator is not an antibody or an antigen-binding derivative thereof. In certain embodiments, the modulator is not a peptide. In certain embodiments, the modulator is a compound mat stimulates, glucose uptake m adipocytes obtained from a mammal, hi certain embodiments, the modulator is a compound that stimulates glucose uptake in adipocytes obtained from the mammal, hi certain embodiments, the modulator is a compound that stimulates glucose uptake in skeletal muscle cells obtained from a mammal. In certain embodiments, the modulator is a compound that stimulates glucose uptake in skeletal muscle cells obtained from the mammal, hi certain embodiments, said modulator is selected from the group consisting of agonist, partial agonist, inverse agonist, and antagonist. In certain preferred embodiments, said modulator is an agonist, hi certain embodiments, said agonist is a compound according
to the second aspect
i hi certain embodiments, said modulator is selective for the GPCR.
hi some embodiments, said modulator is Compound 1, Compound 2, or Compound 3. In some embodiments, said modulator is Compound 1. In seme embodiments, said modulator is Compound 2. In
some embodiments, said modulator is Compound 3.
I
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In certain embodiments, Said modulator is orally bioavailable In some embodiments, said oral bioavailabxiity is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative to intrapentoneal administration. In some embodiments, said oral bioavailablity is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative to intrapentoneal administration.
In certain embodiments, said orally bioavailable modulator is further able to cross the blood-brain barrier.
In some embodiments, said modulator is an agonist with an EC50 of less than 10 uM, of less than 1 \M, of less than 100 nM, or of less than 10 nM. In some embodiments, said modulator is an agonist with an EC50 of less than a value selected fromthe interval of 10 nM to 10 uM. In some embodiments, said modulator is an agonist with an EC50 of less than a value selected from the interval of 10 nM to 1 uM. In some embodiments, said modulator is an agonist wife an EC50 of less than a value selected from the interval of 10 nM to 100 nM. In certain'embodiments, said EC50 is determined using an assay selected from the group consisting of: whole cell cAMP assay carried using transfected HEK293 cells expressing recombmant RUP43 GPCR polypeptide having the ammo acid sequence of SEQ ID NO:2 or 6; and melanophore assay carried out using transfected melanophores expressing recombmant RUP43 GPCR polypepude having the amino acid sequence of SEQ ID NO:2 or 6. In some embodiments, said modulator is an agonist with an EC50ofIess than 10 uM, of less than 1 uM, of less than 100 nM, or of less than 10 nM in said assay. In some embodiments, said modulator is an1 agonist with an EC50 of less than 10 uM in said assay, of less than 9 uM in said assay, of less than 8 uM in said assay, of less than 7 uM in said assay, of less than 6 pM in said assay, of less than 5 ^M in said assay, of less than 4 uM in said assay, of less than 3 uM in said assay, of less than 2 \M in said assay, of less than 1 uM in said assay, of less than 900 nM in ' said assay, of less than 800 nM in said assay, of less than 700 nM in said assay, of less than 600 nM in said assay, of less than 500 nM in said assay, of less than 400 nM in said assay, of less than 300 nM in said assay, of less than 200 sM in said assay, !of less than 100 nM in said assay, of less than 90 nM in said assay, of less than 80 nM in said assay, of less than 70 nM in said assay, of less than 60 nM in said assay, of less than 50 nM in said assay, of less than 40 nM n said assay, of less than 3 0 nM in said assay, of less than 20 nM in said assay, or of less than 10 nM in said assay. In'some embodiments, said modulator is an agonist with an ECS0 in said assay of less than a value selected from the interval of 10 nM to 10 nM. In some embodiments, said modulator is an agonist with an EC50 in said assay of less than a value selected from the interval of 10 nM to 1 uM. In some embodiments, said modulator is an agonist with an ECi0 in said assay of less than a value selected from the interval of 10 nM to =100 nM.
In certain embodiments, said contacting comprises oral administration of said modulator to said individual.
In certain embodiments, said individual is a mammal. In certain embodiments, said individual is a non-human mammal. In certaiii embodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit,
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mouse, rat, non-human primate or human. In certain embodiments, said mammal is a mouse, rat, non-human primate, or human. Most preferred is human.
In an eiglttlt aspect, the invention, features a pharmaceutical or physiologically acceptable composition comprising, consisting essentially of, or consisting of a modulator a RUP43 GPCR, said receptor comprising a GPR131 amino acid sequence.
In. certain embodiments, the modulator is identifiable by a method according to a method of ihe first aspect. In certain embodiments, the modulator is identified according to a method of the first aspect. In certain embodiments, the modulator is not an antibody or an antigen-binding derivative thereof. In certain embodiments, the modulator is not a peptide. ¦ In certain embodiments, the modulator is a compound that stimulates glucose uptake in adipocytes obtained from i mammal. In certain embodiments, the modulator is a compound that stimulates iglucose uptake in skeletal muscle cells obtained from a mammal. In certain embodiments, said modulator is selected from the group consisting of agonist, partial agonist, inverse agonist, and antagonist In certain preferred emboiiiments, said modulator is an agonist. In certain embodiments, said agonist is a compound according to the second aspect
In certain embodiments, said composition is pharmaceutical. In certain embodiments, the
i pharmaceutical composition1 comprises the modulator of a RUP43 GPCR. In certain embodiments, the
pharmaceutical composition consists essentially of the modulator of a RUP43 GPCR. In certain embodiments, the pharmaceutical composition conisists of the modulator of a RUP43 GPCR.
In certain embodiments, said composition is physiologically acceptable. In certain embodiments, the physiologically acceptable composition compnsis the modulator of a RUP43 GPCR. In certain embodiments, the physiologically acceptable composition consists essentially of the modulator of a RUP43 GPCR. In certain embodiments, the physiologically acceptable composition consists of the modulator of a RUP43 GPCR.
In certain embodiments, said modulator is selective for the GPCR.
In some embodiments, said modulator is Compound 1, Compound 2, or Compound 3. In some embodiments, said modulator is Compound 1. In some embodiments, said modulator is Compound 2. In some embodiments, said modulator is Compound 3.
In certain embodiments, said modulator is orally bioavailable. In some embodiments, said oral bioavailabihty is at least 1%, at least 5%, at least 10%j at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative to ihtrapentoneal administration. In some embodiments, said oral bioavailabhty is at least 20%, at least 25%, at least 30°^, at least 35%, at least 40%, or at least 45% lelative to intraperitoneal administration.
In certain embodiments, said orally bioavailable modulator is further able to cross the blood-brain barrier.
In some embodiments, said modulator is an agonist with an ECSo of less than 10 uM, of less than 1 uM, of less than 100 nM, or.of less than 10 nM. In some embodiments, said modulator is an agonist with an EC50 of less than a value selected from the interval; of 10 nM to 10 uM. In some embodiments, said
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modulator is an agonist with aa EC5i of less than a value selected from the interval of 10 nM to 1 uM. In some embodiments, said modulator is an agonist with an EC50 of less than a value selected from the interval of 10 nM to 100 nM In certain embodiments, said EC50 is determined \ising;an assay selected from the group consisting of: whole cell cAMP assay carried using transfected HEK293 cells expressing recombinant RUP43 GPCR polypeptide having the amino acid sequence of SEQ ID NO:2 or 6, and melanophore assay carried out using transfected melanophores expressing recombinant KUP43 GPCR polypeptide having the amino acid sequence'of SEQ ED NO:2 or 6. In some embodiments, said modulator
is an agonist with an EC50 of less than 10 uM, of less than 1 uM, of less than 100 nM, or of less than 10 nM
I in said assay. In some embodiments, said modulator is an agonist with an EC50 of less than 10 u.M in said
assay, of less than 9 uM in said assay, of less than 8 uM in said assay, of lessithan 7 uM in said assay, of less than 6 uM in said assay, of less! than 5 uM in said assay, of less man 4 JJM in said assay, of less than 3 u-M in said assay, of less man 2 jityl in said assay, of less than 1 uM in said assay, of less than 900 nM in
said assay, of less than 800 nM in said assay, of less than 700 nM in said assay, of less than 600 nM in said
1
assay, of less than 500 nM in said [assay, of less than 400 nM in said assay, of less than 300 nM in said
1 ¦
assay, of less than 200 nM in said assay, of less than 100 nM in said assay, of less than 90 nM in said assay,
I of less than 80 nM in said assay, of less than 70 nM in said assay, of less than 60 nM in said assay, of less
than 50 nM in said assay, of less than 40 nM 11 said assay, of less than 30 nM.in said assay, of less than 20
1 nM in said assay, or of less than 10 nM in said assay. In some embodiments, said modulator is an agonist
with ari EC50 in said assay of less than a value selected from the interval of 10 nM to 10 uM. In some embodiments, said modulator is an agonist with an EC50 in said assay of less man a value selected from the interval of 10 nM to 1 uM. In some embodiments, said modulator is an agonist with an ECSQ in said assay of less than a value selected from the interval of 10 nM to 100 nM.
In a ninth aspect, the invention features a melhod of lowering blood glucose concentration comprising providing or administering to an individual in, need of said lowering said pharmaceutical or physiologically acceptable composition of the eighth aspect.
The invention also features a method of preventing or treating a metabolic disorder comprising providing or administering to an individual in need of said prevention or treatment said pharmaceutical or physiologically acceptable composition of the eighth aspect. In certain embodiments, the metabolic disorder is selected from the group consisting of;
(a) diabetes;
(b), impaired glucose tolerance;
(c) insulin resistance; and
(d) hypennsulinemia. '
In some embodiments, diabetes is type 1 diabetes. In certain preferred embodiments, diabetes is type 2 diabetes. In certain embodiments, the metabolic disorder is diabetes. In certain embodiments, the metabolic disorder is type 1 diabetes. In1 certain embodiments, the metabolic disorder is type 2 diabetes. In
certain embodiments, the metabolic disorder is impaired glucose tolerance. In certain embodiments, the
I
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metabolic disorder is insulin resistance. In certain embodiments, the metabolic disorder is hyperinsulinemia. In certain embodiments, the metabolic disorder is related to an elevated blood glucose concentration in the individual.
The invention also features a method of preventing or treating a complication of an elevated blood glucose concentration comprising providing or administering to an individual in need of said prevention or treatment said pharmaceutical or physiologically ace ^ptable composition of the eighth aspect In certain embodiments, the complication is selected from the group consisting of:
(a) Syndrome X; ;
(b) atherosclerosis; ,
(c) atheromatous disease;
(d) heart disease;
(e) hypertension;
(f) stroke;
(g) neuropathy;
(h) retinopathy;
(i) nephropathy; and
(j) peripheral vascular disease. .
Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, coronary artery disease, and high blood pressure. In certain embodiments, the complication is Syndrome X. In certain embodiments, the complication is atherosclerosis. In certain embodiments, the complication is atheromatous disease. In certain embodiments, the complication is heart disease. In certain embodiments, the complication is cardiac insufficiency. In certain embodiments, the complication is coronary insufficiency. In certain embodiments, the complication is coronary artery disease. In certain embodiments, the complication is high blood pressure. In certain embodiments, the complication is hypertension. In certain embodiments, the complication is stroke In certain embodiments, the complication is neuropathy.
In certain embodiments, the complication is retinopathy. In certain embodiments, the complication is
1 neuropathy. In certain embodiments, the complication is peripheral vascular disease In certain
embodiments, the complication is polycystic, ovary syndrome. In certain embodiments, the complication is hyperlipidemia.
In certain embodiments, said modulator is arJ agonist.
In certain embodiments, a therapeutically effective amount of said pharmaceutical or physiologically acceptable composition is provided or administered to said individual.
In certain embodiments, said providing or administering of said pharmaceutical or physiologically acceptable composition is oral.
In certain embodiments, said individual is a mammal. In certain embodiments, said individual is a
non-human mammal. In certain embodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit,
i
i
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mouse, rat, non-human primate or human. In certain embodiments, said mammal is a mouse, rat, non-human primate, or human. .Most preferred xs human
In a tenth aspect, the invention features a modulator of a RUP43 GPCR, said receptor comprising a GPR131 ammo acid sequence, for i se in a method of treatment of the human animal body by therapy.
In certain embodiments, the modulator is identifiable, by a method according to a method of Okie first aspect. In certain embodiments, the modulator is identified according to a method of ihe first aspect. In certain embodiments, the modulator is not an antibody or an antigen-binding derivative thereof. In certain embodiments, the modulator is not a peptide. In certain embodiments, the modulator is a compound that stimulates glucose uptake in adipoc ytes obtained from a mammal. In certain embodiments, the modulator is a compound that stimulates glucose uptake in adipocytes obtained from the human or the animal. In certain embodiments, the modulator is a compound that stimulates glucose uptake in skeletal muscle cells obtained from a mammal, m certain embodiments, the modulator is a compound that stimulates glucose uptake in skeletal muscle cells obtained from the human or the animal. In certain embodiments, said modulator is selected from the group consisting of agonist, partial agonist, inverse agonist, and antagonist. In certain preferred embodiments, said modulator is an agonist. In certain embodiments, said agonist is a compound according to the second aspect
In certain embodiments, said modulator is selective for the GPCR.
In some embodiments, said modulator is Compound 1, Compound 2, or Compound 3. In some embodiments, said modulator is Compound 1. In some embodiments, said modulator is Compound 2. In some embodiments, said modulator is Compound 3.
In certain embodiments, said modulator is orally bioavailable. In some embodiments, said oral bioavailability is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at le ast 45% relative to intraperitoneal administration. In some embodiments, said oral bioavailablity is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative to intraperitoneal administration.
In certain embodiments, said orally bioavailable modulator is further able to cross the blood-brain barrier. ~
In some embodiments, said modulator is an agonist with an EC5o of less than 10 uM, of less than 1
uM, of less than 100 nM, or of less than 10 nM. In some embodiments, said modulator is an agonist with an
] i
ECso of less than a value selected from the interval of 10 nM to 10 uM-r In some embodiments, said
modulator is an agonist .with an BC50 of less than a value selected from the interval of 10 nM to 1 uM. In some embodiments, said modulator is an agonist with an EC50 of less than a value selected from the interval of 10 nM to 100 nM. In certain embodiments, said EC50 is determined using an assay selected from the group consisting of: whole cell cAMP assay carried using transfected HEK293 cells expressing recombinant RUP43 GPCR polypepude having the amino acid sequence of SEQ ID NO:2 or 6; and melanophore assay carried out using transfected melanophores expressing recombinant RUP43 GPCR polypepnde having the amino acid sequence of SEQ ID NO:2 or 6 In some embodiments, said modulator
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is an agonist with an ECS0 of less than 10 [iM, of less than I uM, of less than 100 nM, or of less than 10 nM in said assay In some embodiments, said modulator is an agonist with an EC50 of less than 10 uM in said assay, of less than 9 uM in said assay, of less than 8 uM in said assay, of less than 7 uM in said assay, of less than 6 uM in said assay, of less than 5 uM in said assay, of less than 4 uM in said assay, of less than 3 uM in said assay, of less than 2 uM in said assay, of less than 1 uM in said assay, of less than 900 nM in said assay, of less than 800nM in said assay, of less than 700 nM in said assay, of less than 600 nM in said assay, of less than 500 nM in said assay, of less tfon 400 nM in said assay, of less than 300 nM in said assay, of less than 200 nM in said assay, of less than 100 nM in said assay, of less than 90 nM in said assay, of less than 80 nM in said assay, of less than 70 nM in said assay, of less than 60 nM in said assay, of less than 50 nM in said assay, of less than 40 nM n said assay, of less than 30 nM in,said assay, of less than 20 nM in said assay, or of less than 10 nM in said assay. In some embodiments, said modulator is an agonist with an EC50 in said assay of less than a value selected from the interval of 10 nM to 10 pM. In some embodiments, said modulator is an agonist with an EC50 in said assay of less than a> value selected from the interval of 10 nM to 1 (jMJ In some embodiments, said modulator is an agonist with an EC50 in said assay of less than a value selected from (he interval of 10 nM to 100 nM.
F
In certain embodiments, said animal is a n lammal. : In certain embodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit, mouse, rat, or non-human primate. More preferred of human or animal is human.
In an eleventh aspect, the invention features a modulator of a RUP43 GPCR, said receptor comprising a GPR13I amino acid sequence, for use in a method of lowering blood glucose concentration in the humari animal body by therapy. In certain embodiments, the modulator is an agonist
The invention also features a modulator of a RUP43 GPCR, said receptor comprising a GPR131 amino acid sequence, for use ID a method of [prevention of or treatment for a metabolic disorder in a human or animal body by therapy. In certain embodiments the modulator is an agonist. In certain embodiments, the metabolic disorder is selected from the group consisting of:
(a) diabetes;
(b) impaired glucose tolerance;
(c) insulin resistance; and
(d) hyperinsulinemia.
In some embodiments, diabetes is type 1 diabetes. In certain preferred embodiments, diabetes is type 2 diabetes. In certain embodiments, the metabolic disorder is diabetes. In certain embodiments, the metabolic disorder is type 1 diabetes. In certain embodiments, the metabolic disorder is type 2 diabetes In certain embodiments, the metabolic disorder is impaired glucose tolerance. In certain embodiments, the metabolic disorder is insulin resistance. In certain embodiments, the metabolic disorder is hyperinsulinemia. In certain embodiments,!the metabolic disorder is related to an elevated blood glucose concentration in the individual.
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The invention also features [a modulator of a RUP43. GPCR, said receptor comprising a GPR131 ammo acid sequence, for use m a method of prevention of or treatment for a complication of an elevated blood glucose concentration in a human or animal body by therapy. In certain embodiments, the modulator is an agonist In certain embodiments, the complication is selected from the group consisting of;
(a) Syndrome'X,
(b) atherosclerosis;
(c) atheromatous disease;
(d) heart disease;
(e) hypertension;
(f) stroke;
(g) neuropathy,
(h) rettnopatby;
(i) nephropathy; and
(j) peripheral vasciuju; disease.
Heart disease includes, but is not limited to, cardiac insufficiency, coronaiy insufficiency, coronary artery disease, and high blood pressure. In certain embodiments, the complication is Syndrome X In certain embodiments, the complication is atherosclerosis. ¦ In certain embodiments, the complication is athetomatous disease. In certain embodiments, the complication is heart disease. In certain embodiments, the complication is cardiac insufficiency. In certain embodiments, the complication is coronary insufficiency. In certain embodiments, the complication is coronary artery disease. In certain embodiments, the complication is high blood pressure. In certain embodiments, the complication is hypertension. In certain embodiments, the complication is 'stroke. In certain embodiments, the complication is neuropathy. In certain embodiments; the complication is retinopathy. In certain embodiments, the modulator is identifiable by a method according to a method of fas first aspect. In certain embodiments J the modulator is identified according to a method of tint first aspect. In certain embodiments, the modulator is not an antibody or an antigen-binding derivative thereof In certain embodiments, the modulator is riot a peptide. In certain embodiments, the modulator is a compound that stimulates glucose uptake in adippcytes obtained firom a mammal. In certain embodiments, the modulator is a compound that stimulates glucose uptake in adipocytes obtained firom the human or animal. In certain embodiments, the modulator is a compound that stimulates glucose uptake in skeletal muscle cells obtained from a mammal In certain embodiments, the modulator is a compound that stimulates glucose uptake in skeletal muscle cells obtained from the human or animal. In certain embodiments, said modulator is selected from the group consisting of agonist, partial agonist, inverse agonist, and antagonist In certain
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preferred embodiments, said'modulator is an agomst.| In certain embodiments, said agonist is a compound according to the second aspect.
In certain embodiments, said modulator is selective for the GPCR
In some embodiments, said modulator is Compound,!, Compound 2, or Compound 3. In some embodiments^ said modulator is Compound 1. In sofrie embodiments, said modulator is Compound 2. In some embodiments, said modulator is Compound 3.
In certain embodiments, said modulator is brally bioavailable. In some embodiments, said oral bioavailabihty is at least 1%, at least 5%, at least 10%!, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative to intraperitoneal administration. In some embodiments, said oral bioavailablity is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative to intraperitoneal administration.
In certain embodiments, said orally bioavailable modulator is further able to cross the blood-brain barrier.
In some embodiments, said modulator is an Agonist with an EC50 of less than 10 uM, of less than 1 uM, of less than 100 nM, oil of less than 10 nM. In sbme embodiments, said modulator is an agonist with an EC50 of less than a value selected from the interval of 10 nM to 10 uM. In some embodiments, said modulator is an agonist with an EC50 of less than a value selected from the interval of 10 nM to 1 uM. In some embodiments, said modulator is an agonist with an EC50 of less than a value selected from the interval of 10 nM to 100 nM. In certain embodiments, said EC50 is determined using an assay selected from the group consisting of: whole cell cAMP assay |carried using transfected HEK293 cells expressing recombinant RUP43 GPCR polypeptide having the amino acid sequence of SEQ ID NO:2 or 6; and melanophore assay carried out using transfected melanophpres expressing recombinant RUP43 GPCR polypeptide having tile amtno acid sequence of SEQ ID NO:2 or 6. In some embodiments, said modulator is an agonist with an EC50 of less than 10 uM, of less than 1 uM, of less than 100 nM, or of less than 10 nM in said assay. In some embodiments, said modulatqr is an agonist with an ECSo of less than 10 uM in said assay, of less than 9 uM in said assay, of less than $ pM in said assay, of less than 7 uM in said assay, of less than 6 uM in said assay, of less than 5 \iM in said assay, of less than 4 uM in said assay, of less than 3 uM in said assay, of less than 2 uM in said .assay, of less than 1 uM in said assay, of less than 900 nM in said assay, of less than 800 nM in said assay, of less than 700 nM in said assay, of less than 600 nM in said assay, of less than 500 nM in said assay, of less than 400 nM in said assay, of less than 300 nM in said assay, of less than 200 nMin said assay, of less than1 100 nM in said assay, of less than 90 nM in said assay, of less than 80 nM in said ,assay, of less than 70 nM in said assay, of less than 60 nM in said assay, of less than 50 nM in said assay, of less than 40 nM n saidi assay, of less than 30 nM in said assay, of less than 20 nM in said assay, or of less than 10 nM in said assiy. In some embodiments, said modulator is an agonist with an EC50 in said assay of less than a value selected from the interval of 10 nM to 10 uM. In some embodiments, said modulator is an agonist with an EC50 in said assay of less than a value selected from the
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interval of 10 nM to 1 jiM. in somef embodiments, said modulator is an agonist with an EC50 in said assay ofless than a value selected fi:omthe[interval of 10 nM to 100.nM.
In certain embodiments, said animal is a mammal. In certain embodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit, mouse, rat, or non-human primate More preferred of human or animal is human
In a twelfth aspect, the invention features a method of using a modulator of a RUP43 GPCR, said receptor comprising a GPR131 amifto acid sequence, for the,preparation of a medicament for the lowering of blood glucose concentration. |In certain embodiments, the modulator is an agonist In certain embodiments, the agomst of RUP43 GPCR is an agonist of GPR131 GPCR, where it is understood that GPR131 GPCR is endogenous RUP43 GPCR.
The invention also features a method of using a modulator of a RUP43 GPCR, said receptor comprising a GPR131 amino acidl sequence, for the preparation of a medicament for the prevention or
treatment of a metabolic disorder! In certain embodiments, the modulator is an agonist In certain
i embodiments, the agonist of RUP43 GPCR is an agonist of GPR131 GPCR, where it is understood that
GPR131 GPCR is endogenous RUP43 GPCR. In certain embodiments, the metabolic disorder is selected from the group consisting of:
(a) diabetes;
(b) impaired glucose tolerance;
(c) insulin resistance; |and
(d) hyperinsulinemia.
In some embodiments, diabetes is type 1 diabetes. In certain preferred embodiments, diabetes is type 2 diabetes In certain embodiments, the metabolic disorder is diabetes. In certain embodiments, the metabolic disorder is type 1 diabetes. In certain embodiments, the metabolic disorder is type 2 diabetes. In certain embodiments, the metabolic disorder is impaired glucose tolerance. In certain embodiments, the metabolic disorder is insulin resistance. In certain embodiments, the metabolic disorder is hyperinsulinemia. In certain embodiments, the metabolic disorder is related to an elevated blood glucose concentration in the individual.
The invention also features a method of using a modulator of a RUP43 GPCR, said receptor
t comprising a GPR131 amino ac^d sequence, for the preparation of a medicament for the prevention or
treatment of a complication of pn elevated blood glucose concentration. In certain embodiments, the modulator is an agonist. In certain embodiments, the agonist of R.XJP43 GPCR is an agonist of GPR131 GPCR, where it is understood that GPR131 GPCR is endogenous RUP43 GPCR. In certain embodiments, the modulator is an agonist In certain embodiments, (he complication is selected from the group consisting of:
(a) Syndrome X;
(b) atherosclerosis;
(c) atheromatous disease;
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(d) heart disease;
I
(e) hypertension,
(f) stroke;
(g) neuropathy;
(h) retmopathy;
(1) nephropathy; and
(j) peripheral vascular disease.
Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, coronary artery disease, and high blood pressure. In 'certain embodiments, the complication is Syndrome X. In certaui embodiments, the complication is atherosclerosis. In certain embodiments, the complication is atheromatous disease In certain embodiments, the complication is heart disease. In certain embodiments, the complication is cardiac insufficiency ' In certain embodiments, the complication is coronary insufficiency In certaui embodiments, the complication is coronary artery disease. In certain embodiments, the complication is high blood pressure. In1 certain embodiments, the complication is hypertension, hi certain embodiments, the complication is stroke. In certain embodiments, the complication is neuropathy. In certain embodiments, the complication is retinopathy. In certain embodiments, the complication is neuropathy. In certain embodiments, the complication is peripheral vascular disease. In certain embodiments, the complication is polycystic ovary syndrome. In certain embodiments, the complication is hyperlipidemia
hi certain embodiments, the modulator is identifiable by a method according to a method of Hie first aspect. In certain embodiments, the modulator is identified according to a method of ihe first aspect. In certain embodiments, the modulator is not an antibody or an antigen-binding derivative thereof, In certain embodiments, the modulator is not a peptide. hi ce rtain embodiments, the modulator is a compound that stimulates glucose uptake in adipocytes obtained firon 1 a mammal. In certain embodiments, the modulator is a compound that stimulates glucose uptake in skeletal muscle cells obtained from a mammal. In certain embodiments, said modulator is selected from the group consisting of agonist, partial agonist, inverse agonist, and antagonist In certain preferred embodiments, said modulator is an agonist In certain embodiments, said agonist is a compound according i o the second aspect.
In certain embodiments, said modulator is selective for the GPCR.
In some embodiments, said modulator is Compound 1, Compound 2, or Compound 3. hi some embodiments, said modulator is Compound !l. In some embodiments, said modulator is Compound 2. In some embodiments, said modulator is Compound 3.
In certain embodiments, said modulator is orally bioavailable. In some embodiments, said oral bioavailabiliry is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative tc intrapentoneal administration In some embodiments, said oral bioavailablity is at least 20%, at least 25%, (at least 30%, at least 35%, at least 40%, or at least 45% relative to intrapentoneal administration.
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In certain embodiments, said orally, bioavailable modulator is further able to cross the blood-brain barrier
In some embodiments, said jmodulator is an agonist with an EC5Q of less than 10 \M, of less than 1 uM, of less than 100 nM3 or of less than 10 riM. hi some embodiments, said modulator is an agonist with an ECso of less than a value selected from The interval of 10 nM to 10 uM. In some embodiments, said modulator is an agonist with an EC.io of less than a value selected from,the interval of 10 nM to 1 uM. In some embodiments, said modulator [is an agonist with an ECj'o of less than a value selected from the interval of 10 nM to 100 nM. In certain embodiments, said EC50 is determined using an assay selected from the group consisting of: whole cel| cAMP assay carried using transfected HEK293 cells expressing recombinant RUP43 GPCR polypjeptide having the amino acid sequence of SEQ H> NO:2 or 6, and melanophore assay carried out usjing transfected melanophores expressing recombinant R.UP43 GPCR polypeplide having the amino acid sequence of SEQ ED NQ:2 or 6. In some embodiments, said modulator is an agonist with an EC5D of less than 10 uM, of less than 1 pM, of less man 100 nM, or of less than 10 nM in said assay. In some embodiments, said modulator is an agonist with an EC50 of less than 10 jiM in said assay, of less than 9 uM in said assay, of less than 8 uM in said assay, of less than 7 uM in said assay, of less than 6 uM in said assay, of less than 5 uM in said assay, of less than 4 uM in said assay, of less than 3 uM hi said assay, of less than 2 uM in said assay^ of less than 1 uM in said assay, of less than 900 nM in said assay, of less than 800 nM in (said assay, of less than 700 nM in said assay, of less than 600 nM in said assay, of less than 500 nM in said assay, of less than 400 nM in said assay, of less than 300 nM in said assay, of less than 200 nM in said|assay, of less than 100 nM in said assay, of less than 90 nM in said assay, of less than 80 nM in said assay, pf less than 70 nM in said assay, of less than 60 nM in said assay, of less than 50 nM in said assay, of less than 40 nM n said assay, of less than 30 nM in said assay, of less than 20 nM in said assay, or of less than J10 nM in said assay. In some embodiments, said modulator is an agonist with an EC50 in said assay of less than a value selected from the interval of 10 nM to 10 uM. In some embodiments, said modulator is gn agonist with an EC50 in said assay of less than a value selected from the interval of 10 nM to 1 uM. In sme embodiments, said modulator is an agonist with an EC50 in said assay
of less than a value selected fromithe interval of 10 nM to 100 nM.
[
In a thirteenth aspect, the invention features a metiiod of modulating the activity of a RUP43 GPCR, said receptor comprising a GPR.131 amino acid sequence, wherein said modulation is for lowering blood glucose in an individual | in need of said modulation, comprising contacting said receptor with a therapeutically effective amount of a modulator of the receptor. In certain embodiments, said method comprises first performing a method according to the first aspect to thereby identity the modulator hi certain embodiments, the modulator is an agonist
The invention also features a method of modulating the activity of a RUP43 GPCR, said leceptor comprising a GPR131 amino lacid sequence, wherein said modulation is for preventing or treating a metabolic disorder in an individual in need of said modulation, comprising contacting said receptor with a therapeutically effective amount of a modulator of the receptor, hi certain embodiments, said method
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comprises first performing a method according to thfc first aspect to thereby identify the modulator. In certain embodiments, the modulator is an agonist, |ln certain embodiments, the metabolic disorder is selected from the group consisting of:
(a) diabetes;
(b) impaired glucose tolerance;
(c) insulin resistance; and
(d) hyperinsuliriemia.
i In some embodiments, diabetes is type I diabetes. In certain preferred embodiments, diabetes is
type 2 diabetes. In certain embodiments, theimetabo ic disorder is diabetes. In certain embodimentSj the metabolic disorder is type 1 diabetes. In certain embodiments, the metabolic disorder is type 2 diabetes hi certain embodiments, the metabolic disorder is impaired glucose tolerance, hi certain embodiments, the metabolic disorder is insulin resistance. In certain embodiments, the metabolic disorder is hyperinsulinemia In certain embodiments, the metabolic disorder is related to an elevated blood glucose concentration in the individual.
The invention also features a method of modulating the activity of a RUP43 GPCR, said receptor
comprising a GPR131 amino acid sequence, wherein said modulation is for preventing or treating a
1 (
complication of an elevated blood glucose concentration in an individual in need of said modulation,
i comprising contacting said receptor with a therapeutibally effective amount of a modulator of the receptor.
In certain embodiments, said method comprises first performing a method according to the first aspect to thereby identify the modulator. In certain embodiments, the modulator is an agonist In certain embodiments, the modulator is an agonist. In certain embodiments, the complication is selected from the group consisting of:
(a) Syndrome X;
(b) atherosclerosis;
(c) atheromatous disease;
(d) heart disease;
(e) hypertension;
(f) stroke;
(g) neuropathy;
(h) retinopathy;
(l) nephropathy; and
(j) peripheral'vascular disease.
Heart disease includes, but is not limited to, [cardiac insufficiency, coronary insufficiency, coronary artery disease, and high blood pressure. In certain embodiments, the complication is Syndrome X. In certain embodiments, the complication is atherosclerosis. In certain embodiments, the complication is atheromatous disease. In certain embodiments, the complication is heart disease. In certain embodiments, the complication is cardiac insufficiency. In certain embodiments, the complication is coronary
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insufficiency. In certain embodiments, the complication is coronary artery disease. In certain embodiments, the complication is high blood pressure In certain embodiments, the complication is hypertension In certain embodiments, the complication is stroke. In certain embodiments, the complication is neuropathy In certain embodiments, the complication is retinopathy. In certain embodiments, the complication is neuropathy. In certain embodiments, the complication is peripheral vascular disease. In certain embodiments, the complication is pqlycystic ovary syndrome. In certain embodiments, the complication is hyperhpidemia.
In certain embodiments, the| modulator is not an antibody or an antigen-binding derivative thereof In certain embodiments, said mod\(ilator is not a peptide. In certain embodiments, the modulator is a compound that stimulates glucose uptake in adipocytes obtained from a mammal. In certain embodiments, the modulator is a compound that stimulates glucose uptake in skeletal muscle cells obtained from a mammal. In certain embodiments, $aid modulator is according to the third aspect. In certain embodiments, said modulator is selected from tiie group consisting of agonist, partial agonist, inverse agonist, and antagonist In certain preferred embodiments, said modulator is an agonist.
In certain embodiments, said modulator is selective for the GPCR.
In some embodiments, said modulator is Compound 1, Compound. 2, or Compound 3. In some embodiments, said modulator is Cpmpound 1. In some embodiments, ;said modulator is Compound 2. In some embodiments, said modulator is Compound 3.
In certain embodiments, Said modulator is orally bioavailable. In some embodiments, said oral bioavailability is at least 1%, at leajst 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at le^st 45% relative to intraperitoneal administration. In some embodiments, said oral bioavailablity is at least 30%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative to intraperitoneal administration.
In certain embodiments, Said orally bioavailable modulator is further able to cross the blood-brain barrier.
In some embodiments, said modulator is an agonist with an EC50 of less man 10 uM, of less than 1 uM, of less than lOOnM, orofle^sthan 10 nM. In some embodiments, said modulator is an agonist with an ECJO of less than a value selected from the interval of 10 nM to 10 uM. In some embodiments, said modulator is an agonist with an &C50 of less than a value selected from the interval of 10 nM to 1 uM. In some embodiments, said modulator is an agonist with an ECS0 of less than a value selected fiom the interval of 10 nM to 100 nM. In certain embodiments, said EC50 is determined using an assay selected fiom the group consisting of: whole cell cAMP assay carried using transfected HEK293 cells expressing recombinant RUP43 GPCR polypeptide having the amino acid sequence of SEQ ID NO:2 or 6; and melanophore assay carried outj using transfected melanophores expressing recombinant RUP43 GPCR polypeptide having the amino a&d sequence of SEQ ID NO:2 or 6. In some embodiments, said modulator is an agonist with an EC5o of les^s than 10 uM, of less than 1 uM, of less than 100 nM, or of less than 10 nM in said assay. In some embodiments, said modulator is an agonist with an EC50 of less than 10 uM in said
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assay, of less than 9 uM in said assay, of less than 8 uM in said assay, of less than 7 |oM in said assay, of less than 6 pM in said assay, of less than 5 uM in said dssay, of less than. 4 uM in said assay, of less than 3 (j.M tn said assay, of less than 2 uM in said assay, of less than 1 jiM in said assay, of less than 900 nM in said assay, of less than 800 nM in said assay, of less than 700 nM in said assay, of less than 600 nM in said assay, of less than 500 nM in said assay, of less than 14-00 nM in said assay, of less than 300 nM in said assay, of less than 200 nM in said assay, of less than 10P nM in said assay, of less than 90 nM in said assay, of less than SO nM in said assay, of less than 70 nM injsaid assay, of less than 60 nM in said assay, of less than 50 nM in said assay, of less than 40 nM n said assay, of less than 30 nM in said assay, of less than 20 nM in said assay, or of less than 10 nM in said assay, iln some embodiments, said modulator is an agonist with an EC50 in said assay of less than a value selected from the interval of 10 nM to 10 uM. In some embodiments, said modulator is an agonist with an ECi0 in said assay of less than a value selected from the interval of 10 nM to 1 uM. In some embodiments, said modulator is an agonist with an EC50 in said assay of less than a value selected from the interval of 10 nMjto 100 nM
In certain embodiments, said contacting comprises oral administration of said modulator to said individual.
In certain embodiments, said individual is a mammal. In certain embodiments, said individual is a non-human mammal. In certain embodiments, said rftammal is a horse, cow, sheep, pig, cat, dog, rabbit, mouse, rat, non-human primate or human. In certain embodiments, said mammal is a mouse, rat, non-human primate, or human. Most preferred is human.
In a. fourteenth aspect, the invention features |a method of lowering blood glucose in an individual in need of said lowering, comprising contacting a merapeutically effective amount of a modulator of a RUP43 GPCR with said receptor, said GPCR comprising a,GPR131 amino acid sequence. In certain embodiments, said method comprises first performi4g a method according to the first aspect to thereby identify the modulator In certain embodiments, the mtodulator is an agonist.
The invention also features a method of preventing or treating a metabolic disorder in an individual in need of said prevention or treatment, comprising contacting a therapeutically effective amount of a modulator of a RUP43 GPCR with said receptor, saidl receptor comprising a GPR.131 ammo acid sequence In certain embodiments, said method comprises firstj performing a method according to the first aspect to thereby identify the modulator. In certain embodiments, the modulator is an agonist In certain embodiments, the metabolic disorder is selected from [the group consisting of:
(a) diabetes;
(b) impaired glucose tolerance;
(c) insulin resistance; and
(d) hyperinsuhnemia.
In some embodiments, diabetes is type 1 dikbetes. In certain preferred embodiments, diabetes is type 2 diabetes. In certain embodiments, the metabolic disorder is diabetes. In certain embodiments, the metabolic disorder is type 1 diabetes. In certain embodiments, the metabolic disorder is type 2 diabetes. In
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certain embodiments, the metabolic disorder is impaired glucose tolerance. In certain embodiments, the metabolic disorder is insulin resistance. In certain; embodiments, the metabolic disorder is hyperinsuhnemia. In certain embodiments, the metabolic disorder is,related to an elevated blood glucose concentration in the individual.
The invention also features a method of preventing or treating a complication of an elevated blood glucose concentration in an individual in need of said prevention on treatment, comprising contacting a therapeutically effective amount of a modulator of a RUP43 GPCR with said receptor, said receptor comprising a GPR131 amino acid sequence. In certain embodiments, said method comprises first performing a method according to the first aspect to thereby identify the modulator. In certain embodiments, the modulator is an agonist. In certain embodiments, the modulator is an agonist. In certain embodiments, the complication is ^elected jfrom the group consisting of:
(a) Syndrome X;
(b) atherosclerosis;
(c) atheromatous disease;
(d) heart disease;
(e) hypertension;
(f) stroke;
(g) neuropathy;
(h) retinopathy;
(i) nephropathy; anA
(j) peripheral vascular disease.
Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, coronary artery disease, and high blood pressure. In certain embodiments, the complication is Syndrome X. In certain embodiments, the complication is atherosclerosis. In certain embodiments, the complication is atheromatous disease. In certain embodiments, the complication is heart disease. In certain embodiments, the complication is cardiac insufficiency. In certain embodiments, the complication is coronary insufficiency, in certain embodiments, the complication is coronary artery disease. In certain embodiments, the complication is high blood pressure. In certain embodiments, the complication is hypertension. In certain embodiments, the compication is stroke. In certain embodiments, the complication is neuropathy. hi certain embodiments, the complication is retinopathy. In certain embodiments, the complication is neuropathy. In certain embodiments, the complication is peripheral vascular disease. In certain embodiments, ihe complication] is polycystic ovary syndrome. In certain embodiments, the complication is hyperhpidemia.
In certain embodiment^, the modulator is not an antibody or an antigen-binding derivative thereof. In certain embodiments, said modulator is not a peptide. In certain embodiments, the modulator is a compound that stimulates glucose uptake in adipocytes obtained from a mammal, hi certain embodiments, the modulator is a compound that stimulates glucose uptake in skeletal muscle cells obtained from a
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mammal In certain embodiments, said modulator is according to the third aspect In certain embodiments, said modulator is selected from the group consisting of agonist, partial agonist, inverse agonist, and antagonist. In certain preferred embodiments,,said modulator is, an agonist.
In certain embodiments, said modulator is selective for the GPCR.
In some embodiments, said modulator is Cojmpound .1, Compound 2, or Compound 3. In some embodiments, said modulatbr is Compound 1. In some embodiments, said modulator is Compound 2. In some embodiments, said modulator is Compound 3 ¦
In certain1 embodiments, said modulator is orally bioavailable. In some embodiments, said oral bioavailability is at least 1%, at least 5%, at least 10%j at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative to intrapentoneal administration. In some embodiments, said oral bioavailablity is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative to intrapentoneal administration.
In certain embodiments, said orally bioavailable modulator is further able to cross the blood-brain barrier.
In some embodiments, said modulator is an agonist with an EC50 of less than 10 uM, of less than 1 uM, of less than 100 nM, or of less than 10 nM. In sotne embodiments, said modulator is an agonist with an ECS0 of less than a value selected from the interva} of 10 nM to 10 uM. In some embodiments, said modulator is an agonist with an EC50 of less than a value selected from the interval of 10 nM to 1 uM. In some embodiments, said modulator is an agonist witlil an EC50 of less tiian a value selected from the interval of 10 nM to 100 nM. In certain embodiments, said JEC50 is determined using an assay selected from the group consisting of: whole cell cAMP assay darned using transfected HEK293 cells expressing recombinant RUP43 GPCR polypeptide having thei amino acid sequence of SEQ ID NO:2 or 6; and melanophore assay carried! out using transfected melanophores expressing recombinant RUP43 GPCR polypeptide having the'amino acid sequence of SEQ ID NO:2or 6. In some embodiments, said modulator is an agonist with an EC50 of less than 10 uM, of less Ithan 1 uM, of less than 100 nM, or of less than 10 nM in said assay In some embodiments, said modulator] is an agonist with an EC50 of less than 10 uM in said assay, of less than 9 uM in'said assay, of less than 8|uM in said assay, of less than 7 uM in said assay, of less than 6 uM in said assay, of less than 5 uM in saiil assay, of less than 4 uM in said assay, of less than 3 uM in said assay, of less than 2 uM in said assay, of less than I uM in said assay, of less than 900 nM in said assay, of less than 800 nM in said assay, of less tihan 700 nM in said assay, of less than 600 nM in said assay, of less than 500 nM in said assay, ofiless than 400 nM in said assay, of less than 300 nM in said assay, of less than 200 nM in said assay, of less than 100 nM in said assay, of less than 90 nM in said assay, of less than 80 nM in said assay, of less than 70 nM in said assay, of less than 60 nM in said assay, of less than 50 nM in said assay, of less than 40 nM n said assay, of less than 30 nM in said assay, of less than 20 nM in said assay, or of less than 10 nM in said assay. In some embodiments, said modulator is an agonist with an EC50 in said assay of less than a value selected from the interval of 10 nM to 10 uM. In some embodiments, said modulator is an agonist with an EC50 in said assay of less than a value selected from the
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interval of 10 nM to 1 uM. In some embodiments, said modulator is an agonist with an EC50 in said assay of less than a value selected from the interval of 10 nM to 100 nM.
In certain embodiments, said contacting compnses .oral administration of said modulator to said individual
In certain embodiments, said individual is a mammaL In certain embodiments, said individual is a non-human mammal. In certain embodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit, mouse, rat, non-human primate or human. In certain embodiments, said mammal is a mouse, rat, non-human primate, or human. Most preferred is human.
In a fifteenth aspect, the invention features a pharmaceutical or physiologically acceptable composition comprising, consisting essentially of, or consisting of a modulator a RXJP43 GPCR, said receptor comprising a GPR131 amino acid sequence, hi certain embodiments, said modulator is identifiable by performing a method according to \b& first aspect. In certain embodiments, said modulator is identified by performing a method according to the.Zw-.tf aspect.
In certain embodiments, the modulator is not an antibody or an antigen-binding derivative thereof. In certain embodiments, said modulator is not a peptide. hi certain embodiments, the modulator is a compound that stimulates glucose uptake in adipocytes obtained from a mammal, hi certain embodiments, the modulator is a compound that stimulates glucose uptake in skeletal muscle cells obtained from a mammal, hi certain embodiments, said modulator is according to the third aspect. ID certain embodiments, said modulator is selected frorji the group consisting of agonist, partial agonist, inverse agonist, and antagonist. In certain preferred embodiments, said modulator is an agonist.
In certain embodiments, said composition is pharmaceutical, hi certain embodiments, the pharmaceutical composition comprises the modulator of a RXJP43 GPCR, In certain embodiments, the pharmaceutical composition consists essentially of the modulator of a KUP43 GPCR. In certain embodiments, the pharmaceutical composition consists of the modulator of a RUP43 GPCR.
m certain embodiments!, said composition is physiologically acceptable. In certain embodiments, the physiologically acceptable 1 composition comprises the modulator of a RUP43 GPCR. In certain embodiments, the physiologically acceptable composition consists essentially of the modulator of a RUP43 GPCR. In certain embodiments, the physiologically acceptable composition consists of the modulator of a RTJP43 GPCR.
In certain embodiments, said modulator is selective for the GPCR.
In some embodiments^ said modulator is Compound 1, Compound 2, or Compound 3. hi some embodiments, said modulator js Compound 1. In some embodiments, said modulator is Compound 2. In some embodiments, said modulator is Compound 3.
In certain embodiments, said modulator is orally bioavailable. In some embodiments, said oral bioavailability is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative to mtcaperitoneal administration. In some embodiments,
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said oral bioavaiiabhty is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative to intraperitoneal administration.
In certain embodiments, said orally bioavailable modulator is further able to cross the blood-brain barrier
In some embodiments, said modulator is an ago'nist with' an EC50 of less than 10 uM, of less than 1 uM, of less than 100 nM, or of less than 10 nM.. In some embodiments, said modulator is an agonist with an EC50 of less than a value selected from the interval df 10 nM to 10 uM, In some embodiments, said modulator is an agonist with an EC5o of less than a valijie selected from the interval of 10 nM to 1 \M, In some embodiments, said modulator is an agonist with an EQo of less than a value selected from the interval of 10 nM to 100 nM. In certain embodiments, said EC50 is determined using an assay selected from the group consisting of: whole cell cAMP assay carried using transfected HEK293 cells expressing recombinant RUP43 GPCR polypeptide having the arnino acid sequence of SEQ ID NO:2 or 6; and melanophore assay carried out using transfected mel|anophores expressing recombinant RUP43 GPCR polypeptide having the amino acid sequence of SEQ H| NO:2 or 6. In some embodiments, said modulator is an agonist with an EC5o of less than 10 uM, of less thbn 1 uM, of less than 100 nM, or of less than 10 nM in said assay. In some embodiments, said modulator is! an agonist with an EC50 of less than 10 uM in said assay, of less than 9 uM in said assay, of less than 8 pJM in said assay, of less than 7 uM in said assay, of less than 6 uM in said assay, of less man 5 uM in said assay, of less than 4 uM in said assay, of less than 3 uM in said assay, of less than 2 uM in said assay, of less than 1 uM in said assay, of less than 900 nM in said assay, of less than 800 nM in said assay, of less than 700 nM in said assay, of less than 600 nM in said assay, of less than 500 nM in said assay, of less than|400 nM in said assay, of less than 300 nM in said assay, of less than 200'nM in said assay, of less than 100 nM in said assay, of less than 90 nM in said assay, of less than 80 nM in said assay, of less than 70 nM in said assay, of less than 60 nM in said assay, of less than 50 nM in said assay, of less than 40 nM n said as$ay, of less than 30 nM in said assay, of less than 20 nM in said assay, or of less than 10 nM in said assay., In some embodiments, said modulator is an agonist with an EC50 in said assay of less than a value selected from the' interval of 10 nM to 10 uM. In some embodiments, said modulator is an agonist with an EC|so in said assay of less than a value selected from the interval of 10 nM to 1 uM. In some embodiments, said modulator is an agonist with an ECS0 in said assay of less than a value selected from the interval of 10 nM|to 100 nM.
In a sixteenth aspect, the invention features]a method of lowering blood glucose concentration comprising providing or administering to an individual in need of said reduction said pharmaceutical or physiologically acceptable composition of ihefifteenthlaspecL
The invention also features a method of preventing or treating a metabolic disorder comprising providing or administering to an individual in need of said prevention or treatment said pharmaceutical or physiologically acceptable composition of the fifteenth aspect. In certain embodiments, the metabolic disorder is selected from the group consisting of:
(a) diabetes;
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(b) impaired glucose tolbrance; '
(c) insulin resistance, and
(d) hyperinsulinemia
In some embodiments, diabetes is type 1 diabetes. In certain preferred embodiments, diabetes is type 2 diabetes. In certain embodiments,'1 the metabolic disorder is diabetes. In certain embodiments, the metabolic disorder is type 1 diabetes In certain embodiments, the metabolic disorder is type 2 diabetes. In certain embodiments, the metabolic disorder is impaired glucose tolerance. In certain embodiments, the metabolic disorder is insulin resistance. In certain embodiments, the metabolic disorder is hyperinsulinemia. In certain embodiments, the metabolic disorder is related to an elevated blood glucose concentration in the individual.
The invention also features la method of preventing or treating a complication of an elevated blood glucose concentration comprising providing or administering to an individual in need of said prevention or treatment said pharmaceutical or physiologically acceptable composition of the fifteenth aspect In certain embodiments, the complication is selected from the group consisting of:
(.a) Syndrome X;
(b) atherosclerosis;
(c) atheromatous disease;
(d) heart disease;
(e) hypertension;
(f) stroke;
(g) neuropathy;
(h) retinopathy;
(I) nephropathy; and
(j) peripheral vascular disease.
Heart disease includes, but is not limited to, cardiac rinsufficiency, coronary insufficiency, coronary artery disease, and high blood pressure, hi certain embodiments, the complication is Syndrome X. In certain embodiments, the complication is atherosclerosis. In certain embodiments, the complication is atheromatous disease. In certain embodiments, the complication is heart disease. In certain embodiments, the complication is cardiac insufficiency. In certain i embodiments, the complication is coronary insufficiency. In certain embodiments, the In certain embodiments, Said modulator is an agonist.
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In certain embodiments, a therapeuticallyj effective amount of said pharmaceutical or physiologically acceptable composition is provided or a iministered to said individual.
In certain embodiments, said providing or administering of said pharmaceutical or physiologically acceptable composition is oral.
In certain embodiments, said individual is a mammal. In certain embodiments, said individual JS a non-human'mammal. In certain embodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit, mouse, rat, non-human primate or human. In certain embodiments, said mammal is a mouse, rat, non-human primate, or human Most preferred is human,
In an seventeenth aspect, the invention features a modulator of a RUP43 GPCR, said receptor comprising a GPR131 amino acid sequence, for use in a method of treatment of the human or animal body by therapy. ¦ In certain embodiments, said modulator is identifiable by performing a method according to the first aspect. In certain embodiments, said modulator is identified by performing a method according to the first aspect
In certain embodiments, the modulator is not An antibody or an antigen-binding derivative thereof In certain embodiments, said modulator is not a peptide In certain embodiments, the modulator is a compound that stimulates glucose uptake in adipocytes obtained from a mammal. In certain embodiments, the modulator is a compound that stimulates glucose uptake in skeletal muscle cells obtained from a mammal. In certain embodiments, said modulator is according to the third aspect. In certain embodiments, said modulator is selected from the group consisting of agonist, partial agonist, inverse agonist, and antagonist. In certain preferred embodiments, said modulator is an agonist
In certain embodiments, said modulator is selective for the GPCR.
In some embodiments, said modulator is Compound 1, Compound 2, or Compound 3. In some embodiments, said modulator is Compound 1. In some embodiments, said modulator is Compound 2. In some embodiments, said modulator is Compound 3.
In certain embodiments, said modulator is otally bioavailable. In some embodiments, said oral bioavailability is at least l%Jat least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative to htraperitoneal administration. In some embodiments, said oral bioavailablity is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative to intraperitoneal administration
In certain embodiments, said orally bioavailable modulator is further able to cross the blood-brain barrier.
In some embodiments, said modulator is an agonist with an ECS0 of less than 10 uM, of less than 1 uM, of less than 100 nM, or of less than 10 nM. In sone embodiments, said modulator is an agonist with an ECS0 of less than a value selected from the interval of 10 nM to 10 \JM. In some embodiments, said modulator is an agonist with an EC50 of less than a vslue selected from the interval of 10 nM to 1 uM. In some embodiments, said modulator is an agonist with an EC50 of less than a value selected from the interval of 10 nM to 100 nM. In certain embodiments, said EC50 is determined using an assay selected from the
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group consisting of: whole cell cAMP assay earned using transfected HEK293 cells expressing recombinant RUP43 GPCR polypeptide having the amino acid sequence of SEQ ID NO:2 or 6; and melanophore assay earned out using transfected melanophores expressing recombinant RUP43 GPCR polypeptide having the arrono acid sequence of SEQ ED NO:2 or 6. hi some embodiments, said modulator is an agonist with an EC50ofless than 10 uM, of less than 1 pM, of less than 100nM,orof less than 10 nM ni said assay. In some embodiments, said modulator is an agonist with an EC50 of less than 10 uM in said assay, of less man 9 \iM in said assay, of less than 8 uM in:said assay, of less than 7 uM in said assay, of less than 6 uM in said assay, of less than 5 uM m said assay', of less than 4 uM in said assay, of less than 3 (iM in said assay, of less than 2 uM in said assay, of less than 1 uM in said assay, of less than 900 nM in said assay, of less than 800 nM in said assay, of less than 700 nM in said assay, of less than 600 nM in said assay, of less than 500 nM in said assay, of less than 400 nM in said assay, of less than 300 nM in said assay, of less than 200 nM in said assay, of less than 100 nM in said assay, of less than 90 nM in said assay, of less than 80 nM in said assay, cf less than 70 nM in said.assay, of less than 60 nM in said assay, of less than 50 nM in said assay, of less than 40 nM n said assay, of less than 30 nM, in said assay, of less than 20 nM in said assay, or of less than 10 nM in said assay. In some embodiments, said modulator is an agonist with an EC50 in said assay of less than a value selected from the interval of 10 nM to 10 uM. In some embodiments, said modulator is an agonist with an ECS0 in said assay of less than a value selected from the interval of 10 nM to 1 |JM. In some embodiments, said modulator is an agonist with an ECS0 in said assay of less than a value selected from the interval of 10 nM to 100 nM.
In certain embodiments, paid animal is a mammal. In certain embodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit, mouse, rat, or non-human primate. More preferred of human or animal is human.
In an eighteenth aspect, the invention features a modulator of a KUP43 GPCR, said receptor comprising a GPR131 amino acid sequence, for use in a method of lowering blood glucose concentration in the human or animal body by therapy. In certain embodiments, said modulator is identifiable by performing a method according to 1hs first aspect. In certain embodiments, said modulator is identified by performing a method according to the first aspect. In certain embodiments, the modulator is an agonist
The invention also features a modulator of a RUP43 GPCR, said receptor comprising a GPR131 amino acid sequence, for use in almethod of prevention of or treatment for a metabolic disorder in a human or animal body by therapy. In certain embodiments, said modulator is identifiable by performing a method according to the first aspect. In certain embodiments, said modulator is identified by performing a method according to the/iref aspect. In certain embodiments, the modulator is an agonist. In certain embodiments, the metabolic disorder is selected from the group consisting of:
(a) diabetes;
(b) impaired glucose tolerance;
(c) insulin resistanc s; and
(d) hyperinsuUnemia.
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In some embodiments, diabetes is type 1 diabetes. In certain preferred embodiments, diabetes is type 2 diabetes. In certain embodiments, the [metabolic disorder is diabetes. In certain embodiments, the metabolic disorder is type 1 diabetes. In certain embodiments, the metabolic disorder is type 2 diabetes. In certain embodiments, the metabolic disorder is impaired glucose tolerance. In certain embodiments, the metabolic disorder is insulin resistance. ' In certain embodiments, the metabolic disorder is hyperinsulinemia In certain embodiments, the metabolic disorder is related to an elevated blood glucose concentration in the individual
The invention also features a modulator of ajRUP43,GPCR, said receptor comprising a GPR131 ammo acid sequence, for use in a method of prevention of or treatment for a complication of an elevated blood glucose concentiation in a human or animal body by therapy. In certain embodiments, said modulator is identifiable by performing a method according to the first aspect In certain embodiments, said modulator is identified by performing a method according to ihe first aspect. In certain embodiments, the modulator is an agonist In certain embodiments, the complication is selected from the group consisting of:
(a) Syndrome X;
(b) atherosclerosis;
(c) atheromatous disease;
(d) heart disease;
(e) hypertension;
(f) stroke;
(g) neuropathy;
(h) retinopathy;
(I) nephropathy; and
(j) peripheral vascular disease.
Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, coronary artery disease, and high blood pressure. In certain embodiments, the complication is Syndrome X. In certain embodiments, the complication is atherosclerosis. In certain embodiments, the complication is atheromatous disease. In certain embodiments, the complication is heart disease. In certain embodiments, the complication is cardiac insufficiency. In certain embodiments, the complication is coronary insufficiency. In certain embodiments, the complication is coronary artery disease. In certain embodiments, the complication is high blood pressure. In certain embodiments, the complication is hypertension In certain embodiments, the complication is stroke. In :ertain embodiments, the complication is neuropathy fii certain embodiments, the complication is retinopathy. In certain embodiments, the complication is neuropathy. In certain embodiments, the complication is peripheral vascular disease. In certain embodiments, the complication is polycystic ovary syndrome. In certain embodiments, the complication is hyperlipidemia.
In certain embodiments, the modulator is not an antibody or an antigen-binding derivative thereof. In certain embodiments, said modulator is not a p 5ptide. In certain embodiments, the modulator is a
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compound that stimulates glucose uptkke in adipocytes obtained from a mammal. In certain embodiments, the modulator is a compound that stimulates glucose uptake in skeletal muscle cells obtained from a mammaL In certain embodiments, said modulator is according to the third aspect En certain embodiments, said modulator is selected; from the group consisting of agonist, partial agonist, inverse agonist, and antagonist In certain preferred embo diments, said modulator is an agonist.
In certain embodiments, said modulator is selective for the GPCR.
In some embodiments, said modulator is Compound 1, Compound 2, or Compound 3. hi some embodiments, said modulator is Compound 1. In some embodiments, said modulator is Compound 2, In some embodiments, said modulator is Compound 3.
hi certain embodiments, said modulator is orally bioavailable. In some embodiments, said oral bioavailabihty is at least 1%, at least! 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative to intraperitoneal administration. In some embodiments, said oral bioavailablity is at least 20?/o, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative to intraperitoneal administration.
In certain embodiments, said orally bioavailable modulator is further able to cross the blood-brain barrier.
In some embodiments, said modulator is an agonist with an EC50 of less than 10 uM, of less Than 1 uM, of less than 100 nM, or of less than 10 nM. In some embodiments, said modulator is an agonist with an ECso of less than a value selected from the interval of 10 nM to 10 u.M. In some embodiments, said modulator is an agonist with an ECso of less than a value selected from the interval of 10 nM to 1 uM. In some embodiments, said modulator is an agonist with an EC50 of less than a value selected from the interval of 10 nM to 100 nM. In certain embodiments, said EC50 is determined using an assay selected from the group consisting of: whole cell cAMP assay carried using transfected HEK293 cells expressing recombinant KUP43 GPCR polypeptide having the amino acid sequence of SEQ ID NO:2 or 6; and melanophore assay carried out using transfected melanophores expressing recombinant RUP43 GPCR polypeptide having the ammo acid sequence of SEQ ID NO:2 or 6. In some embodiments, said modulator is an agonist with an EC50 of less than IOJJM, of less than 1 uM, of less than 100 nM, or of less than 10 nM in said assay. In some embodiments, said modulator is an agonist with an EC30 of less than 10 uM in said assay, of less than 9 uM in said assay, of less than 8 uM in said assay, of less than 7 uM in said assay, of less than 6 jiM in said assay, of i ess than 5 uM in said assay, of less than 4 uM in said assay, of less than 3 uM in said assay, of less than 2 uM in said assay, of lessj than 1 uM in said assay, of less than 900 nM in said assay, of less than 800 nM in said assay, of less man 700 nM in said assay, of less than 600 nM in said assay, of less than 500 nM in said assay, of less than 400 nM in said assay, of less than 300 nM in said assay, of less than 200 nM in said assay, of less than 100 nM in said assay, of less than 90 nM in said assay, of less than 80 nM in said assay, of less tiaan 70 nM in said assay, of less than 60 nM in said assay, of less than 50 nM in said assay, of less than 40 nM n said assay, of less than 30 nM in said assay, of less than 20 nM in said assay, or of less than 10 nM in said assay. In some embodiments, said modulator is an agonist
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with an EC50 in said assay of less than a value selected from the interval of 10 nM to 10 uM. in some embodiments, said modulator is an agonist with an EC50 m said assay of less than a value selected from the interval of 10 nM to 1 |iM. In some embodiments, sai 1 modulator is an agonist with an EC50 in said assay of less than a value selected from the interval of 10 nM to 100 nM.
In certain embodiments, said animal is a mammal. In certain embodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit, mouse, rat, or non-human primate. More preferred of human or animal is human.
In a nineteenth aspect, the invention featuresja method of using a modulator of a RUP43 GPCR, said receptor comprising a GPR131 ammo acid sequence, for the preparation of a medicament for the lowering of blood glucose concentration. In certain embodiments, said method comprises first performing a method according to the first aspect to thereby identify the modulator. In certain embodiments, the modulator is an agonist (
The invention also features a method of using a modulator of a RUP43 GPCR, said receptor comprising a GPR.131 ammo acid sequence, for the preparation of a medicament for the prevention or treatment of a metabolic disorder. In certaini embodiments, said method comprises performing a method according to tb&Jirsl aspect to thereby identify a modulator In certain embodiments, the modulator is an agonist. In certain embodiments, the metabolic disorder is selected from the group consisting of:
(a) diabetes;
(b) impaired glucose tolerance;
(c) insulin resistance; and
(d) hyperinsulinernia.
In some embodiments, diabetes is type 1 diabetes. In certain preferred embodiments, diabetes is type 2 diabetes. In certain embodiments, the metabolic disorder is diabetes. In certain embodiments, the metabolic disorder is type 1 'diabetes. In certain embodiments, the metabolic disorder is type 2 diabetes. In certain embodiments, the metabolic disorder, is impaired glucose tolerance. In certain embodiments, the metabolic disorder is insulin resistance.1 In certain embodiments, the1 metabolic disorder is hyperinsulinemia. hi certain embodiments^ the metabolic disorder is related to an elevated blood glucose concentration in the individual.
The invention also features a method of using a modulator of a RUP43 GPCR, said receptor comprising a GPR131 amino acid sequence, for the preparation of a medicament for the prevention or treatment of a complication of an elevated blood glucose concentration. In certain embodiments, said method comprises performing a method according to Htm first aspect to thereby identify a modulator. In certain embodiments, the modulator is an agonist. In certain embodiments, the complication is selected from the group consisting of:
(a) Syndrome X;
(b) atherosclerosis;
(c) atheromatous disease;
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(d) heart disease;
(e) hypertension;
(f) stroke;
(g) neuropathy,
(h) retinopathy;
(i) nephropathy; and
(j) peripheral vascular disease.
Heart disease includes, but is not limited to, cardiac insufficiency, coionary insufficiency, coronary artery disease, and high blood pressure, hi certain embodiments, the complication is Syndrome X In certain embodiments, the complication is atherosclerosis, hi certain embodiments, the complication is atheromatous disease. In certain embodiments, the complication is heart disease, hi certain embodiments, the complication is cardiac insufficiency, hi certain embodiments, the complication is coronary insufficiency. In certain embodiments, the complication xs coronary artery disease, hi certain embodiments, the complication is high blood pressure, hi certain embodiments, the complication is hypertension hi certain embodiments, the complication is stroke. In certain embodiments, the complication is neuropathy. hi certain embodiments, the complication, is retinopathy. In certain embodiments, the complication is neuropathy. In certain embodiments, the complication is peripheral vascular disease, hi certain embodiments, the complication is p olycystic ovary syndrome, hi certain embodiments, the complication is hyperlipidemia.
hi certain embodiments, the modulator is not an antibody or an antigen-binding derivative thereof, hi certain embodiments, said modulator is not a peptide. ; hi,certain embodiments, the modulator is a compound mat stimulates glucose lptake in adipocytes obtained from a mammal, hi certain embodiments, the modulator is a compound th;.t stimulates glucose uptake in skeletal muscle cells obtained from a mammal, hi certain embodiments, said modulator is according to the third aspect hi certain embodiments, said modulator is selected from the group consisting of jagonist, partial agonist, inverse agonist, and antagonist. In certain preferred embodiments, said modulator is an agonist.
hi certain embodiments, said modulator is selective for the GPCR
hi some embodiments, seid modulator is Compound 1, Compound 2, or Compound 3. In some embodiments, said modulator is Compound 1. In some embodiments, said modulator is Compound 2. In some embodiments, said modulator is Compound 3.
hi certain embodiments, said modulator is orally bioavailable. hi some embodiments, said oral bioavailability is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative to intraperitoneal administration, hi some embodiments, said oral bioavailabhty is at least! Wo, at least 25%, at least:30%, at least 35%, at least 40%, or at least 45% relative to intraperitoneal adminis ration.
hi certain embodiments, said orally bioavailable modulator is further able to cross the blood-brain barrier.
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In some embodiments, said modulator is an agbnist with an EC50 of less than 10 uM, of less than 1 uM, of less than 100 nM, or of less than lOnM, In sonfe embodiments, said modulator is an agonist with an EC3Q of less than a value selected from Hie interval of 10 nM to 10 uM. In some embodiments, said modulator is an agonist with an ECS0 of less Uian a va.ue selected from the interval of 10 nM to 1 uM. In some embodiments, said modulator is an agonist with zn EGso of less than a value selected from the interval of 10 nM to 100 nM, In certain embodiments, said EC50 is determined using an assay selected from the group consisting of: whole cell cAMP assay cakried using tansfeeted B0BK293 cells expressing recombinaat RUP43 GPCR'potypeptide having fee iimino acid sequence of SEQ IB NO;2 or 6; and melanophore assay carried out using transfected me lanophores expressing recombinant RUP43 GPCR polypeptide having the amino acid sequence of SEQ B) NO;2 or 6, In some embodiments, said modulator is an agonist with an EC50 of tess than 10 uM, of less than 1 jiM, of less than 100 nM, or of less than 10 nM in said assay. In some embodiments, said modulator i s an agonist with an EC50 of less than. 10 uM in said assay, of less than 9 uM in said assay, of less than 8 |iM in said assay, of less than 7 uM in said assay, of less than 6 uM in said assay,; of less than 5 uM in said assay, of less than 4 uM in said assays of less than 3 uM in said assay, of less than 2 uM in said assay, of less than 1 uM in said assay, of less than 900 nM in said assay, of less than 800 nM in said assay, of less tfc an 700 nM in said assay, of less than 600 nM in said assay, of less titan 500 nM in said assay, of less than 400 nM in said assay, of less than 300 nM in said assay, of less than 200 nM in said assay, of less than 100 nM in said assay, of less than 90 nM in said assay, of less man 80 nM in said assay, of less than 70 nM h i said assay, of less than 60 nM in said assay, of less than 50 nM in said assay, of less than 40 nM n said assay, of less than 30 nM in said assay, of less than 20 nM in said assay, or of less than 10 nM in said assay. In some embodiments, said modulator is an agonist with an ECS0 in said assay of less than a value selected from the interval of 10 nM to 10 uM. In some ¦ embodiments, said modulator is an agonist with an ECL) in said assay of less than a value selected from the interval of 10 nM to 1 uM. fa. some embodiments, sap modulator is an agonist with an EC50 in said assay of less than a value selected from the interval of 10 nM to 100 nM.
In a Uventkth aspect, the invention feattres a method of preparing a pharmaceutical or physiologically acceptable composition comprising jadm&ing a compound according according to the second aspect and a carrier.
In certain embodiments, said composition [is pharmaceutical. In certain embodiments, said composition is physiologically acceptable.
In certain embodiments, said compound is c rally bioavailable. In some embodiments, said oral bioavailability is at least 1%; at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative to ititraperitoneal administration. In some embodiments, said oral bioavailablity is at least 20%, at Ieast25%, a{ least 30%, at least 35%, at least 40%, or at least 45% relative to intraperitoneal administration.
In certain embodiments, said orally bioavaila|5le compound is forte able to cross the blood-brain
barrier.
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In a twenty-first aspect, the invention features a pharmaceutical or physiologically acceptable composition comprising, consisting (essentially of, or consisting of a compound according to the second aspect.
In certain embodiments, said composition is pharmaceutical, tn certain embodiments, the pharmaceutical composition compnses the compound according to the second aspect. In certain embodiments, the pharmaceutical composition consists essentially of the compound according to the second aspect. In certain embodiments, the pharmaceutical composition consists of the compound according to the second aspect.
In certain embodiments, said composition is physiologically acceptable. In certain embodiments, the physiologically acceptable composition (comprises the compound according to the second aspect. In certain embodiments, the physiologically acceptable composition consists essentially of the compound according to the second aspect. In certain embodiments, the physiologically acceptable composition consists of the compound according to the second aspect
In certain embodiments, said compound is orally bioavailable. In some embodiments, said oral bioavailability is at least 1%, at leas: 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative to intraperitoneal administration. In some embodiments, said oral bioavailablity is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative to intrapentoneal administration.
In certain embodiments, said orally bioavailable compound is further able to cross the blood-brain barrier.
In a twenty-second aspect, the invention features a method of modulating the activity of a RUP43 GPCR, said receptor comprising a GPR131 amino acid sequence, wherein said modulation is for lowering blood glucose level in an individual in need of said modulation, comprising contacting said receptor with a therapeutically effective amount of a compound according (to the second aspect or with a therapeutically effect amount of a pharmaceutical or physiologically acceptable composition according to the twenty-first aspect. In certain embodiments, siid contacting is with a therapeutically effective amount of a compound according to the second aspect. IIL certain embodiments, said contacting is with a therapeutically effective amount of a pharmaceutical or physiologically acceptable composition according to the twenty-first aspect.
In certain embodiments, said compound is orally bioavailable. In some embodiments, said oral bioavailability is at least 1%, at lee st 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative to intraperitoneal administration, hi some embodiments, said oral bioavailablity is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative to intraperitoneal administration.
In certain embodiments, said orally bioavailable compound is further able to cross the blood-brain barrier.
In certain embodiments, feaid individual is a mammal. In certain embodiments, said individual is a non-human mammal. In certain (embodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit,
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mouse, rat, non-human primate or human In certain lembodiments, said mammal is a mouse, rat, non-human primate, or human. Most preferred is human
In a tyvmty-third aspect, the invention features a method of modulating the activity of a RUP43 GPCR, said receptor comprising a GPR131 amino acid sequence, wherein said modulation is for preventing or treating a metabolic disorder in an individual in need of said modulation, comprising contacting said receptor with a therapeutically effective amount of a compound according to the second aspect or with a therapeutically effect amount! of a pharmaceutical or physiologically acceptable composition according to the twenty-first aspect In certain embodiments, said contacting is with a therapeutically effective amount of a compound according to the second aspect. In certain embodiments, said contacting is with a therapeutically effective amount of a pharmaceutical or physiologically acceptable composition according to the twenty-first aspect In certain embodiments, the metabolic disorder is selected from the group consisting of;
(a) diabetes;
(b) impaired glucose tolerance;
(c) insulin resistance; and
(d) hyperinsulinemia.
In some embodiments, diabetes is type 1 diabetes. In certain preferred embodiments, diabetes is type 2 diabetes, hi certain embodiments, the metabolic disorder is diabetes. In certain embodiments, the metabolic disorder is type 1 diabetes. In certain embodiments, the metabolic disorder is type 2 diabetes. In certain embodiments, the metabolic disorder is impaired glucose tolerance. In certain embodiments, the metabolic disorder is insulin resistance. In csrtam embodiments, the metabolic disorder is hyperinsulinemia. In certain embodiments, the metabolic disorder is related to an elevated blood glucose concentration in the individual.
The invention also features a method of modulating the activity of a RUP43 GPCR, said receptor comprising a GPR131 amino acid sequence, wherein said modulation is for preventing or treating a complication of an elevated blood glucose concentrition in an individual in need of said modulation, comprising contacting said receptor with a therapeutically effective amount of a compound according to the second aspect or with a therapeutically effect amour t of a pharmaceutical or physiologically acceptable composition according to the twenty-first aspect n certain embodiments, said contacting is with a therapeutically effective amount of a compound according to the second aspect. In certain embodiments, said contacting is with a therapeutically effective amount of a pharmaceutical or physiologically acceptable composition according to the twenty-first aspect. In certain embodiments, the complication is selected from the group consisting of:
(a) Syndrome X,
(b) atherosclerosis;
(c) atheromatous disease;
(d) heart disease;
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(e) hypertension;
(f) stroke;
(g) neuropathy;
(h) retinopafhy;
(i) nephropathy; and
(j) peripheral vascular disease.
Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, coronary artery disease, and high blood pressure. In certain embodiments, the complication is Syndrome X. In certain embodiments, the complication is atherosclerosis. In certain embodiments, the complication is atheromatous disease. In certain embodiments, the complication is heart disease. In certain embodiments, the complication is cardiac insufficiency. In certain embodiments, the complication is coronary insufficiency. In certain embodiments, the complication is coronary artery disease. In certain embodiments, the complication is high blood pressure. In certain embodiments, the complication is hypertension. In certain embodiments, the complication is stroke. In certain embodiments, the complication is neuropathy In certain embodiments, the complication, is retinopathy. In certain embodiments, the complication is neuropathy. In certain 'embodiments, the complication is peripheral vascular disease, In certain embodiments, the complication is polycystic ovary syndrome. In certain embodiments, trie complication is hyperlipiderma.
In certain embodiments, skid compound is orally bioavailable. In some embodiments, said oral bioavailability is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative to intraperitoneal administration. In some embodiments, said oral bioavailablity is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative to intraperitoneal administration.
In certain embodiments, said orally bioavailable compound is further able to cross the blood-brain barrier.
In certain embodiments, skid individual is a mammal. In certain embodiments, said individual is a non-human mammal. In certain embodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit, mouse, rat, non-human primate or human. In certain embodiments, said mammal is a mouse, rat, non-human primate, or human. Most preferred is human
In a twenty-fourth aspectj the invention features a method of lowering blood glucose concentration in an individual in need of said lowering, comprising contacting said receptor with a rherapeutically effective amount of a compound according to the second aspect or with a therapeutically effective amount of a pharmaceutical or physiologically acceptable composition according to the twenty-first aspect with a RUP43 GPCR, said receptor comprising a GPR131 amino acid sequence. In certain embodiments, said contacting is with a therapeutically effective amount of a compound according to the second aspect. In certain embodiments, said conts cting is with a therapeutically effective amount of a pharmaceutical or physiologically acceptable composition according to the twenty-first aspect.
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In certain embodiments, said compound is orally bioavailafale In some embodiments, said oral bioavailabihty is at least 1%, at least 5%, at least 10%, it least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative to lntrapentoneal administration. In some embodiments, said oral bioavadabkty is at least 20%, at least 25%, at east 30%, at least 35%, at least 40%, or at least 45% relative to intrapentoneal administration In certain Embodiments, said oraUy bioavailable compound is further able to cross the blood-brain barrier
In certain embodiments, said individual is a mammal. In certain embodiments, said individual is a non-human mammal. In certain embodiments, said n-ammal is a horse, cow, sheep, pig, cat, dog, rabbit, mouse, rat, non-human primate or human. In certain embodiments, said mammal is a mouse, rat, non-human primate, or human. Most preferred is human.
In a twenty-fifth aspect, the invention features a method of preventing or treating a metabolic disorder in an individual in need of said reducing, comprising contacting said receptor with a therapeutically effective amount of a compound according to the second aspect or with a therapeutically effect amount of a pharmaceutical or physiologically acceptable composition according to the twenty-first aspect with a RUP43 GPCR, said receptor comprising a GPR131 amino acid sequence. In certain embodiments, said contacting is with a therapeutically effective amount of a compound according to the second aspect. In certain embodiments, said contacting is with a therapeutically effective amount of a pharmaceutical or physiologically acceptable composition according to the twenty-first aspect. In certain embodiments, the metabolic disorder is selected from the group consisting of:
(a) diabetes;
(b) impaired glucose tolerance,
(c) insulin resistance; and
(d) hyperinsulinemia.
In some embodiments, diabetes is type 1 diabetes In certain preferred embodiments, diabetes is type 2 diabetes. In certain embodiments, the metabchc disorder is diabetes. In certain embodiments, the metabolic disorder is type 1 diabetes. In certain ernbc diments, the metabolic disorder is type 2 diabetes. In certain embodiments, die metabolic disorder is impaired glucose tolerance. In certain embodiments, the metabolic' disorder is insulin resistance. ¦ In Certain embodiments, the metabolic disorder is hyperinsulinemia. In certain embodiments, the metapolic disorder is related to an elevated blood glucose concentration in the individual.
The invention also features a method of preventing or, treating a complication of an elevated blood glucose concentration in an jindividual in need of said prevention or treatment, comprising contacting said receptor with a therapeutically effective amount of d compound according to the second aspect or with a therapeuttcaUy effect amount of a pharmaceutical on physiologically acceptable composition according to the twenty-first aspect with a RUP43 GPCR, said redeptor comprising a GPR131 amino acid sequence. In certain embodiments) said contacting is with a therapeutically effective amount of a compound according to the second aspect. In certain embodiments, said contacting is with a therapeutically effective amount of a
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pharmaceutical or physiologically acceptable composition according to the twenty-first aspect. In certain embodiments, the complication is selected from the group consisting of:
(a) Syndrome X;
(b) atherosclerosis;
(c) atheromatous disease;
(d) heart disease;
(e) hypertension;
(f) stroke;
(g) neuropathy;
(h) retinopatiiy;
(1) nephropathy; and
0) peripheral vasculaij disease.
Heart disease includes, but! is not limited to, cardiac insufficiency, coronary insufficiency, coronary artery disease, and high blood pressure In certain embodiments, title complication is Syndrome X. In certain embodiments, the complication is atherosclerosis In certain embodiments, the complication is atheromatous disease. In certain embodiments, the complication is heart disease. In certain embodiments, the complication is cardiac insufficiency. In certain embodiments, the complication is coronary insufficiency. In certain embodiments, the complication is coronary artery disease. In certain embodiments, the complication is high blood pressure. In certain embodiments, the complication is hypertension, In certain embodiments, the complication is stroke. In certain embodiments, the complication is neuropathy. In certain embodiments, the complication is retinopathy. In certain embodiments, the complication is neuropathy. In certain embodiments, the complication is peripheral vascular disease In certain embodiments, the complication isjpolycystic ovary syndrome. In certain embodiments, the complication is hyperlipidemia.
In certain embodiments,!said compound is orally [bioavailable. In some embodiments, said oral bioavailability is at least 1%, at leLst 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative to intraperitoneal administration. In some embodiments, said oral bioavailabliry is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative to intraperitoneal administration.
In certain embodiments,[said orally bioavailable compound is further able to cross the blood-brain barrier.
In certain embodiments,] said individual is a mammaL In certain embodiments, said individual is a non-human mammal. In certain embodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit, mouse, rat, non-human primatei or human. In certain embodiments, said mammal is a mouse, rat, non-human primate, or human. Most preferred is human.
In a tiventy-sixth aspect, the invention features ai method of lowering blood glucose concentration comprising providing or administering to an individual in need of said reducing a compound according to
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the second aspect or with a therapeutically effective amount of a pharmaceutical or physiologically acceptable composition according to the twenty-first aspect. In certain embodiments, said providing or administering a compound isiproviding or administering a compound according to the second aspect In certain embodiments, said providing or administering a pharmaceutical or physiologically acceptable composition is providing or administering a pharmaceutical or physiologically acceptable composition according to the twenty-first aspect.
In certain embodiments, said compound is orally bioavailable. In some embodiments, said oral bioavailability is at least 1 %, at least 5%, at least 10%, It least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or,at least 45% relative to inltrapentoneal administration. In some embodiments, said oral bioavailablity is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative to intraperitoneal administration.
In certain embodiments, said orally bioavailabjle compound is further able to cross the blood-brain barrier.
In certain embodiments, said individual is a mkmmal. In certain embodiments, said individual is a non-human mammal. In certain embodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit, mouse, rat, non-human primate or human. In certain embodiments, said mammal is a mouse, rat, non-human primate, or human. Most preferred is human.
In a twenty-seventh aspect, the invention features a method of treating a metabolic disorder comprising providing or administering to an individual in need of said treating or preventing a compound according to (he second aspect or with a therapeutically effective amount of a pharmaceutical or physiologically acceptable composition according to ihe twenty-first aspect La certain embodiments, said providing or administering a compound is providing or administering a compound according to the second aspect In certain embodiments, said providing on administering a pharmaceutical or physiologically acceptable composition is providing or administering a pharmaceutical or physiologically acceptable composition according to the twenty-first aspect In certain embodiments, the metabolic disorder is selected from the group consisting of:
(a) diabetes;
(b) impaired glucose tolerance;
(c) insulin resistance; and
(d) hyperinsulinerrua.
Ill some embodiments, diabetes is type 1 diabetes. In certain preferred embodiments, diabetes is type 2 diabetes. In certainiembodiments, the metabolic disorder is diabetes. In certain embodiments, the metabolic disorder is type 1 diabetes. In certain embodiments, the metabolic disorder is type 2 diabetes. In certain embodiments, the metabolic disorder, is impaired glucose tolerance. In certain embodiments, the metabolic disorder is insulin resistance. In icertain embodiments, the metabolic disorder is hyperinsulinemia. In certain embodiments, the metabolic disorder is related to an elevated blood glucose concentration in the individual.
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The invenUon also features a method of treating a complication of an elevated glucose concentration comprising providing or administering to an individual in need of said treating or preventing a compound according to the second aspect or with a therapeuucally effective amount of a pharmaceutical or physiologically acceptable composition according to the twenty-first aspect. In certain embodiments, said providing or administering a compoimd is providing or administering a compound according to the second aspect In certain embodiments, said providing or administering a pharmaceutical or physiologically acceptable composition is providing or administering a pharmaceutical or physiologically acceptable composition according to the twenty-first aspect In certain embodiments, the complication is selected from the group consisting of:
(a) Syndrome X;
(b) atherosclerosis;
(c) atheromatous disease;
(d) heart disease;
(e) hypertension;
(f) stroke;
(g) neuropathy;
(h) retbopathy;
(1) nephropathy; and
(j) peripheral vascular disease:
Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, coronary artery disease, and high blood pressure. In certain embodiments, the complication is Syndrome X. In certain embodiments, the complication is i atherosclerosis. In certain embodiments, the complication is atheromatous disease. In certain e ribodiments, the complication is heart disease. In certain embodiments, the complication is cardiac insufficiency. In certain embodiments, the complication is coronary insufficiency, hi certain embodiments, the complication is coronary artery disease. In certain embodiments, the complication is high blood pressure. In certain embodiments, the complication is hypertension. In certain embodiments, the complication is stroke. In certain embodiments, the complication is neuropathy. In certain embodiments, the complication is retinopathy. In certain embodiments, the complication is neuropathy. In certain embodiments, the complication is peripheral vascular disease. In certain embodiments, the complication is (polycystic ovary syndrome. In certain embodiments, the complication is hyperlipidemia.
In certain embodiments, [said compound is orally bioavailable. In some embodiments, said oral bioavailability is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%," at least 35%, at least 40%, or at least 45% relative to intraperitoneal administration. In some embodiments, said oral bioavailablity is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative to intraperitoneal administration,
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In certain embodiments, said orally bioavailable compound is further able to cross the blood-brain barrier.
In certain embodiments, said individual is a mammal. In certain embodiments, said mdividual is a non-human mammal In certain embodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit, mouse, rat, non-human primate or human. In certain embodiments, said mammal is a mouse, rat, non-human primate, or human. Most preferred is human.
In a twenty-eighth aspect, the invention features a compound according to the second aspect for use in a method of treatment of the human or animal body oy therapy.
In certain embodiments, said compound is orally bioavailable. In some embodiments, said oral bioavailabihty is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative to intraperitoneal administration. In some embodiments, said oral bioavailablity is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative to intrapcritoneal administration
In certain embodiments, said orally bioavailable compound is further able to cross the blood-brain barrier.
In certain embodiments, said animal is a minimal In certain embodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit, mouse, rat, or non-human primate More preferred of human or animal is human.
In a tyvetity-ninth aspect, the invention features a compound according to the second aspect for use tn a method of lowering blood glucose concentration in the human or animal body by therapy.
In certain embodiments, said compound is c rally bioavailable. In some embodiments, said oral bioavailability is at least 1%, at least 5%, at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative to Intraperitoneal administration In some embodiments, said oral bioavailablity is at least 20%, at least 25%, a least 30%, at least 35%, at least 40%, or at least 45% relative to intrapentoneal administration.
In certain embodiments, said orally bioavailable compound is further able to cross the blood-brain barrier.
In certain embodiments, said animal is a rriammal. In certain embodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit, mouse, rat, pt non-human primate. More preferred of human or animal is human.
In a thutieth aspect, the invention features alcompound according to the second aspect for use in a method of prevention or treatment for a metabolic disorder m the human or animal body by therapy In certain embodiments, the metabolic disorder is selected from the group consisting of:
(a) diabetes;
(b) impaired glucose tolerance;
(c) insulin resistance; and
(d) hypennsuljnemia.
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In some embodiments, diabetes is type 1 diabetes In certain preferred embodiments, diabetes is type 2 diabetes In certain embodiments, the metabolic disorder is diabetes. In certain embodiments, the metabolic disorder is type 1 diabetes] In certain embodiments, the metabolic disorder is type 2 diabetes In certain embodiments, the metabolic disorder is unpaired glucose tolerance. In certain embodiments, the metabolic disorder is insulin resistance. In certain embodiments, the metabolic disorder is hyperinsulinemia. In certain embodiments, the metabolic disorder is related to an elevated blood glucose concentration in die individual.
The invention also features/ a compound according ,to the second aspect for use in a method of prevention or treatment for a complication of an elevated blood glucose concentration in the human or animal body by therapy. Iu certain Embodiments, the complication is selected from the group consisting of:
(a) Syndrome X;
(b) atherosclerosis;
(c) atheromatous disease;
(d) heart disease;
(e) hypertension;
(f) stroke;
(g) neuropathy;
(h) retirtopathy;
(i) nephropathy; and
(j) peripheral vasculaV disease.
Heart disease includes, bu: is not limited to, cardiac insufficiency, coronary insufficiency, coronary artery disease, and high blood prsssure. In certain embodiments, the complication is Syndrome X. In certain embodiments, the complication is atherosclerosis, fa certain embodiments, the complication is atheromatous disease. In certain embodiments, the complication is heart disease, In certain embodiments, the complication is cardiac insufficiency. In certain embodiments, the complication is coronary insufficiency. In certain embodim ents, the complication is coronary artery disease. In certain embodiments, the complication is high blood pressure, In certain embodiments, the complication is hypertension hi certain embodiments, the complication is stroke. In certain embodiments, the complication is neuropathy. In certain embodiments, the coijjpiication is retinopathy. In certain embodiments, the complication is neuropathy. In certain embodiments, the complication is peripheral vascular disease. In certain embodiments, the complication is polycystic ovary syndrome, m certain embodiments, the complication is hyperlipidemia.
hi certain embodiments' said compound is orally bioavailable. In some embodiments, said oral bioavaUability is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative to intraperitoneal administration. In some embodiments, said oral bioavailablity is at least! 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative to intraperitoneal administration.
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In certain embodiments, said orally bioavailafcle compound is further able to cross the blood-brain barrier.
In certain embodiments, said animal is a mammal. In certain embodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit, mouse, rat, or non-human primate. More preferred of human or animal is human.
In a thirty-first aspect, the invention features a method of using a compound according to the second aspect for the preparation of a medicament for the reduction of blood glucose concentration.
In certain embodiments, said compound is orally bioavailable. In some embodiments, said oral bioavailabm'ty is at least 1%, at least 5%, at least 10%; at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative to ntraperitoneal administration. In some embodiments3 said oral bioavailablity is atleast 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative to intrapentoneal administration.
In certain embodiments, said orally bioavaila'ble compound is further able to cross the blood-brain barrier.
In a thirty-second aspect, the invention features a method of using a compound according to the second aspect for the preparation of a medicament for the prevention of or treatment of a metabolic disorder, hi certain embodiments, the metabolic disorder is selected from the group consisting of;
(a) diabetes;
(b) impaired glucose tolerance;
(c) insulin resistance; and
(d) hyperinsulinemia.
In some embodiments, diabetes is type 1 di sbetes. In certain preferred embodiments, diabetes is type 2 diabetes. In certain embodiments, the metabolic disorder is diabetes, hi certain embodiments, the metabolic disorder is type 1 diabetes. In certain embadiments, the metabolic disorder is type 2 diabetes, hi certain embodiments, the metabolic disorder is impaired glucose tolerance. In certain embodiments, the metabolic disorder is insulin resistance. In certain embodiments, the metabolic disorder is hyperinsulinemia. in certain embodiments, the metabolic disorder is related to an elevated blood glucose concentration in the individual.
The invention also features a method of usiiig a compound according to the second aspect for the preparation of a medicament for the prevention of] or treatment of a complication of an elevated blood glucose concentration. In certain embodiments, the qomplication is selected from the group consisting of.
(a) Syndrome X;
(b) atherosclerosis;
(c) atheromatous disease;
(d) heart disease;
(e) hypertension;
(f) stroke;
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(g) neuropathy,
(h) retinopathy;
(i) nephropathy; and
(j) peripheral vascular disease.
Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, coronary artery disease, and high blood pressure. In certain embodiments, the complication is Syndrome X. In certain embodiments, the complication is atherosclerosis. In certain embodiments, the complication is atheromatous disease, hi certain embodiments, the complication is heart disease. In certain embodiments, the complication is cardiac insufficiency. In certain embodiments, the complication is coronary insufficiency, in certain embodiments, the complication is coronary artery disease. In certain embodiments, the complication is high blood pressure. In certain embodiments, the complication is hypertension. In certain embodiments, the complicatmn is stroke. In certain embodiments, the complication is neuropathy. In certain embodiments, the complication is retinopathy. In certain embodiments, the complication is neuropathy. In certain embodiments, the complication is peripheral vascular disease. -In certain embodiments, the complication is polycysuc ovary syndrome. In certain embodiments, the complication is hyperlipidemia.
In certain embodiments, sa!id compound is orally bioavailable. hi some embodiments, said oral bioavailability is at least 1%, at leasj 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%,'or at least 45% relative to intraperitoneal administration. In some embodiments, said oral bioavailablity is at least 20P/o, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative to intraperitoneal administration.
In certain embodiments, sa^d orally bioavailable compound is further able to cross the blood-brain barrier
In a thirty-third aspect, tie invention features a method of modulating a RUP43 GPCR, said receptor comprising a GPR131 amino acid sequence, comprising contacting said receptor with a compound according to the second aspect or with, a pharmaceutical or physiologically acceptable composition according to the twenty-first aspect In certain embodiments, said contacting is with a compound according to the second aspect In certain embodiments, said contacting is with a pharmaceutical or physiologically acceptable composition according t D the twenty-first aspect
In certain embodiments, £ aid compound is orally bioavailable. hi some embodiments, said oral bioavailability is at least 1%, at leak 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at last 45% relative to intraperitoneal administration. In some embodiments, said oralbioavailabhty is at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% relative to intraperitoneal adminisa ation.
In certain embodiments, said orally bioavailable compound is further able to cross the blood-brain barrier.
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In a thirty-fourth aspect, the invention features a method of identifying one or more candidate compounds as a compound that binds to a RUP43 GPCR, said receptor comprising a GPR131 ammo acid sequence, comprising the steps of:
(a) contacting the receptor with a det^ctably labeled known ligand of the GPCR in the
presence or absence of the candidate compound; and
i
(b) determining whether the binding of Said labeled ligand is inhibited in the presence of the
candidate compound; '
wherein said inhibition is indicative of the candidate compound being a compound that binds to a
RUP43 GPCR. !
In certain embodiments, the GPR131 ammo acid sequence is selected from the group consisting of:
(a) the1 amino acid sequence of ££EQ ID NO:2;
(b) amino acids 2-330 of SEQ Dp NO.2;

(c) amino acids 2-330 of SEQ ID NO:^, with the proviso that the RUP43 G protein-coupled
receptor does not comprise the methionine residue at ^imino acid position 1 of SEQ ED NO:2;
(d) the amino acid sequence of a G protein-coupled receptor encoded by a polynucleotide
comprising a nucleic acid sequence, said nucleic aci{3 sequence being obtainable by a process comprising
performing PCR on a human DNA sample using printers SEQID NO:3 and SEQ ID NO:4;
. (e) the amino acid sequence of SEQ ID WO:6;
(f) the amino acid sequence of a G protein-coupled receptor encoded by a polynucleotide
comprising a nucleic acid sequence, said nucleic acii sequence being obtainable by a process comprising
performing PCR on a human DNA sample using prirr ers SEQ.ID NO:7 and SEQ ID NO:8;
(g) the amino acid sequence of SEQ ID NO:2 wherein the alanine at amino acid position 223
of SEQ ID NO:2 is substituted with lysine;
(h) amino acids 2-330 of SEQ ID NO:2 jwherein the alanine at amino acid position 223 of SEQ ID NO:2 is substituted with lysine;
(i) amino acids 2-330 of SEQ ID NO:2 [wherein the alanine at amino acid position 223 of SEQ ID NO:2 is substituted with lysine, with the proviscj that the RUP43 G protein-coupled receptor does not comprise the methionine residue at amino acid position 1 of SEQ ED NO:2; and
(j) the amino acid sequence of a G protein-coupled receptor encoded by a polynucleotide that hybridizes under stringent conditions to the complement of SEQ ED NO:1.
In certain embodiments, the RUP43 GPCpR is recombinant. In certain embodiments, said contacting comprises contacting with a host cell or vfdth membrane of a host cell that expresses the GPCR. In certain embodiments, said host cell that expressesi the GPCR comprises an expression vector comprising a polynucleotide encoding the receptor.
In some embodiments, the GPR131 amino acid sequence is the amino acid sequence of SEQ ID NO:2. In some embodiments, the GPR131 amino a£id sequence is a variant of the amino acid sequence of SEQ ID NO:2. In some embodiments, said variarit of the amino acid sequence of SEQ ID NO:2 is an
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allehc variant or mammalian ortholcjg of said amino acid sequence. In some embodiments, said variant of the amino acid sequence of SEQ ID NO:2 is a non-endogenous, constitutively activated mutant of said ammo acid sequence or of an allehc variant or mammalian ortholog of said amino acid sequence In certain embodiments, said variant of the am no acid sequence of SEQ ID NO:2 is a biologically active fragment of said amino acid sequence or of an allehc variant or mammalian ortholog of said amino acid sequence. In certain embodiments, said biologically active fragment of the amino acid sequence of SEQ ID NO:2 or of an allehc variant or mammalian ortholog of said amino acid! sequence is (he ammo acid sequence of SEQ ID NO:2 or of an allehc variant or mammalian ortholog of said amino acid sequence absent the N-terminal methdonine. In certain embodiment^, said variant of the amino acid sequence of SEQ BO NO:2 is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about S9% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, said variantof the air ino acid sequence of SEQ ID NO:2 is at least about 90%, at least about 9,1%, at least about 92%, at least about 93%, at least about 94%, at least about 95%s at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to the amino acid sequence of SEQ ID NO:2.
In certain embodiments, said membrane preparation is made by homogenization of the cells with a Brinkman Polytron™. In certain embodiments, said membrane preparation is made by homogenization wife. 3 bursts of 10-20 secduration each of said polytron.
In certain embodiments, said candidate compound is not an antibody or derivative thereof.
In certain embodiments, SE id candidate compound is not a peptide.
In certain embodiments, sc id known ligand is a compound according to me second aspect.
In certain embodiments, said known ligand is a modulator according to the third aspect.
In certain embodiments, said known ligand is Compound 1, Compound 2, or Compound 3. In certain embodiments, said known hgand is Compound 1.: In certain embodiments, said known ligand is Compound 2. In certain embodiments, said known ligand is Compound 3.
In certain embodiments, said known ligand is an antibodyspecificifor the GPCR, or an antigen-binding derivative of the antibody.
In certain embodiments, said label is selected from.the group consisting of:
(a) radioisotbpe;
(b) enzyme; and
(c) fluorophore.
In certain embodiments, said label is a radioisotope. In certain embodiments, said label is selected from the group consisting of3H, "C, 3SS, and 125I.
Compound 1, Compound 2, or Compound 3 can be radiolabelled using techniques known in the art, infra. In certain embodiments, Cpmpound 1, Compound 2, or Compound 3 is radiolabelled with 3H or 14C.
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In other embodiments, said method further comprises the step of comparing the level of inhibition of binding of a labeled first known hgand by the candidate compound to a second level of inhibition of binding of said labeled first known hgand by a.second ligand known to bind to the GPCR.
In a thirty-fifth aspect, the invention features |a method for detecting ligands that bind to a RUP43 GPCR, said receptor comprising a GPR131 amino aci4 sequence, comprising the steps of:
contacting a test ligand with a host cell or with* membrane of a host cell that expresses said receptor, under conditions which permit interaction between said receptor and said test hgand; and
detecting a ligand bound to said receptor.
In certain embodiments, the GPR131 amino acid sequence is selected from the group consisting of
(a) the arruno acid sequence of SEQ ID NO:2;
(b) amino acids 2-330 of SEQ ID NO:2;

(c) amino acids 2-330 of SEQ ID NO:2| with the proviso that the RUP43 G protein-coupled
receptor does not comprise the methionine residue at ahuno acid position 1 of SEQ ID NO:2,
(d) the amino acid sequence of a G prfotein-coupled receptor encoded by a polynucleotide
comprising a nucleic acid sequence, said nucleic aci4 sequence being obtainable by a process comprising
performing PCR on a human DNA sample using pnmfers SEQ ID NO:3 and SEQ ID NO:4;
(e) the amino acid sequence of SEQ ID NO:6;
(f) the amino 'acid sequence of a G protein-coupled receptor encoded by a polynucleotide
comprising a nucleic acid sequence, said nucleic acid sequence being obtainable by a process comprising
performing PCR on a human DNA sample using primers SEQ ID NO:7 and SEQ ID NO:8;
(g) the ammo acid sequence of SEQ ID |NO:2 wherein the alanine at amino acid position 223
of SEQ ID NO:2 is substituted with lysine;
(h) amino acids 2-330 of SEQ ID NO:2 wherein the alanine at amino acid position 223 of SEQ ID NO:2 is substituted with jysine;
(i) amino acids 2-330 of SEQ ID N02 ^herein the alanine at amino acid position 223 of SEQ ID NO:2 is substituted with lysine, with the proviso | that the RUP43 G protein-coupled receptor does not comprise the methionine residue at amino acid position 1 ofSEQIDNO:2;and
(j) the amino acid sequence of a G protein-coupled receptor encoded by a polynucleotide that hybridizes under stringent conditions to the complement of SEQ ID NO: 1.
In some embodiments, the GPR13I amino a!cid sequence is the amino acid sequence of SEQ ID NO:2. In some embodiments, the GPR131 amino ac&d sequence is a variant of the ammo acid sequence of SEQ ID NO:2. In some embodiments, said variant of the amino acid sequence of SEQ ID NO:2 is an allelic variant or mammalian ortholog of said amino jacid sequence. In some embodiments, said variant of the amino acid sequence of SEQ ID NO:2 is a non-endogenous, constitutively activated mutant of said amino acid sequence or of an allelic variant or mamirialian ortholog of said amino acid sequence. In certain embodiments, said variant of the amino acid sequence of SEQ ID NO:2 is a biologically active fragment of said amino acid sequence or of an allelic variant or jfnammalian ortholog of said amino acid sequence. In
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certain embodiments, said biologically active fragment of tiie amino acid sequence of SEQ ID NO:2 or of an allehc variant or mammalian ortiiolog of said amino acid sequence is the amino acid sequence of SEQ ID NO:2 or of an allehc variant or njiammalian ortholog of said ammo acid sequence absent the N-terminal methionine. In certain embodiment^, said variant of the amino acid sequence of SEQ H> NO:2 is at least about 75%, at least about 80%, at ikast about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, said variant of the amjino acid sequence of SEQ ID NO:2 is at least about 90%, at least about 91%, at least about 92%, at least abjout 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98°/ In certain embodiments, jthe RUP43 GPCR is recombinant In certain embodiments, said
contacting comprises contacting wijh a host cell or with membrane of a host cell that expresses the GPCR.
In certain embodiments, said host cjell that expresses the GPCR. comprises an expression vector comprising
a polynucleotide encoding the recerjtor. ,
In certain embodiments, sajd test hgand is not an antibody or an antigen-binding derivative thereof. In certain embodiments, said test ligand is not a peptide.
In certain embodiments, s^id membrane preparation is made by homogenization of the cells with a Brinkman Polytron™. In certain [embodiments, said membrane preparation is made by homogenization with 3 bursts of 10-20 sec durationjeach of said polytron.
In certain embodiments, jsaid test hgand is labeled In certain embodiments, said label is a radioisotope. In certain embodimbnts, said label is selected from the group consisting of 3H, 14C, 35S, and 125I.
Applicant reserves the ri^ht to exclude any one or more candidate compounds from any of the embodiments of the invention. Applicant also reserves the right to exclude,any one or more modulators from any of the embodiments qf the invention. Applicant further reserves the right to exclude any polynucleotide or polypeptide frpm any of the embodiments of the invention. Applicant additionally reserves the right to exclude any metabolic disorder or any complication of elevated blood glucose concentration. It is also expressly contemplated that metabolic disorders of the invention can be included in an embodiment either' individually or iin any combination It is also expressly contemplated that complications of elevated blood glucose concentration of the invention can be included in an embodiment either individually or in any combination.
Throughout this application, various publications, patents and published patent applications are cited. The disclosures of these jpublicatxons, patents and published patent applications referenced in this application are hereby incorporated by reference in their entirety into the present disclosure. Citation herein by Applicant of a publication, patent, or published patent application is not an admission by Applicant of said publication, patent, or published patent application as prior art.
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Modifications and extension of the disclosed inventions that are within the purview of the skilled artisan are encompassed within the above disclosure ana the claims that follow.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. By way of example and not limitation, Figure 1 depicts results from a primary screen of
candidate compounds againsfca "target receptor" which is a GsccFusion Protein construct of an endogenous,
constitutively active Gs-coupled GPCR unrelated to KUP43. Results for "Compound A" are provided in
wellA2. Results for "Compound "B" are provided in vellG9. (See, Example 7.)
Figure 2. RT-PCR analysis of RUP43 expression by adipocytes and skeletal muscle cells. Human
and mouse adipocytes express RUP43. Human and Imouse skeletal muscle cells express RUP43. (See,
Example 11)
Figure 3. Endogenous RUP43 couples to Gs (See, Example 14.)
Figure 4. Identification of Compound I as an lagonist of RXJP43. (See, Example 15.)
Figure 5. Identification of Compound 2 as aniagonist of RUP43. (See, Example 16.)
Figure 6. Compound 2 stimulates glucose Uptake in mouse 3T3L1 adipocytes by Compound 2.
(See, Example 18.)
Figure 7. Compound 2 enhances insulin-stimulated glucose uptake in mouse 3T3L1 adipocytes.
(See, Example 19.)
Figure 8. Compound 2 stimulates glucose uptake in primary human adipocytes. (See, Example
20.)
Figure 9. Compound 2 stimulates glucose uptake in rat L6 myoblast cells (See, Example 21.) Figure 10. Compound 2 enhances insulin-stuhulated glucose uptake in rat L6 myoblast cells. (See,
Example 22.)
Figure 11. Compound 2 stimulates glucose uptake in primary human skeletal muscle cells. (See,
Example 23.)
DETAILED DESCRIPTION Definitions
The scientific literature that has evolved around receptors has adopted a number of terms to refer to ligands having various effects on receptors. For clarity and consistency, the following definitions will be used throughout this patent document To the,extent that these definitions conflict with other definitions for these terms, the following definitions shall control:
AGONISTS shall mean materials (e.g, ligands, candidate compounds) that activate an intracellular response when they bind to the receptor. In some embodiments, AGONISTS are those materials not previously known to activate the intracellular response when they bind to the receptor (e g. to enhance GTPyS binding to membranes or to elevate intracellujar cAMP level). In some embodiments, AGONISTS
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are those materials not previously known to, stimulate glucose uptake in adipocytes or in skeletal muscle cells obtained from a mammal when they bind to the receptor i
AMINO ACID ABBREVIATIONS used herein are set out in Table A: TABLEA

ALANINE ALA ; A i
ARGININE ARG R '
ASPARAGINE - ASMi N
ASPARTIC ACID , ASP D :
CYSTEINE CYS C
GLUTAMIC ACID GLU I E . .
GLUTAMINE GLN Q i
GLYCBME 1 GLY ¦ G "
HISTIDINE i HIS i H
ISOLEUCINE ' ILE ' I
LEUCINE ¦ LEU L
LYSINE LYS K
METHIONINE MET M
PHENYLALANTNE PHE F
PROLINE PRO P
SERINE SER S '
THREONINE THR T
TRYPTOPHAN TRP . w
TYROSINE TYR Y
VALINE VAL V
ANTAGONISTS shall rriean materials (e.g., ligands, candidate compounds) that competitively bind to the receptor at the same sit; as the agonists but which do not activate an intracellular response, and can thereby inhibit the intracellular responses elicited by agonists. ANTAGONISTS do not diminish the baseline intiacellular response in the absence of an agonist, hi some embodiments, ANTAGONISTS are those materials not previously knolvn to compete with an agonist to inhibit ihe cellular response when they bind to the receptor, e.g. wherein jhe cellular response is GTPyS binding to membranes or the elevation of
intracellular cAMP level.
ANTIBODIES are intended herein to encompass monoclonal antibodies and polyclonal antibodies. Antibodies are further intended to encompass IgG, IgA, IgD, IgE, and IgM. Antibodies include whole antibodies, including single-chain whole antibodies, and antigen binding fragments thereof, including Fab, Fab*, F(ab)2 and F(ab')2. Antibodies may be from any animal origin. Preferably, antibodies are human, murine, rabbit, goat, guinea pig, hamster, camel, donkey, sheep, horse or chicken. Preferably antibodies
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have binding affinities with a dissociation constant orlKd value less than 5xlO"*M, LO^M, 5xlO"7M, 10'7M, SXKT'M, 10*M, 5xlO^M, lp"9M, 5xlO'IOM Wi0M, 5 jlO'nM, 10*uM, 5xlO'12M, l(Tl2M, 5xl0"!3M, 10-13M, 5xlO'14M 1O"I4M, 5xlO"lsMland 10"l5M. Antibodies of the present invention may be prepared by any suitable method known in the art. Derivatives of antipodies are intended to encompass, but not be limited to, antigen-binding fragments.
BIOLOGICALLY, ACTIVE FRAGMENT of a GPCR polypeptide or amino acid sequence shall mean a fragment of the polypeptide or amino acid sequence having structural and biochemical functions of a naturally occurring GPCR. In certain embod.imer.ts, the biologically active fragment couples to a G protein. In certain embodiments, the biologically activ e fragment binds to an endogenous ligand.
CANDIDATE COMPOUND shall mean a molecule (for example, and not limitation, a chemical compound) lhat is amenablejto a screening technique.
CHEMICAL GROUP, MOIETY OR RADJICAL:
i The term "Cw alkyl" denotes a straight or branched carbon radical containing the number of
carbons as indicated, for examples, in some embodiments, alkyl is a "CM alkyl" and the group contains 1 to 4 carbons, in still other embodiments, alkyl is a "Q^ ¦ The term "halogen", or "halo" denotes to a fluoro, chloro, bromo or iodo group.
CODON shall mean a grouping of three nuclcotides (or equivalents to nucleoudes) which generally comprise a nucleoside [adenosine (A), guanosine (G), cytidine (C), uridine (U) and thymidine (T)] coupled to a phosphate group and which, when translated, encc des an amino acid.
COMPOSITION means a material comprising at least one component. A "pharmaceutical composition" is an example of a composition.
COMPOUND EFFICACY shall mean a measurement of the ability of a compound to inhibit or stimulate receptor functionality; i.e. the ability to activate/inhibit a signal transduction pathway, in contrast to receptor binding affinity. Exemplary means of deteptiing compound efficacy are disclosed in the Example section of this patent document.
COMPRISING, CONSISTING ESSENTIALLY OF, and CONSISTING OF are defined herein according to their standard meaning. A defined meaning set forth in the M.P.E.P. controls over a defined meaning in the art and a defined meaning setj forth in controlling Federal Circuit case law controls over a meaning set forth in the M P.E.P.
CONSTITUTTVELY ACTIVE RECEPTOR shall mean a receptor stabilized in an active state
by means other than through binding of the receptor to its ligand or a chemical equivalent thereof. A
i CONSTITUTTVELY ACTIVE RECEPTOR may be endogenous or non-endogenous.
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CONSTITUTIVELY AClTVATED RECEPTOR shall mean an endogenous receptor that has been modified so as to be constitutivply active.
CONSTITUTIVE RECEPTOR ACTIVATION [shall mean activation of a receptor in the absence of binding to its Ugand or a Chemical equivalent thereof
CONTACT or CONTACTING shall mean bringing at least two moieties together, whether in an in vitro system or an in vivo system.
DECREASE is used to re: er to a reduction in a measurable quantity and is used synonymously with the terms "reduce", "dimmish", "lower", and "lessen".
ELEVATED BLOOD GLUCOSE CONCENTRATION shall mean a fasting blood glucose concentration in a mammal greater than the normal fasting jblood glucose concentration for the mammal By way of example, normal human fasting blood glucose concentration is less than 100 mg/dl. As used herein, an elevated human blood glucose concentration is a fasting blood glucose concentration of 100 mg/dl or greater. By way of illustration and not limitation, an elevated blood glucose concentration encompasses hyperglycerma.
ENDOGENOUS shall m^an a material that a mammal naturally produces. ENDOGENOUS in reference to, for example and not Ui nitation, the term "receptor," shall mean that which is naturally produced by a mammal (for example, and not limitation, a human). ENDOGENOUS shall be understood to encompass allelic variants of a gen; as well as the allelic polypepti.de variants, so encoded. As used herein, "endogenous GPCR" and "native GPCR." are used interchangeably. By contrast, the term NON-ENDOGENOUS in this context shall mean that which is not naturally produced by a mammal (for example, and not limitation, a human). For example, iand not limitation, a receptor which is not constitutively active in its endogenous form, but when manipulated becomes constitutively active, is most preferably referred to herein as a "r on-endogenous, constitutively activated receptor."
EXPRESSION VECTOR is defined herein as a DNA sequence that is required for the transcription of cloned DNA and the translation of the transcribed noRNAs in an appropriate host cell recombinant for said EXPRESSION VECTOR. An appropriately constructed EXPRESSION VECTOR should contain an origin of replica ion for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters. Said cloned DNA to be transcribed is operably linked to a constitutively or conditionally active promoter within said expression vector By way of illustration and not limitation, pCMV is an,expression vector.
G PROTEIN COUPLED RECEPTOR FUSION PROTEIN and GPCR FUSION PROTEIN, in the context of the invention disclosed herein, each mean a non-endogenous protein comprising an endogenous, constitutively active GPCR or a non-endogenous, constitutively activated GPCR fused to at least one G protein, most preferably the alpha (a) subunit of such G protein (this being the subunit that binds GTP), with the G protein preferably being of the same type as the G protein that naturally couples with endogenous GPCR. For example, and not limitation, in an endogenous state, if the G protein "Gsa" is the predominate G protein that douples with the GPCR, a GPCR Fusion Protein based upon the specific
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GPCR would be a non-endogenous protein comprising) the GPCR fused to Gscc, in some circumstances, as will be set forth below, a non-predominant G protein can be fused to the GPCR. The G protein can be fused directly to the C-terminus of the constitutively active G?CR or there may be spacers between (he two.
HOST CELL shall mean a cell capable of having a vector incorporated therein. In certain embodiments, the vector is an expression vector. Exenplary host cells include but are not limited to 293, 293T, CHO, MCB3901, and COS-7 cells, as well as mdanophoie cells
IN NEED OF PREVENTION OR TRE ATI 1ENT as used herein refers to a judgement made by a caregiver (e.g. physician, nurse, nurse practitioner, e:c. m the case of humans; veterinarian in the case of animals, including non-human mammals) that an individual or animal requires or will benefit from treatment. This judgement as made based on a variety of factors that are in the realm of a caregiver's expertise, but that include the knowledge that the indr idual or animal is ill, or will be ill, as the result of a condition that is treatable by the compounds of the invc ntion.
INDIVIDUAL as used herein refers to any an mal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and most preferably humans.
INHIBIT ]or INHIBITING, in relationship to the term "response" shall mean that a response is decreased or prevented in the presence of a compound is opposed to in the absence of the compound.
IMPAIRED GLUCOSE TOLERANCE (IGT) as, used herein is intended to indicate that condition associated with insulin-resistance that is intermediate between frank, type 2 diabetes and normal glucose tolerance (NGT). IGT is diagnosed by a procedure wherein an affected person's postprandial glucose response is determined to be abnormal as assessed by 2-hour postprandial plasma glucose levels. In this test, a measured amount of glucose is given to tie patient and blood glucose levels are measured at regular intervals, usually every half hour for the first tvo hours and every hour thereafter. In a "normal" or non-IGT individual, glucose levels rise during the first two hours to a level less than 140 mg/dl and then drop rapidly. In an IGT individual, the blood glucose levels are higher and the drop-off level is at a slower rate.
INSULIN RESISTANCE as used herein is intended to encompass the usual diagnosis of insulin resistance made by any of a number of methods, inc.uding but not restricted to: the intravenous glucose tolerance test or measurement of the fasting insulin level. It is well known that there is an excellent correlation between the height of the fasting insulin Uvel and the degree of insulin resistance. Therefore, one could use elevated fasting insulin levels as a surrogate marker for insulin resistance for the purpose of identifying which normal glucose tolerance (NGT) individuals have insulin resistance. A diagnosis of insulin resistance can also be made using the euglycennc glucose clamp test
INVERSE AGONISTS shall mean materials (e.g., ligand, candidate compound) that bind either to the endogenous form or to the constitutively activated form of the receptor so as to reduce the baseline intracellular response of the receptor observed in the absence of agonists.
ISOLATED shall mean that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For examplp, a naturally occurring polynucleotide or polypeptide
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present in a living animal is not isolated, but the same polynucleotide or DNA or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such a polynucleotide could be part of a vector and/or such a polynucleotide or polypeptide could be part of a composition, and still be isolated in that the vector or'composition is not part of its natural environment
LIGAND shall mean a molecule that specifically binds to a GPCR. A hgand may be, for example, a polypeptide, a lipid, a small molecule, an antibody. An endogenous ligand is a hgand that is an endogenous, natural ligand for a nat ve GPCR. A ligand may; be a GPCR "antagonist", "agonist", "partial agonist", or "inverse agonist", or the like.
As used herein, the terms MODULATE or MODIFY are meant to refer to an increase or decrease
in the amount, quality, or effect of a particular activity, function or molecule By way of illustration and not
v limitation, agonists, partial agonists, inverse agonists, and antagonists of a G protein-coupled receptor are
modulators of the receptor.'
PARTIAL AGONISTS shjall mean materials (e g., hgands, candidate compounds) that activate the intracellular response when they binu to the receptor to a lesser degree/extent than do full agonists
PHARMACEUTICAL COMPOSITION shall mean a composition comprising at least one
active ingredient, whereby the composition is amenable to investigation for a specified, efficacious outcome
in a mammal (for example, and no. limitation, a human). Those of ordinary skill in the art will understand
and appreciate the techniques appropriate for determining whether an active ingredient has a desired
efficacious outcome based upon the needs of the artisan. ,
POLYNUCLEOTEDES shall mean RKA, DNA, or KNA/DNA hybrid sequences of more than one .nucleotide in either single chain or duplex form The polynucleotides of the invention may be prepared by any known method, including synthetic, recombinant, ex vivo generation, or a combination thereof, as well as utilizing any purification methods known in the art
POLYPEPTIDE shall r:fer to a polymer of amino acids without regard to the length of the polymer. Thus, PEPTIDES, oligopepudes, and proteins are included withurthe definition of polypeptide. This term also does notspecify or exclude post-expression modifications of polypeptides. For example, polypeptides that include the covalent attachment of glycosyl groups, acetyl groups, phosphate groups, hpid groups and the like are expressly PRIMER is used herein to denote a specific oligonucleotide sequencp which is complementary to a
target nucleotide sequence and used to hybridize to the target nucleotide sequence. A primer serves as an
initiation point for nucleotide polymerization catalyzed by DNA polymerase, RNA polymerase, or reverse
transcriptase. f
PURIFIED is used herein to describe a polynucleotide or polynucleotide vector of the invention that has been separated from other compounds including, but not limited to, other nucleic acids, carbohydrates, hpids and proteins (such as the enzymes used in the synthesis of the polynucleotide). In certain embodiments, a polynuc' eotide is substantially pure when at least about 50%, at least about 60%, at least about 75%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least
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about 97%, at least about 98%, at least about 99%, orlat least about 99.5% of a sample contains a single polynucleotide sequence. In some embodiments, a substantially pure polynucleotide typically comprises about 50%, about 60%, about'70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.5% weight/weight of a polynucleotide sample.
Similarly, the term PURIFIED is used herein to describe a polypeptide of the invention that has been separated from other compounds including, but not limited to, nucleic acids, lipids, carbohydrates and other proteins In certain embodiments, a polypeptide l > substantially pure when at least about 50%, at least about 60%, at least about 75%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99 5% of the polypeptide molecules of a sample have1 a single amino acid sequence. In some embodiments, a substantially pure polypeptide typically comprises about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or aboui 99.5% weight/weight of a protein sample.
Similarly, the term PURIFIED is used herein to describe a modulator of the invention. In certain embodiments, a substantially pure modulator typically comprises at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 9 Further, as used herein, the term PURIFIED (foes not require absolute purity, rather, it is intended as a relative definition.
RECEPTOR FUNCTIONALITY shall refeV to the normal operation of a receptor to receive a stimulus and moderate an effect in the cell, i includii g, but not limited to regulating gene transcription, regulating the influx or efflux of ions, effecting a catalytic reaction, and/or modulating activity through G-proteins
SECOND MESSENGER shall mean an intraceilular response produced as a result of receptor activation. A second messenger can include, for example, inositol tnphosphate QP3), diacylglycerol (DAG), cyclic AMP (cAMP), cyclic GMP (cGMP), MAP kirase acitiyity, and Ca2+. Second messenger response can be measured for a determination of receptor activation. In addition, second messenger response can be measured for the identification of candidate cpmpounas as, for example, inverse agonists, partial agonists, agonists, and antagonists.
SIGNAL TO NOISE RATIO shall mean the signal generated in response to activation, amplification, or stimulation wherein the signal is above the background noise or the basal level in response to non-activation, non-amplificanon, or non-stimulation.
SPACER shall mean a translated number of iimino acids that are located after (he last codon or last amino acid of a gene, for example a GPCR. of interest, but before the start codon or beginning regions of the G protein of interest, wherein the translated number ^imino acids are placed in-frame with the beginnings
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regions of the G protein of interest. The number of translated ammo acids can be one, two, three, four, etc, and up to twelve.
STIMULATE or STIMULATING, in relationship to the term "response" shall mean that a response is increased in the presence o "a compound as opposed.to in the absence of the compound.
SUBJECT shall mean pnmai es, including but not limited to humans and baboons, as well as pet animals such as dogs and cats, labora ory animals such as rats and mice, and farm animals such as horses, sheep, and cows.
THERAPEUTICALLY EFFECTIVE AMOtJNT as used herein refers to the amount of active
compound or pharmaceutical agent ihat elicits the biological or medicinal response in a tissue, system,
animal, individual or human that is being sought by a researcher, veterinarian, medical doctor or other
clinician, which includes one or more of the following: j
(1) Preventing the dise se; for-example, preventing a disease, condition or disorder in an
individual that may be predisposed to the diseases conditioni or disorder but does not yet experience or
display the pathology or symptomatology of the disease,
(2) Inhibiting the disease; for'example, inhibiting a disease, condition or disorder in an
individual that is experiencing or displaying the pathology or symptomatology of the disease, condition
or disorder (i.e.s arresting further development of the pathology and/or symptomatology), and
(3) Ameliorating the disease; for example, ameliorating a disease, condition or disorder in an
individual that is experiencing or displaying the pathology or symptomatology of the disease, condition or
disorder (i.e., reversing the pathology and/or symptomatology).
VARIANT as the term is. used herein, is a polynucleoUde or polypeptide mat differs from a reference polynucleotide or polypeptide respectively, but retains essential properties. A typical variant of a polynucleotide differs in nucleotidu sequence from another, reference polynucleotide. Changes in the nucleoti.de sequence of the1 variant nay or may not alter the aniino acid sequence of a polypeptide encoded by the reference polynucleotide. A typical variant of a polypeptide differs in amino acid sequence from another,-reference polypeptide. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A variant of a polynucleotide or polypeptide may be a naturally occurring one such as an ALLELIC VARIANT, or it may be a variant that is not known to occur naturally. N Introduction i
The order of the following sections is set forth for presentational efficiency and is not intended, nor should be construed, as a limitation on the disclosure or the claims to follow.
B. Receptor Expres; iion
1. GPCR polypeptides of interest ,
A RHP43 GPCR of the invention comprises a GPR131 amino acid sequence. As used herein, "a GPR131 amino acid sequence" is intended to encompass the endogenous human GPR131 amino acid
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sequence of SEQ ID NO:2 as well as a variant ammo acid sequence at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about ?1%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at. least about 98% or at least about 99% identical to the ammo acid sequence of SEQ ID VO:2 In other words, a GPCR comprising a variant of the amino acid sequence of SEQ DD NO:2 also may be used in the subject methods. In certain embodiments, a GPCR that1 may be used in the subject methods may comprise an allelic variant of the amino acid sequence of SEQ ID NO:2 In certain embodiments, an aliehc variant of the amino acid sequence of SEQ ID NO:2 is encoded by an endogenous GPR131 nucleotide sequence obtainable by performing polymerase chain reaction (PCR) on a human DNA sample using specific primers SEQ ID NO:3 and SEQ ID NO:4. In some embodiments, an allelic variant of the amino acid sequence of SEQ ID NO:2 is encoded by an endogenous GPR131 nuclectide sequence obtainable by performing polymerase chain reaction (PCR) on a Human DNA sample using a specific primer comprising SEQ ID NO:3 and a specific primer comprising SEQ ID NO:4. In certain embodiments, the human DNA sample is human genomic DNA. In certain embodiments, the process is RT-PCR (reverse transcription-polymerase chain reaction) RT-PCR techniques are well known to the skilled artisan, hi certain embodiments, the human cDNA sample is human mbnocyte or macrophage ;DNA. In certain embodiments, the human cDNA sample is human adipocyte cDNA. In certain embo iiments, the human cDNA sample is human skeletal muscle cell cDNA. In certain embodiments, the humiin DNA sample is provided. In certain embodiments, the human DNA sample is obtained from a commercial source. In certain embodiments, a variant ammo acid sequence that may be used in the subject method i is a mammalian ortholog of the amino acid sequence of SEQ ID NO:2. By way of illustration and not limitation, the GFR131 amino acid sequences of rabbit (GenBank® Accession No.; BAC55237, e.g), cow (GenBank® Accession No. NP_778219, e.g.), mouse (GenBank® Accession No.,NP__778150, e.g.), and r.it (GenBank® Accession No. NP_808797, e.g) are envisioned to be within the scope of "a GPR131 amiro acid sequence". It is understood that as used herein "GPR131 GPCR" is endogenous RUP43 GPCR; by way of illustration and not limitation, endogenous human RUP43 GPCR is human GPR131 of GenBank® Accession No NM_170699 (having an amino acid sequence identical to SEQ ID NO:2) and alleles theieif, endogenous rabbit RUP43 GPCR is rabbit GPR131 of GenBank® Accession No. BAC55237 and alleles thereof, endogenous cow RUP43 GPCR is cow GPR131 of GenBank® Accession No. NP_778219 arid alleles ;thereof, endogenous mouse RUP43 GPCR is mouse GPR131 of GenBank® Accession No. NP_7'815O and alleles thereof, and endogenous rat RUP43 GPCRisratGPR131 of GenBank® Accession No. >P_808797 and alleles thereof.
In certain embodiments, a GPCR that may JQ used in the subject methods may comprise a non-endogenous, constitutively activated mutant of the arrino acid sequence of SEQ ID NO:2, an allele of SEQ ID NO:2, or a mammalian1 ortholog of SEQ ID NQ:2. As is known in the art, a constitutively activated GPCR may be made using a variety of methods (sse, e.g., PCT Application Number PCT/US98/07496 published as WO 98/46995 on 22 October 1998, and US patent no. 6,555,339; the disclosure of each of which is hereby incorporated by reference in its entiri ;ty.) A biologically active fragment of the amino acid
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sequence of SEQ ID NO:2, of an aljele of SEQ ID NO:2, of a mammalian ortholog of SEQ ED NO:2, of a non-endogenous, constitutively activated mutant of endogenous GPR131, or of an amino acid sequence at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 9,5%, at least about 96%, at least about 97%, at least about 98% or at least ibout 99% identical to the ammo acid sequence of SEQ ED NO:2 may be used in the subject invention. By way of illustration and not limitation, deletion of an N-termina)
!
methionine or an N-terminal signal peptide is envisioned to provide a biologically active fragment that may be used in the subject methods. By way of further illustration and not limitation, a RUP43 GPCR that may be used in the subject methods may comprise amino acids 2-330 of SEQ ID NO:2, with the proviso that the R.UP43 G protein-coupled receptor does not comprise the methionine residue at amino acid position 1 of SEQH>NO:2;
In certain embodiments, a GPCR that may be used in the subject methods may comprise an amino acid sequence at least about 75%, lit least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%,;at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to the amiuo acid sequence of SEQ ID NO:2. In certain embodiments, a GPCR that may be used in me subject methods may comprise an amino acid sequence at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to the amino acid sequence of SEQ ID NO:2. In certain embodiments, a GPCR that may be used in the subject methods may comprise an amino acid sequence at least about 75% identical to the amino acid sequence of SEQ ID NO:2. In certain embodiments, a GPCR that may be used in the subject methods may comprise an amino acid sequence atjeast about 80% identical to the amino acid sequence of SEQ ID NO:2. In certain embodiments, a GPCR that may be usedjin the subject methods may comprise an amino acid sequence at least about 85% identical to the amino acid sequence of SEQ ID NO;2. In certain embodiments, a GPCR mat may be used in the subject methods may comprise an amino acid sequence at least about 90% identical to the amino acid sequence of SEQ ID NO:2. In certain embodiments, a GPCR that may be used in the subject methods may comprise an amino acid sequence at least about 91% identical to the amino acid sequence of SEQ ID NO:2. In certain embodiments, a GPCR that may be used in the subject methods may comprise an amino acid sequence at least about 92% identical to the amino acid sequence of SEQ ID NO:2. In certain embodiments, a GPCR 1hat may be used in 1he subject methods may comprise an amino acid sequence at least about 93% identical to the amino acid sequence of SEQ ID NO:2. In certain embodiments, a GPCR that may be used in the subject methods may comprise an aituno acid sequence at least about 94%, identical to the amino acid sequence of SEQ ID NO:2. In certain embodiments, a GPCR that may be used in the subject methods may comprise an amino acid sequence at least about 95% identical to the ammo acid sequence of SEQ ID NO:2 hi certain embodiments, a GPCR that may be used in the subject methods may comprise an araino acid sequence at least about 96% identical to the amino acid sequence of SEQ ED NO:2. In certain embodiments, a GPCR that may be used in the subject methods may' comprise an amino acid sequence at least about 97% identical to the ammo acid
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sequence of SEQ ID NO:2 In certain embodiments, a 3PCR that may be used in the subject methods may comprise an amino acid sequence at least about 98% identical to the arnino acid sequence of SEQ ID NO:2. In certain embodiments, a GPCR that may be used in the subject methods may comprise an amino acid sequence at least about 99% 'identical to the amino acid sequence of SEQ DD NO:2. By an amino acid sequence having at least, for example, 95% "identity" to the amino acid sequence of SEQ ED NO.2 is meant that the amino acid sequence is identical to the amino acid sequence of SEQ ID NO:2 except that it may include up to five ammo acid alterations per each 100 imino acids of the amino acid sequence of SEQ ID NO:2. Thus, to obtain an amino acid sequence having at least 95% identity to that of SEQ ID NO:2, up to 5% (5 of 100) of the amino acid residues in the sequence may be inserted, deleted, or substituted with another amino acid compared with the amino acid sequence of SEQ ID NO:2. These alternations may occur at the ammo or carboxy termini or anywhere tetween those terminal positions, interspersed either individually among residues in the sequence or in one or more contiguous groups within the sequence.
In some embodiments, aGPR131 amino acid sequence that may be used in the subject methods is the airuno acid sequence of a G protein-coupled rectptor encoded by a complementary sequence to the sequence of a polynucleotide that hybridizes under s nngent conditions to filter-bound DNA having the sequence set forth in SEQ ED NO:1. By way of ill jstration and not limitation, a GPR131 amino acid sequence that may be used in the subject methods : s the amino acid sequence of a G protein-coupled receptor encoded by a polynucleotide that hybridizes under stringent conditions to the complement of SEQ IDNO:1. Hybridization techniques are well known tc the skilled artisan. Preferred stringent hybridization conditions include overnight incubation at 42°C in a solution comprising: 50% formamide, 5xSSC (150mM NaCl, 15mM trisodium citrate), 50mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran siilfate, and 20ug/ml denatured, sheared salmon sperm DNA; followed by washing the filter in O.lxSSC at about 65°C
a. Sequence identity
A preferred method for determining the bes: overall match between a query sequence (eg., the amino acid sequence of SEQ ID NO:2) and a sequence to be interrogated, also referred to as a global sequence alignment, can be determined using the FASTDB computer program'based on the algorithm of Brutlag et al. [Corop App Biosci (1990) 6:237-245 the disclosure of which is hereby incorporated by reference in its entirety]. In a sequence alignment the query and interrogated sequences are both amino acid sequences. The results of said global sequence alignj nent is in percent identity. Preferred parameters used in a FASTDB amino acid alignment are: i Matrb=PAM 0, k-tuple=2, Mismatch. Penalty=l, Joining Penalty=20, Randomization Group=25, Length=0, Cutoff Score=l, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=24' or the lenth of the interrogated amino acid sequence, whichever is shorter.
If the interrogated sequence is shorter than t le query sequence due to N- or C-terminal deletions, not because of internal deletions, the results, in percent identity, must be manually corrected because the FASTDB program does not account for N- and C-terminal truncations of the interrogated sequence when
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calculating global percent identity. Fcr interrogated sequences truncated at the N- and C-termini, relative to
the query sequence, the percent identity is corrected by calculating the number of residues of the query
sequence that are N- and C-termina! of the, interrogated sequence, that are not matched/aligned with a
corresponding interrogated sequence residue, as a percent of the total bses of the query sequence. Whether
a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage
is then subtracted from the perecent identity, calculated by the above FASTDB program using the specified
parameters, to arrive at a final percen: identity score This final percent identity score is what is used for the
purposes of the present invention. Only residues to the N- and C-termini of the interrogated sequence,
which are not matched/aligned with the query sequence, are considered for the purposes of manually
adjusting the percent identity score. [That is, only querey ammo acid residues outside the farthest N- and C-
terminal residues of the interrogated sequence. ,
For example, a 90 amino acid residue interrogated sequence is aligned with a 100-residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the interrogated sequence and therefore, the FASTDB alignment does-not match/align with the first residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C- termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched, the final percent identity would be 90%.
In another example, a 90-residue interrogated sequence is compared with a 100-residue query sequence. This time the deletions are internal so there are no residues at the N- or C-termini of the interrogated sequence, which are not matched/aligned with the query, hi this case, the percent identity calculated by FASTDB is not manually corrected. Once again, only residue positions outside the N-and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequence are manually corrected. No other corrections are made for the purposes of the present invention.
b. Fusion proteins
In certain embodinents, a polypepude of mterest is a fusion protein, and may contain, for example, an affinity tag domain or a reporter domain. Suitable affinity tags include any amino acid sequence that may be specifically bound to another moiety, usually another pplypeptide, most usually an antibody. Suitable affinity tags include epitope tags, for example, the the V5 tag, the FLAG tag, the HA tag (from hemagglutinin influenza virus), ths myc tag, and the like, as is known in the art Suitable affinity tags also include domains for which, bindrc g substrates are known, e.g., HIS, GST and MBP tags, as is known in the art, and domains from other prcteins for which specific binding partners, e.g., antibodies, particularly monoclonal antibodies, are available. Suitable affinity tags also include any protein-protein interaction domain, such as a IgG Fc region, which may be specifically bound and detected using a suitable binding partner, e.g the IgG Fc receptoi. It is expressly contemplated that such a fusion protein may contain a
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heterologous N-terminal domain (e.g., an epitope tag) fused in-frame with a GPCR that has had its N-terminal methionine residue either deleted or substituted with an alternative ammo acid.
Suitable reporter domains include any domain that can report the presence of a polypeptide While it is recognized that an affinity tag may be used to report the presence of a polypeptide using, e g, a labeled antibody that specifically binds to the tag, light emitting reporter domains are more usually used Suitable light emitting reporter domains include luciferase (from, e.g., firefly, Vargula, Renilla reniformis orRenilla muelleri), or light emitting variants thereof. Other suitable reporter domains include fluorescent proteins, (from e.g.,1 jellyfish, comls and other coelenterates E« such those from Aequona, Renilla, Pnlosarcus, Stylaiula species), or light emitting variants thereof. Light emitting variants of these reporter proteins aie very well known in the art: and may be brighter, dimmer, or have different excitation and/or emission spectra, as compared to a native reporter protein. For example, some variants are altered such that they no longer appear green, and may appear blue, cyan, yejow, enhanced yellow red (termed BFP, CFP, YFP eYFP and RFP, respectively) or have other emission iipectra, as is known in the art Other suitable reporter domains include domains that can report the presence of a polypeptide through a biochemical or color change, such as (3-galactosidase, p-glucuronidase, chloramphenicol acetyl transferase, and secreted embryonic alkaline phosphatase.
Also as is known in ithe art, an affinity tags 01 a reporter domain may be present at any position in a polypeptide of interest However, in most embodime its, they are present at the C- or N-terrrunal end of a polypeptide of interest
2. Nucleic acids encoding GPCR poly peptides of interest
Since the genetic code and recombinant tectruques for manipulating nucleic acid are known, and the amino acid sequences of GPCR polypeptides of i nterest described as above, the design and production of nucleic acids encoding a| GPCR polypeptide of interest is well within the skill of an artisan. In certain embodiments, standard recombinant DNA technology (Ausubel, et al, Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995; Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.) methods are used. For example, GPCR coding sequences may be isolated from a library of' GPCR coding sequence using any one or a combination of a variety of recombinant methods that do not need to be described herein. Subsequent substitution, deletion, and/or addition of nucleotides in the nucleic acid sequence encoding a protein may also be done using standard recombinant DNA techniques.
For example, site directed mutagenesis and subcloning may be used to inrroduce/delete/substitute nucleic acid residues in a polynucleotide encoding ;L polypeptide of interest. In other embodiments, PCR may be used. Nucleic acids encoding a polypeptida of interest may also be made by chemical synthesis entirely from oligonucleotides (e.g., Cello et al., Scieice (2002) 297:1016-8).
In some embodiments, the codons of the nucleic acids encoding polypeptides of interest are optimized for expression in cells of a particular species, particularly a mammalian, e g., mouse, rat, hamster, non-human primate, or human, species. In some embodiments, the codons of the nucleic acids encoding
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polypeptides of interest are optimized for expression in cells of a particular species, particularly an amphibian species.
a. Vectors ,
The invention further provides vectors (also.referred to as "constructs") comprising a subject nucleic acid. In many embodiments o F the invention, the subject nucleic acid sequences will be expressed in a host after the sequences have been operably linked to an expression control sequence, including, e g. a promoter The subject nucleic acids are also typically placed in an expression vector that can replicate in a host cell either as an episome or as am integral part of the host chromosomal DNA. Commonly, expression vectors will contain selection markers, e.g., tetracyckne or neomycin, to permit detection of those cells transformed with the desired DNA sequences (see, e.g., U.S. Pat No. 4,704,362, which is incorporated herein by reference). Vectors, including single and dual expression cassette vectors are well known in the art (Ausubel, et al, Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995; Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.). Suitable vectors include viral vectors, plasmds, cosrrnds, artificial chromosomes (human artificial chromosomes, bacterial artificial chromosomes, yeast artificial chromosomes, etc.), mini-chromosomes, and the like. Retroviral, adenoviral and adeno-asspciated viral vectors may be used.
A variety of expression vectors are available to those in the art for purposes of producing a polypeptide of interest in a cell One suitable vector is pCMV, which is used in certain embodiments. This vector was deposited with the American Xype Culture Collection (ATCC) on October 13, 1998 (10801 University Blvd., Manassas, VA 2CillO-2209 USA) under the provisions of the Budapest Treaty for the International Recognition of the Der osit of Microorganisms for the Purpose of Patent Procedure. The DNA was tested by the ATCC and determined to be viable. The ATCC has assigned the following deposit number to pCMV; ATCC #2033 51.
The subject nucleic acids usually, comprise an single open reading frame encoding a subject polypeptide of interest, however, in certain embodiments, since the host cell for expression of the polypeptide of interest may be a eikaryotic cell, e.g., a mammalian cell, such as a human cell, the open reading frame may be interrupted by introns. Subject nucleic acid are typically part of a transcriptional unit which may contain, in addition to the subject nucleic acid 3' and 5' untranslated regions (UTRs) which may direct KNA stability, translational efficiency, etc. The subject nucleic acid may also be part of an expression cassette which contains, in addition to the subject nucleic acid a promoter, which directs the transcription and expression of a polypeptide of :nterest,,and a transcriptional terminator.
Eukaryotic promoters can be any promoter that is ifunctional in a eukaryotic host cell, including viral promoters and promoters deiived from eukaryotic genes. Exemplary eukaryotic promoters include, but are not limited to, the followin g: the promoter of the mouse metallothionein I gene sequence (Hamer et al., J. Mol. Appl. Gen. 1:273-288, 1982); the TK promoter of Herpes virus (McKnight, Cell 31:355-365, 19S2), the SV40 early promoter (Benoostet al, Nature (London) 290:304-310, 1981); the yeast gall gene sequence promoter (Johnston et ah, Proc. Natl. Acad Sci. (USA) 79.6971-6975, 1982); Silver et al, Proc.
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Nati Acad Scu (USA) S1-5951-59SS, 1984), the CMV promoter, the EF-1 promoter, Ecdysone-responsive
i promoters), tetracycline-respbnsive promoter, and the 'ike. Viral promoters may be of particular interest as
they are generally particularly strong promoters In certain embodiments, a promoter is used that is a ptomoter of the target pathogen. Promoters for use in ihe present invention are selected such that they are functional in the cell type (and/or animal) into which they are being introduced. In certain embodiments, the promoter is a CMV promoter1
In certain embodiments, a subject vector may also provide for expression of a selectable marker. Suitable vectors and selectable markers are well-known in the art and discussed in Ausubel, et al, (Short Protocols in Molecular Biology, 3rd ed., Wiley, & Sons', 1995) and Sambrook, et al, (Molecular Cloning: A Laboratory Manual, Third Edition, (2001) Cold Sprirg Harbor, N.Y.). A variety of different genes have been employed as selectable markers, and the particular gene employed in the subject vectors as a selectable marker is !chosen primarily las a matter of convenience. Known selectable marker genes include: the thymidine kinase gene, the dihydrofolate reductase gene, the xantmne-guanine phosphonbosyl transferase gene, CAD, the adenosine deanunase gene, the asparagine synthetase gene, (he antibiotic resistance genes, e.g. tetr, ampr, Cmr or cat, kanr or neor (amnioglycoside phosphotransferase genes), the hygromycin B phosphotransferase gene, and the like
As mentioned above, polypeptides of interest i nay be fusion proteins that contain an affinity domain and/or a reporter domain. Methods for making fusions between a reporter or tag and a GPCR, for example, at the C- or N-terminus of the GPCR, are well within the skill of one of skill in the art (e.g. McLean et al, Mol. Pharma. Mol Pharmacol 1999 56:1182-91; Ramsay et al., Br. J. Pharmacology, 2001, 315-323) and will not be described any further. It is expressly cor templated that such a fusion protein may contain a hetcrologous N-terminal domain (eg., an epitope tag) fused in-frame with a GPCR that has had its N-terminat methionine residue either deleted or substitute d with an alternative amino acid. It is appreciated that a polypeptide of interest may first be made from a nat ive polypeptide and then operably linked to a suitable reporter/tag as described above.
The subject nucleic! acids may also contain restriction sites, multiple cloning sites, primer binding sites, Ugatable ends, recombination sites etc., usually n order to facilitate the construction of a nucleic acid encoding a polypeptide of interest b'. Host cells
The invention further provides host cells co uprising a vector comprising a subject nucleic acid. Suitable host cells include prokaryotic, e.g., bacterial cells (for, example E. coli), as well as eukaryotic cells e g. an animal cell (for example an insect, mammal, fish, amphibian, bird or reptile cell), a plant cell (for example a maize or Arabidopsis cell), or a funga cell (for example a S. cerevisiae cell). In certain embodiments, any cell suitable for expression of a nolypeptide of interest-encoding nucleic acid may be used as a host cell. Usually, an animal host cell line is used; examples of which are as follows: monkey kidney cells (COS cells), monkey kidney CVI cells transformed by SV40 (COS-7, ATCC CRL 165 1); human embryonic kidney cells (HEK-293 ["293"], Graham et al. J. Gen Virol. 36:59 (1977)); HEK-293T
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["293T"] cells; baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary-cells (CHO, Urlaub and Chasm, Proc. Natl Acai. SCL (USA) 77.4216, (1980); Syrian golden hamster cells MCB3901 (ATCC CRL-9595); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey bdney cells (CVT ATCC CCL 70), afheak green ¦monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75); human liver cells (hep G2, KB 8065), mouse mammary timor (MMT 060562, ATCC CCL 51), TRI cells (Mather et al., Annals N. Y Acad Sci 383:44-68 (1982));jNIH/3T3 cells (ATCC CRL-1658); and mouse L cells (ATCC CCL-1). In certain embodiments, melanophores are used. Melanophores are skin cells found in lower vertebrates Relevant materials and methods will be followed according to the disclosure of U.S. Patent Number 5,462,856 and U.S. Patent Numfcer 6,051,386. These patent disclosures are hereby incorporated by reference in their entirety. Additional cell lines will become apparent to those of ordinary skill in the art, and a wide variety of cell lines are available from The American Type Culture Collection, 10S01 University Boulevard, Manassas, Va. 20110-2209.
C. Screening of Candidate Compounds
1. Generic (SPCR screening assay techniques
"When a G protein receptor becomes active, it binds to a G protein (e.g., Gq, Gs, Gi, Gz, Go) and stimulates the binding of GTP to the G piotein. The G protein then acts as a GTPase and slowly hydrolyzes the GTP to GDP, whereby the receptor, under normal conditions, becomes deactivated. However, activated receptors continue to exchange GE P to GTP. A non-hydrolyzable analog of GTP, [35S]GTPyS, can be used to monitor enhanced binding to membranes which express activated receptors. It is reported that [33S]GTPyS can be used to monito:: G protein coupling to membranes in the absence and presence of ligand. An example of this monitoring, anong other examples well-known and available to those in the art, was reported by Traynor and Nahorski in 1995. A preferred use of this assay system is for initial screening of candidate compounds because the system is generically applicable to allrG protein-coupled receptors
r
regardless of the particular G protein that interacts with the intracellular domain of the receptor.
2. Specific GPCR screening assay techniques
Once candidate compounds are identified using the "generic" G protein-coupled receptor assay
{i.e., an assay to select compounds that are agonists or inverse agonists), in some embodiments further
screening to confirm that the compounds have interacted at the receptor site is preferred. For example, a
compound identified by the "generic" assay may not bind to the receptor, but may mstead merely
"uncouple" the G protein from the intracellular domain. •,
Gs, Gz and Gi.
Gs stimulates the enzyrie adenylyl cyclase Gi> (and Gz and Go), on the other hand, inhibit adenylyl cyclase . Adenylyl cychse catalyzes the conversion of ATP to cAMP; thus, activated GPCRs that couple the Gs protein are associs ted with increased cellular levels of cAMP. On the other hand, activated GPCRs that couple Gi (or Gz, (So) protein are associated with decreased cellular levels of cAMP. See,
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generally, 'Tndiiect Mechanisms of Synaptic Transmission," Chpt 8, From Neuron To Brain (3rf Ed) Nichols, J.G. et al eds Sinauer Associates, Inc. (1992). Thus, assays that detect cAMP can be utilized to determine if a candidate compound is, e g., an inverse igonist to the receptor (i e, such a compound would decrease the levels of cAMP) A variety of approaches known in the art for measuring cAMP can be utilized, in some embodiments a preferred approach relies upon the use of anti-cAMP antibodies in an ELISA-based format Another type of assay that can be utilized is a whole cell second messenger reporter system assay. Promoters on genes drive the expression of the proteins that a particular gene encodes. Cyclic AMP drives gene expression by promoting tfie binding of a cAMP-responsive DNA binding protein or transcription factor (CREB) that then binds to the promoter at specific sites called cAMP response elements and drives the expression of the gene Reporter systems can be constructed which have a promoter containing multiple cAMP response elements 'before the reporter gene, e.g., p-galactosidase or luciferase Thus, an activated Gs-linked'receptor causes the accumulation of cAMP that then activates the gene and expiession of the reporter protein. The reporter protein such as p-galactosidase or luciferase can then be detected using standard biochemical assays (Chen et al 1995).
Go and Gq.
Gq and Go are associated with activation of tl ie enzyme phospholipase C, which in turn hydrolyzes the phospholipid PIP2, releasing two intracellular messengers: diacyclglycerol (DAG) and inositol 1,4,5-triphosphate (IP3). Increased accumulation of EP3 is associated with activation of Gq- and Go-associated receptors. See, generally, "Indirect Mechanisms of Synaptic Transmission," Chpt. 8, From Neuron To Brain (3* Ed.) Nichols, J.G. et al eds. Sinauer Associates, Ire. (1992). Assays that detect IP3 accumulation can be utilized to determine if a candidate compound is, e.g., an inverse agonist to a Gq- or Go-associated receptor (i.e., such a compound would decrease the levels of IP3). Gq-associated receptors can also been examined using an API reporter assay in that Gq-dependent phospholipase C causes activation of genes containing API elements; thus, activated Gq-associated receptoi s will evidence an increase in the expression of such genes, whereby inverse agonists thereto will eviderce a decrease in such expression, and agonists will evidence an increase in such:expression. Commercially available assays for such detection are available.
3. GPCR Fusion Protein
The use of an endogenous, constitutively active GPCR or a non-endogenous, constitutively activated GPCR, for use in screening of candidate compounds for the direct identification of inverse agonists or agonists provides an interesting screening challenge in that, by definition, the receptor is active even in the absence of an endogenous ligand bound thereto. Thus, in order to differentiate between, e.g., the non-endogenous receptor in the presence of a candidate compound and the non-endogenous receptor in the absence of that compound, with an aim of such a difi erentiation to allow for an understanding as to whether such compound may be an inverse agonist or agonist or have no affect on such a receptor, in some embodiments it is preferred that an approach be ut lized that can enhance such differentiation. In some embodiments, a preferred approach is the use of a GICR Fusion Protein.
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Generally, once it is determijied that a non-endogenous GPCR has been constitutively activated using the assay techniques set forth ibove (as well as others (known to the art-skilled), it is possible to determine the predominant G protein t bat couples with the endogenous GPCR. Coupling of the G protein to the GPCR provides a signaling pathway that can be assessed In some embodiments it is preferred that screening take place using a mammalian expression system, as such a system will be expected to have endogenous G protein therein. Thus, by definition, in such a system, the non-endogenous, constitutively activated GPCR will continuously signal In some embodiments it is preferred that this signal be enhanced such that in the presence of, eg., an inverse agonist to the receptor, it is more likely that it will be able to more readily differentiate, particularlv in the context of screening, between the receptor when it is contacted with the inverse agonist
The GPCR Fusion Protein is intended to enhance the efficacy of G protein coupling with the non-endogenous GPCR. The GPCR Fusion Protein may be preferred for screening with either an endogenous, constitutively active GPCR or a non- endogenous, constitutively activated GPCR because such an approach increases the signal that is generate in such screening techniques. This is important in facilitating a significant "signal to noise" ratio; such a significant ratio is preferred for the screening of candidate compounds as disclosed herein
The construction of a construct useful for expression of a GPCR Fusion Protein is within the purview of those having ordinary ssill in the art. Commercially available expression vectors and systems offer a variety of approaches that can fit the particular needs of an investigator. Important criteria in the construction of such a GPCR Fusion Protein construct include but are not limited to, that the GPCR
sequence and the G protein sequence both be in-frame (preferably, the sequence for the endogenous GPCR
i
is upstream of the G protein sequence), and that the "stop" codon of the GPCR be deleted or replaced such that upon expression of the GPCR, ihe G protein can also be expressed. The GPCR can be linked directly to (he G protein, or there can be spacer residues between the two (preferably, no more than about 12, although this number can be readily ascertained by one of ordinary skill in the art). Based upon convenience, it is preferred to use a spacer. In some embodiments, it is preferred that the G protein that couples to the non-endogenous GPCR will have beer identified prior to the creation of the GPCR Fusion Protein construct. Because there are only a few G prc terns that have been identified, it is preferred that a construct comprising the sequence of the G protein (i.e.f a universal Gprotein construct, see Example 5(a) below) be available for insertion of an endogenous GPCR sequence therein; this provides for further efficiency in the context of large-scale screening of a variety of different endogenous GPCRs having different sequences.
As noted above, activated GPCRs that couple to Gi, Gz and Go are expected to inhibit the formation of cAMP making assays based upon these types of GPCRs challenging [i.e., the cAMP signal decreases upon activation, thus making the direct identification of, e.g., agonists (which would further decrease this signal) challenging]. As will be disclosed herein, it has been ascertained that for these types of receptors, it is possible to create i. GPCR Fusion Protein that is not based upon the GPCR's endogenous G protein, in an effort to establish a viable cyclase-based assay. Thus, for example, an endogenous Gi coupled
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receptor can be fused to a Gs protein -such a fusior, construct, upon expression, "drives" or "forces" the endogenous GPCR to couple with, e.g., Gs rather thin the "natural" Gi protein, such that a cyclase-based assay can be established Thus, for Gi, Gz and Go coupled receptors, in some embodiments it is preferred that when a GPCR Fusion Protein is used and the assay is based upon detection of adenylyl cyclase activity, that the fusion construct be established with Gs (or an equivalent G protein that stimulates the formation of the enzyme adenylyl cyclase).
^ABLEB

G protein Effect on cAMP Effect on IP3 Effect on cAMP Effect on EP3
Production upon Accumulation Production upon Accumulation upon
Activation of upon Activate nof contact with an contact with an
GPCR (£&, GPCR (Le., Inverse Agonist Inverse Agonist
constitutive i constitutive
activation or activation or
agonist binding) agonist bindin 8)
Gs Increase N/A Decrease N/A
Gi Decrease N/A Increase N/A
Gz 1 Decrease 1 N/A Increase N/A
Go Decrease Increase Increase Decrease
Gq N/A' ¦ Increa e N/A Decrease
Equally effective is'a G Protein Fusion construct that utilizes a Gq Protein fused with a Gss Gi, Gz or Go Protein. In some embodiments a preferred fusion construct can be accomplished with a Gq Protein wherein the first six (6) arriino acids of the'G-protein a-subunit ("Gaq") is deleted and the last five (5) ammo acids at the C-terminal end of Gaq is replace* I with the corresponding amino acids of the Ga of the G protein of interest. For example, a fusion construct can have a Gq (6 amino acid deletion) fused with a Gi Protein, resulting in a "Gq/Gi Fusion Construct" This fusion construct will forces the endogenous Gi coupled receptor to couple to its non-endogenous 3 protein, Gq, such that the second messenger, for example, inositol triphosphate or diacylgycerol, can b 3 measured in lieu of cAMP production.
Co-transfechon of a Target Gi Coupled GPCR with a1 Signal-Enhancer Gs Coupled GPCR (cAMP Based Assays)
A Gi coupled receptor is known to inhibit adenylyl cyclase, and, therefore, decreases the level of cAMP production, which can make the assessment of cAMP levels challenging. In certain embodiments, an effective technique in measuring the decrease in production of cAMP as an indication of activation of a receptor that predominantly couples Gi upon activation can be accomplished by co-transfecting a signal enhancer, e.g., a non-endogenous, constitutively activ ited receptor that predominantly couples with Gs upon activation (e.g., TSHR-A623I; see z'n/m), with the Gi linked GPCR. As is apparent, activation of a Gs coupled receptor can be determined based upon an increase in production of cAMP. Activation of a Gi
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WO 2005/116653 PCT/US2005/012447
coupled receptor leads to a decrease in production cAMP. Thus, the co-transfection approach is intended to advantageously exploit these "opposite" affects For example, co-transfection of a non-endogenous, consututively activated Gs coupled ieceptor(the "signal enhancer") with expression vector alone provides a baseline cAMP signal (i.e, althouga the Gi coupled receptor will decrease cAMP levels, this "decrease" will be relative to the substantial increase in cAMP levels established by constitutively activated Gs coupled signal enhancer). By then co-transf acting the signal enhancer with the "target receptor", an inverse agonist of the Gi coupled target receptor will increase the measured cAMP signal, while an agonist of the Gi coupled target receptor will decrease this signal.
Candidate compounds that are directly identified using this approach should be assessed independently to ensure that these do not target the signal enhancing receptor (this can be done prior to or after screening against the co-transfi:cted receptors).
D ' Medicinal Chemisiry - ,
Candidate Compounds
Any molecule known in tt e art can be tested for its ability to modulate (increase or decrease) the activity of a GPCR of the present invention. For identifying ia compound that modulates activity, candidate compounds can be directly providei 1 to a cell expressing the receptor
This embodiment of the invention is well suited to screen chemical libraries for molecules which modulate, e.g., inhibit, antagonize, or agonize, the amount of, or activity of, a receptor. The chemical libraries can be peptide libraries, peptidomimetic libraries, chemically synthesized libraries, recombinant, e.g., phage display libraries, and in vitro translation-based; libraries, other non-peptide synthetic organic libraries, etc. This embodiment of the invention is also well suited to screen endogenous candidate compounds comprising biological materials, including but not limited to plasma and tissue extracts, and to screen libraries of endogenous compounds known to have biological activity.
In some embodiments direct identification of candidate compounds is conducted in conjunction with compounds generated via combinatorial chemistry techniques, whereby thousands of compounds are randomly prepared for such analysis. The candidate compound may be a member of a chemical library. This may comprise any; convenient number of individual members, for example tens to hundreds to thousand to millions of suitabl; compounds, for example peptides, peptoids and other oligomeric compounds (cyclic or linear), ;ind template-based smaller molecules, for example benzodiazepines, hydantoins, biaryls, carbocyclic and polycyclic compounds (e.g., naphthalenes, phenothiazines, acndines, steroids etc.), carbohydrate and :imino acid derivatives, dihydropyridines, benzhydryls and heterocycles (e.g., trizines, indoles, thiazolidines etc.). The numbers quoted and the types of compounds listed are illustrative, but not limiting. Prei erred chemical libraries comprise chemical compounds of low molecular weight and potential therapeutic agents.
Exemplary chemical libraries are commercially available from several sources (ArQule, Tripos/PanLabs, ChemDesign, PI .armacopoeia). hi some cases, these chemical libraries are generated using combinatorial strategies that enc -79-

WO 2005/116553 PCT/US2005/012447

member compound is attached, thus allowing' direct and immediate identification of a molecule that is an effective modulator Thus,' in many combinatorial i pproaches, the position on a plate of a compound specifies that compound's composition. Also,1 in one example, a single plate position may have from 1-20 chemicals'that can be screened by administration to a well containing the interactions of interest Thus, if modulation is detected, smaller and smaller pools of interacting pairs can be assayed for the modulation activity By such methods, many candidate molecules can be screened.
Many diversity libraries suitable for use are known in the art and can be used to provide compounds to be tested according to the present invention. Alte natively, libraries can be constructed using standard methods. Further, more general, structurally constrained, organic diversity (e.g., nonpeptide) libraries, can also be used. By way of example, a benzodiazepine library (see e.g., Bunin et al, 1994, Proc. Natl. Acad Sci. USA 91:4708-4712) may be used.
In another embodiment of the present invention, combinatorial chemistry can be used to identify modulators of the GPCRs of the present invention. C Dmbinatonal chemistry is capable of creating libraries containing hundreds of thousands of compounds, many of which may be structurally similar. While high throughput screening programs are capable of screening these, vast libraries for affinity for known targets, new approaches have been developed that1 achieve libraries of smaller dimension but which provide maximum chemical diversity. (See e.g., Matter, 1997, Journal of Medicinal Chemistry 40*1219-1229).
One method of combinatorial chemistry, affinity fingerprinting, has previously been used to test a discrete library of small molecules for binding affinities for a defined panel of proteins. The fingerprints obtained by the screen are used to predict the affinity of the individual library members for other proteins or receptors'of interest (in the instant invention, the receptors of the present invention). The fingerprints are compared with fingerprints obtained from other compounds known to react with the protein of interest to predict whether the library compound might similarly react For example, rather than testing every hgand in a large library for interaction with a complex or protein component, only those Hgands having a fingerprint similar to other compounds known to have that activity could be tested. (See, e.g., Kauvar et al., 1995, Chemistry and Biology 2:107-118; Kauvar, 1995, Affinity fingerprinting, Pharmaceutical Manufacturing International. 8:25-28; andiKauvar, Toxic-Chemical Detection by Pattern Recognition in New Frontiers in Agrochermcal Immunoassay, D. Kurtz. L. Stanker and J.H. Skerritt Editors, 1995, AOAC: Washington, D.C., 305-312).
Candidate Compounds Identified as Modulators
Generally, the results of such screening will be compounds having unique core structures; thereafter, these compounds may be subjected to additional chemical modification around a preferred core structure(s) to further enhance the medicinal properties thereof. Such techniques are known to those in the art and will not be addressed in detail in this patent document. ¦
In certain embodiments, said identified me dulator is bioavailable. A number of computational approaches available to those of ordinary jskill in the art have been developed for prediction of oral bioavailability of a drug [Ooms et al., Biochim Bio'phys Acta (2002) 1587:118-25; Clark & Grootenhuis,
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WO 2005/116653 PCT/US2005/012447
Curr OpinDrug Discov Devel (2002) 5:3S2-90; Cheng et al, J Comput Chem (2002) 23:172-83; Nonnder & Haeberiein, Adv Drug Dehv Rev (2002) 54:291-313; Matter et aL, Comb Chem High Throughput Screen (2001) 4:453-75; Podlogar & Muegge, Cuir Top Med Chem (2001) 1.257-75; the disclosure of each of which is hereby incorporated by reference in its entirety). Furthermore, positron emission tomography (PET) has been successfully used by a number of groups to obtain direct measurements of drug distribution, including an assessment of oral bioa vailabmty, in the mammalian body following oral administration of the drug, including non-human primate and human body [Noda et aL, J Nucl Med (2003) 44:105-8; Gulyas et al., Eur JNucl Med Mol Imaging (2002) 29:1031-8; Kanerva et al., Psychopharmacology (1999) 145:76-81; the disclosure of each of which is hereby incorporated byj reference in its entirely]. Also, see infra, including Example 25.
In certain embodiments, said bioavailable identified Jmodulator further is able to cross the blood-brain barrier. A number of computational .approaches available to those of ordinary skill in the art have been developed for prediction of the permeation of the blood-brain barriei [Ooms et al., Biochim Biophys Acta (2002) 1587:118-25;.Clark & Grootenhuis, Curr OpinDrug Discov Devel (2002) 5:382-90; Cheng et aL, J Comput Chem (2002) 23:172-83, Nonnder & Haeberiein, Adv Drug Dehv Rev (2002) 54:291-313; Matter et al., Comb Chem High Throughput Screen (2001) 4:453-75; Podlogar & Muegge, Curr Top Med Chem (2001) 1257-75; the disclosure of each of which is hereby incorporated by reference in its entirety). A number of in vitro methods have been developed to predict blood-brain barrier permeability of durgs [Lohmann et al., J Drug Target (2002) 10:263-76; Hansen et al., J Pharm Biomed Anal (2002) 27:945-58, Otis et al, J Pharmocol Toxicol Methods (2001) 45:71-7; Dehouck et al, JNeurochem (1990) 54:1798-301; the disclosure of each of which is hereby incorporated by reference in its entirety]. Furthermore, a number of strategies have been developed to enhance drug delivery across the blood-brain barrier [Scherrmann, Vascul Pharmacol (2002) 38:349-:>4; Pardridge, Arch Neurol (2002) 59:35-40; Pardridge, Neuron (2002) 36:555-8; the disclosure of each of which is hereby incorporated by refrence in its entirety]. Finally, positron emission tomography (PET) has ;been successfully used by a number of groups to obtain direct measurements of drug distribution, including that within brain, in the mammalian body, including non-human primate and human body [Noda et al, J Nucl Med (2003) 44:105-8; Gulyas et al., Eur J Nucl Med Mol Imaging (2002) 29:1031-8; Kanerva et aL, Psychopharmacology (1999) 145:76-81; the disclosure of each of which is hereby incorporated by reference in its entirety]. Also, see infra, including Example 26.
E. Compounds of tl le Invention
One aspect of the present invention pertains to a compound of Formula (H):

or a pharmaceuticaUy acceptable salt thereof,
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WO 2005/116653 PCT/US2005/012447
wherein'
Ri is H or C[^ alkyl;
R2 is a 2-methyI-4)5,6,7-tetrahydro-2H-indazo -3-yl group; or
Ri' and R2 together with the nitrogen to which they are bonded form a 3,4-dihydro-2H-quinoline-l-
yl group; and ' Rio and Rj t are each independently H or halogen.
F. Synthetic Methods for Making Compounds,of the Invention Preparation of Compounds of the Present Invention - General Synthetic Methods
The novel compounds of the present inventi Dn can be readily prepared according to a variety of synthetic methods, all of which would be familiar to one skilled in the art Certain methods for the preparation of compounds of the present invention include, but are not limited to, those described in Schemes 1-3, infra.
The intermediate (AD) of the novel 2-piperid in-4-yl-thiazoles can be prepared as shown in Scheme 1 The thioamide (AA), protected at the nitrogen witt a suitable protecting group (l e. PG), is cyclized via a Hantzsch-Iike reaction with 3-halo-2-oxo-propionic asid (AB), protected at the carboxyhc acid, to give di-protected 2-piperidin-4-yl-thiazoIe (AC). Generally the two protecting groups are different Suitable solvents for the cyclization include, for example, alcohols (such as, methanol, ethanol, and propanol), lower halocarbons (such as, dichloromethane, dichloroethiine and chloroform), DMF, and the like. Reaction temperatures for the cychzation can range from about room temperature to about the boiling point of the solvent used; generally the temperature range is about 50°C to about 90°C.
Suitable protecting groups for thioamide (AA) include *-butyl carbamate (BOC), benzyl carbamate (Cbz), /Mnethoxybenzyl carbamate (Moz), and the like. Various methods can be used to protect the nitrogen of thioamide (AA)! For example, the /-bury, carbamate group can be introduced using a variety of reagents, such as (BOQ2O, with a suitable base (such as, NaOH, KOH, or Me^NOH) and in a suitable solvent® (THF, CH3CN)DMF, EtOH, MeOH, H2O, or mixtures thereof) at a temperature of about 0°C to about50°C.
Suitable protecting groups for 3-halo-2-oxo-propionic acid (AB) include alkyl esters (such as methyl, ethyl, propyl, ' and f-butyl), substituted methyl esters (such as, methoxymethyl, methoxyethoxymethyl, and benzyloxymethyl), optionally substituted benzyl esters (such as, benzyl, 4-methoxybenzyl, and 2,6-dimethoxybenzyl), and the like. One particular useful protected 3-halo-2-oxo-propionic acid (AB) is 3-bromo-2-oxo~propionic acid ethyl ester, also commonly referred to as ethyl bromopyruvate.
Other representative protecting groups suitable for a wide variety of synthetic transformations are disclosed in Greene and Wuts, Protective Groups in Organic Synthesis, third edition, John Wiley & Sons, New York, 1999, the disclosure of which is incorporated herein by reference in its entirety.
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For convenience, the two protecting groups in 2~pipendin-4-yl-thiazole (AC) are selected so one protecting group can be substantially1 removed without substantially affecting the other protecting group. This type of strategy is referred to as orthogonal protection One example includes, protecting the nitrogen with a BOC group and protecting the carboxylic acid as a methyl or ethyl ester., In this example, the BOC group can be removed under acidic conditions without substantially affecting the ester group. Alternatively, since (lie BOC group is not substantially hydrolyzed under) basic conditions the ester can be removed without substantially affecting the BOC group. Many orthogonal protection schemes are known in the art and can be applied herein.
Subsequently, as shown m ! Scheme 1, the nitrogen protecting group for 2-pipendin-4-yl-thiazoles (AC) is removed (i.e. deprotected), while substantially maintaining the carboxylic acid protection, to give common intermediate (AD). In the case when the nitrogen is protected with a BOC group effective cleavage can be achieved in the presence of an acid and optionally in a suitable solvent Suitable acids include, HC1 (aqueous or anhydrous), HBr (aqueous or anhydrous), H2SO4, trifluoroacetic acid, p~ toluenesulfonic acid, and the like When present, suitable solvents include, ester solvents (such as, ethyl acetate), alkyl alcohols (such as, me hanol, ethanol, i-propanol, n-propanol and 71-butanol), ethereal solvents (such as, tetrahydrofuran and dioxene), and the like or mixtures thereof. Optionally a scavenger can be added to capture the liberated caions. Suitable scavengers include, thiophenol, anisole, thioanisole, thiocresol, cresol, dimethyl sulfide and the like. Reaction temperature ranges for the deprotection of the nitrogen in 2-piperidin-4-yl-tbiazolcs (AC) can range from about -20°C to about the boiling point of the solvent used; generally the temperature range is about -1O°C to about 50°C.
SCHEME 1 :

The intermediate (AD) Is coupled with a carboxylic acid in the presence of a dehydrating
condensing agent and an,inert sob ent with or without abase to provide amide (AE) as illustrated in Scheme
2, Method A. Suitable dehydrating condensing agents include dicyclohexylcarbo-diinude (DCC), l-ethyI-3-
(3-dimemylaminopropyl)carboduinide hydrochloride (EDOHCI), bromo-tns-pyrxohdino-phosmum
hexafluorophosphate (ijyBroP), O^V-azabenzotriazol'l-y^-ia^^-tetramethyiuroniuni
hexafluorophosphate (HATU), liyclohexyl-3-methylpolystyrene-carbodiiimde and the like. Suitable bases
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WO 2005/116653 PCT/US2005/012447
include tertiary amines (such as, AyV-dnsopropyl-et lylamine, W-methylmorpholine, and tnethylamine) Suitable inert solvents include lower halocarbon solve nts (preferably dichloromethane, dichloroethane, and chloroform), ethereal solvents (such as, tetrahydrofuran and dioxane), mtrile solvents (such as acetomtnle), amide solvents (such as, WjV"-dimethylformarnide, and j^-dimethylacetamide), or mixtures thereof. Optionally, other reagents: can be used in the i :ouphng - reaction and these reagents include, 1-hydroxybenzotnazole (HOBT), HOBT-6-carboxaamidomethyl polystyrene, l-hydroxy-7-azabenzotriazole (HOAT) and tlie like. Suitable reaction temperature ringes from about -25°C to about 60°C, and about 0°C toabout35°C.
SCHEME 2

Alternatively, amide (AE) can be obtained by an armdation reaction using an acid hahde with intermediate (AD) in the presence of a base and an in :rt solvent as shown in Scheme 2, Method B. Suitable acid halides, include, acid chlorides or acid bromides. Suitable bases include alkali metal carbonates (such as, sodium carbonate and potassium carbonate), alkali metal hydrogencarbonates (such as, sodium hydrogencarbonate and potassium hydrogencarbonaf s), alkali hydroxides (such as, sodium hydroxide and potassium hydroxide), tertiary amines (such as, AyV-diisopropylethylamine, trietfaylanune, and N-methylmorpholine), and aromatic amines (such as, pyridine, imidazole, and poly-(4-vinylpyridme)). Suitable inert solvents include lower halocarbon solvents (such as, dichloromethane, dichloroethane, and chloroform), ethereal solvents (such as, tetrahydrofuran, and dioxane), amide solvents (such as, NJf-dimethylformamide, and Ay/-dimethylacetamide), end aromatic solvents (such as toluene, benzene, and pyridine). Suitable reaction temperature ranges from about -25*C to about 55°C, preferably about -5°C to about 40°C.
The protected acid group in amide (AE) is removed to give the corresponding carboxylic acid as shown in Scheme 3. Suitable methods for deprotectiig the carboxylic acid are known to those of originally skill in the art For example, alkyl esters1 (such E:S, methyl, ethyl, and n-piopyl) can be converted to carboxylic acids via hydrolysis in the presence of a base and in a suitable solvent Suitable bases include, alkali metal carbonates (such as, sodium carbonate and potassium carbonate), alkali metal hydrogencarbonates (such ' -S4-

WO 2005/116653 PCT/US2005/012447
hydroxides (such as, lithium hydroxide, sodium hydroxide and potassium hydroxide). Suitable solvents for the deprotection include, alkyl alcohols (such as, methanol, ethanol, j'-propanol, w-propanol and ;i-butanol), etliereal solvents (such asa tetrahydi'01111011 and dioxane), and the like or mixtures thereof, preferably the hydrolysis is conducted in the pres :nce of H2O Reaction temperatures for the deprotection of the acid group in amide (AE) can range from about room temperature ,to about the boiling point of the solvent used, generally (he temperature range is about 50°C to about 90°C. Other deprotection methods for esters as well as other additional suitable protecting groups are described in Greene and Wuts, Protective Groups in Organic Synthesis, third edition. Join Wiley & Sons, New York, 1999, supra. The carboxyhc acid (AF) is coupled with either methyl-(2-mi;thyl-4,5,637-tetrahydro-2H-indazol-3-yl)-amine or 1,2,3,4-tetrahydro-quinoline to give compounds of Formulae (AG) and (AH) respectively. Generally the coupling can be conducted in the presence of a deh> drating condensing agent and an inert solvent with or without a base, or by an amidation reaction using an a ;id hahde generated from carboxyhc acid (AF) in the presence of a base and an inert solvent, each method is as described for Scheme 2, supra.
SCHEME 3
Some embodiments of thi present invention include compounds illustrated in TABLE 1 as shown below.
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G. Pharmaceutical compositions
The invention provides methods of treatment (and prevention) by administration to an individual in need of said treatment (or prevention) a therapeutically effect amount of a modulator of the invention [also see, e.g., PCT Application Number PCT/IB02/01461 published as WO 02/066505 on 29 August 2002; the disclosure of each of which is hereby incorporated by reference in its entirety]. In a preferred aspect, the modulator is an agonist. In a preferred aspect, the modulator is substantially purified. The individual is preferably an animal including, but not limited to animals such as cows, pigs, horses, chickens, non-human primates, cats, dogs, rabbits, rats, mice, etc., and is preferably a mammal, and most preferably human.
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Modulators of the'invention can be administered to non-human animals [see Examples, infra] and/or humans, alone or in pharmaceutical or physiologically acceptable compositions where they are mixed with suitable earners or excipient(s) using techniques well known to those in the art Suitable pharmaceutically-acceptable carrier^ are available to those in the art; for example, see Remington's Pharmaceutical Sciences, 16th Edition, 1980, Mack Publishing Co , (Oslo et al., eds.)
The pharmaceutical or physiologically acceptable composition is then provided at a therapeutically effective dose A therapeutically ef fective dose refers to that amount of a modulator sufficient to result in prevention or amelioration of symptoms or physiological status of a disorder as determined illustratively and not by limitation by the methods described herein, wherein the prevention or amelioration of symptoms or physiological status of a disorder includes but is not limited to lowering of blood glucose concentration, prevention or treatment of certaiA metabolic disorders, such as insulin resistance, impaired glucose tolerance, and diabetes, and prevention or treatment of a complication of an elevated blood glucose concentration, such as atheroscleros is, heart disease, stroke, hypertension and peripheral vascular disease.
It is expressly considered that the modulators of the invention may be provided alone or in combination with other pharmaceuiically or physiologically iacceptable compounds. Other compounds for the treatment of disorders of the invention, wherein the treatment of disorders,of the invention includes but is not limited to lowering of blood glucose concentration, prevention or treatment of certain metabolic disorders, such as insulin resistance, impaired glucose tolerance, and diabetes, and prevention or treatment of a complication of an elevated blood glucose concentration, such as atherosclerosis, heart disease, stroke, hypertension and peripheral vascular disease, are currently well known in, the art One aspect of the invention encompasses the use according to embodiments disclosed herein further comprising one or more agents selected from the group consisting of sulfonylurea (e.g., ghbenclamide, glipizide, gliclazide, glimepiride), meghtinide (e.g., re|paghnide, nateglinide), biguanide (eg., metformin), alpha-glucosidase inhibitor (eg., acarbose, epalrestat miglitol, voglibose), thizaolidmedione (e g,, rosiglitazone, pioglitazone), insulin analog (e.g., insulin hspro, insulin aspart, insulin glargine), chromium picolinate/biotin, and biological agent {e.g, adiponectin or a fragment comprising the C-terminal globular domain thereof, or a mummer of adiponectin or said fragment thereof; or an agonist of adiponectin receptor AdipoRl or AdipoR2, preferably wherein said agonist is orally bioavailable). Additionally, it is expressly contemplated that the modulators of the invention, e.g. agonists and partial agonists of the invention, may be provided alone or in combination with a ahosphodiesterase (PDE) inhibitor (inclusive of an inhibitor selective for type 4 cAMP-specific PDE (PDE4), e.g roflurrulast; an inhibitor selective for PDE4B; and an inhibitor selective for PDE4B2).
In certain embodiments, (he metabolic disorder is selected from the group consisting of impaired glucose tolerance, insulin resistance, hyperinsuhnemia, and diabetes. In some embodiments, diabetes is type 1 diabetes In certain prefened embodiments, diabetes is type 2 diabetes. In certain embodiments, the metabolic disorder is diabetes. In certain embodiments, the metabolic disorder is type 1 diabetes. In certain embodiments, the metabolic disorder is type 2 diabetes. In certain embodiments, the metabolic disorder is
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WO 2005/116553 PCT/US2005/012447

impaired glucose tolerance In certain embodiments, tie metabolic disorder is insulin resistance In certain embodiments, the metabolic] disorder is hyperinsulineinia. In certain embodiments, the metabolic disorder
is related to an elevated blood glucose concentration in the individual.
i '
In certain embodiments, the complication of an elevated blood glucose concentration is selected from the group consisting of Syndrome X, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, neuropathy, retinopathy, nephropathy, and peripheral vascular disease. Heart disease includes, but is not limited to, cardiac insufficiency, coronary, insufficiency, coronary artery disease, and high blood pressure In certain embodiments, the comolication is Syndrome X. In certain embodiments, the complication is atherosclerosis In certain embodiments, the complication is atheromatous disease hi certain embodiments, the complication is heart disease. In certain embodiments, the complication is cardiac insufficiency. In certain embodiments, the complication is coronary insufficiency. In certain embodiments, the complication is coronary artery disease In certain embodiments, the complication is high blood pressure. In certain embodiments, the complication is hypertension. In certain embodiments, the complication is stroke. In certain embodiments, the complication is neuropathy. In certain embodiments, the complication is retinopathy. In certain embodments, the complication is neuropathy. In certain embodiments, the complication is peripheral vascular disease. In certain embodiments, the complication is polycystic ovary syndrome, hi certain embodiments, the complication is hyperlipidemia. Routes of Administration
Suitable routes of administration include oral, nasal, rectal, transmucosal, transdermal, or intestinal administration, parenteral delivery, including intrarruscular, subcutaneous, lntrarnedullary injections, as
well as intrathecal, direct intraventricular, intravenous, intrapentoneal, lntranasal, intrapuimonary (inhaled)
i
or intraocular injections using methods known in the art. Other particularly preferred routes of administration are aerosol and depot formulation. Sustained release formulations, particularly depot, of the invented medicaments are expressly contemplated. In certain embodiments, route of administration is oral Composition/Formulation .
Pharmaceutical or physiologically acceptable compositions and medicaments for use in accordance with the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliar es. Proper formulation is dependent upon the route of administration chosen. '
Certain of the medicaments described herein will include a pharmaceutically or physiologically acceptable carrier and at least one modulator of the invention. For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer such as a phosphate or bicarbonate buffer For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in he art
Pharmaceutical or physiologically acceptable preparations that can be taken orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol
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WO 2005/116653 PCT/US2005/012447
or sorbitol. The push-fit capsules can contain the active ingredients in admixture with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be, dissolved 01 suspended in suitable;liquids, such as fatty oils, liquid paraffin, or liquid polyethylen; glycols In addition, stabilizers may be added All formulations for oral administration should be jn dosages suitable for such administration
For buccal administration, the compositions may take,the form of tablets or lozenges formulated in
conventional manner. ~ i
For administration by inhalation, the compounds for use according to the present invention are
conveniently delivered in the form of an aerosol spray presentation from piessurized packs for a nebulizer,
with the use of a suitable gaseous p ropellant, e.g., carbon dioxide. In the case of a pressurized aerosol the
dosage unit may be determined by r iroviding a valve to deliver a metered amount. Capsules and cartridges
of, e g., gelatin, for ue in an inhaler or insufflator, may be forumulated containing a powder mix of the
compound and a suitable powder base such as lactose or starch. •-
The compounds may be formulated for parenteral administration by injection, e g., by bolus
injection or continuous infusion, formulations for injection may be presented in unit dosage for, e.g., in
ampoules or in muti-dose container i, with an added preservative. The compositions may take such forms as
suspension, solutions or emulsions in aqueous vehicles, and may contain formulatory agents such as
suspending, stabilizing and/or dispe rsing agents. ,
Pharmaceutical or physiologically acceptable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form.! Aqueous suspension may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
Alternatively, (he active ingredient may be in powder or lyophilized form for constitution with a suitable vehicle, such as sterile pwogen-free water, before use.
In addition to the formulations described previously, the compounds may also be formulated as a
depot preparation Such long a;tmg formulations may be administered by implantation (for example
subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may
be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an
acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly
soluble salt |
In a particular embodiment, the compounds can beidelivered via a controlled release system. In one embodiment, a pump maybe used (Langer, supra; Sefton, J1987, CRC Crit Ref. Biomed. Eng. 14:201-240; Buchwald et al, 1980, Surgery 85:507-516; Saudek et aL, 1989, N. Engl. X Med. 321:574-579). In another embodiment, polymeric materia s can be used (Medical Applications of Controlled Release, Langer and Wise, eds, CRC Press, Boca Rz.ton, Florida, 1974; Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Hall, eds., Wiley, New York, 1984; Ranger and Peppas, 1983, Macromol
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Sci. Rev. Macromol. Chem. 23:61; Levy et al., 1985, Science 228:190-192, During et ah, 1989, Ann. Neurol 25-351-356; Howardjetal, 1989, J. Neurosurg 71:858-863). Other controlled release systems are discussed in the review by Longer (1990, Science 249 1527-1533)
Additionally, the compounds may be deli ered using a sustained-release system, such as
semipemieable matrices of solid hydrophobic polymers containing the therapeutic agent Various sustained
release materials have been established and are well kiown by those skilled in the art. Sustained-release
capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100
days. '
Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for modulator stabilization may be employed.
The pharmaceutical or physiologically acceptable compositions also may comprise suitable solid or gel phase carriers or excipients Examples of such carriers or escipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulos derivatives, gelatin, and polymers such as polyethylene glycols.'
Effective Dosage
Pharmaceutical or physiologically acceptable compositions suitable for use in the present invention include compositions wherein die active ingredients are contained hi an effective amount to achieve their intended purpose More specifically, a therapeutically effective amount means an amount effective to prevent development of or toi alleviate the existing sym ptoms of the subject being treated. Determination of the effective amounts is wll within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell ^culture assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes or encompasses a concentration point or range shown to stimulate glucose uptake in a cell, to prevent or treat certain metabolic disorders, or to prevent or treat a complication of elevated blood glucose concentration. [See Examples, infra, for m vitro assays and in vivo animal models.] Such information can be used to more accurately determine useful doses in humans.
A therapeutically effective dose refers to that imount of the compound that results in amelioration of symptoms m a patient. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the test population) and the JiD50 (the dose therapeutically effective in 50% of the test population) The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LDsl) and ED30. Compounds that exhibit high therapeutic indices are preferred.
The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the EDS0, with little or no toxicity. The dosage may vary within this
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range depending upon the dosage form employed and the route of .administration utibzed. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e g., Fingl it al, 1975, in "The Pharmacological Basis; of Therapeutics", Ch. 1)
Dosage amount and interval may be adjusted individually to provide, plasma levels of the active compound which are sufficient to prevent or treat a disorder of the invention,, depending on the particular situation. Dosages necessary to ach eve these effects will depend on individual characteristics and route of administration.
Dosage intervals can also b; determined using the value for the minimum effective concentration
Compounds should be administered using a regimen that maintains plasmaj levels above the minimum
effective concentration for'10-90% of the time, preferably between 30-99%, and most preferably between
50-90%. In cases of local administration or selective uptake, the effective local concentration of me drug
may not be related to plasma concentration.. i
The amount of composition administered will, of course, be dependent on fhe subject being treated, on the subject's weight, the seventy of the affliction, the manner of administration, and the judgement of the prescribing physician.
A preferred dosage range for the amount of a modulator of the invention, which can be
administered on a daily or regular! basis to achieve desired results is 0.1-100 mg/kg body mass. Other
preferred dosage range is 0.1-30 rr.gfleg body mass. Other preferred dosage, range is 0.1-10 mg/kg body
mass. Other preferred dosage range is 0.1-3.0 mg/kg bodyimass. Of course, these daily dosages cart be
delivered or administered in small amounts periodically during the course of a day. It is noted that these
dosage ranges are only preferred rmges and are not rneant to be limiting to the invention. Said desired
results include, but are not limited to, lowering blood glucose concentration, .preventing or treating certain
metabolic disorders, such as insulin resistance, impaired glucose tolerance, and diabetes, and preventing or
treating a complication of an elevated blood glucose concentration, such as atherosclerosis, heart disease,
stroke, hypertension and peripheral vascular disease. (
H. Methods of Treatment i
The invention is drawn HI ter alia to methods including, but not limited to, methods of lowering blood glucose concentration, methods of preventing or treating certain metabolic disorders, such as insulin resistance and diabetes, and methods of preventing or treating a complication of an elevated blood glucose concentration, such as atherosclerosis, heart disease, strokej hypertension and peripheral vascular disease, comprising providing an individua 1 in need of such treatment with a modulator of the invention, hi certain embodiments, the modulator is an agonist In some embodiments, said modulator is orally bioavailable. In some embodiments, said orally bioavailable modulator is further able to cross the blood-brain barrier. In certain embodiments, the modula:or is provided to the individual in a pharmaceutical or physiologically acceptable composition, hi cerain embodiments, the modulator is provided to the individual in a pharmaceutical composition. In certain embodiments, the modulator is provided to the individual in a physiologically acceptable composition. In certain embodiments, the modulator is provided to the
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individual in a pharmaceutical or physiologically acceptable composition thai is taken orally. In certain embodiments, the individual is a non-human mammal In certain embodiments, the individual is a mammal. In certain embodiments, the individual or mammal is a human.
In certain embodiments, the metabolic disordsr is selected from the group consisting of impaired glucose tolerance, insulin resistance, hypennsulinemia, and diabetes. In some embodiments, diabetes is type 1 diabetes. In certain preferred embodiments, diabetes is type 2 diabetes In certain embodiments, the metabolic disorder is diabetes. In certain embodiment:;, the metabolic disorder is type 1 diabetes. In certain embodiments, die metabolicldisorder is type 2 diabetes. In certain embodiments, the metabolic disorder is impaired glucose tolerance. In certain embodiments, the metabolic disorder is insulin resistance. In certain embodiments, the metabolic, disorder is hyperinsuline nia. In certain embodiments, the metabolic disorder is related to an elevated blood glucose concentration in the individual.
In certain embodiments, the complication of an elevated blood glucose concentration is selected from the group consisting of Syndrome X, athsrosclerosis, atheromatous disease, heart disease, hypertension, stroke, neuropathy, retinopathy,nephropathy, and peripheral vascular disease. Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, coronary artery disease, and high blood pressure. In certain embodiments, the com plication is Syndrome X. In certain embodiments, the complication is atherosclerosis. In certain embodiments, the complication is atheromatous disease. In certain embodiments, the complication is heart disease. In certain embodiments, the complication is cardiac insufficiency. In certain embodiments, the complication is coronary insufficiency. In certain embodiments, the complication is coronary artery disease. In certain embodiments, the complication is high blood pressure. In certain embodiments, the complicate an is hypertension. In certain embodiments, the complication is stroke. In certain embodiments, the :omplication is neuropathy In certain embodiments, the complication is retinopathy. In certain, embodments, the complication is neuropathy. In certain embodiments, the complication is peripheral vascular disease hi certain embodiments, the complication is polycystic ovary syndrome In certain embodiments,' he complication is hyperhpidemia.
I. Other Utility
Agents that modulate (i.e, increase, decrease, or block) RUP43 receptor functionality may be identified by contacting a candidate compound with a RUP43 receptor and determining the effect of the candidate compound on RUP43 receptor functionality. The selectivity of a compound that modulates the functionality of the RUP43 receptor can be evaluated by comparing its effects on the RUP43 receptor to its effects on other G protein-coupled receptors. By way of illustration and not limitation, a modulator of an endogenous RUP43 receptor can be shown to be selective in comparison with one or more other endogenous G protein-coupled receptors from the same species. By way of illustration and not limitation, an agonist of an endogenous RXJP43 receptor can be shown to be a selective R.UP43 agonist if the EC50 of the agonist on the endogenous RUP43 receptor is at least 100-fold lower than the EC50 of the agonist on one or more other endogenous G protein-coupled receptors from the same species. Following identification of compounds that modulate RUP43 receptor func ionahty, such candidate compounds may be further
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tested in other assays including, bu: not limited to, in vivo models, in order to confirm or quantitate their activity. Modulators of RUP43 receptor functionality are therapeuti'cally useful in treatment of diseases and physiological conditions in which normal or aberrant R.UP43 receptor functionality is involved.
Agents that are ligands ofIUJP43 receptor may be identified by contacting a candidate compound with a RUP43 receptor and determining whether the candidate compound binds to the RUP43 receptor The selectivity of a compound that binds to the RUP43 receptor can be evaluated by comparing its binding to the RUP43 receptor to its binding on other receptors. By way of illustration and not limitation, a hgand of an endogenous RUP43 receptor can be shown to be selective m comparison with one or more other endogenous G protein-coupled receptors from the same species. Ligands that are modulators of RUP43 receptor functionality are therapeutically useful in treatment of diseases and physiological conditions in which normal or aberrant RUP43 receptor functionality is involved.
The present invention also relates to radioisotope-labeled versions of compounds of the invention identified as modulators or liganda of RUP43 receptor that would be useful not only in radio-imaging but also in assays, both in vitro and in vivo, for localizing and quantitating RXJP43 receptor in tissue samples, including human, and for identifying RUP43 receptor ligands by inhibition binding of a radioisotope-labeied compound. It is a further object of this invention to develop novel RUP43 receptor assays which comprise such radioisotope-labeled compounds.
The present invention embraces(radioisotope-labeled versions of compounds of the invention identified as modulators or hgands of RUP43 receptor.
The present invention also relates to radioisotope-labeled versions of test ligands that are useful for detecting a ligand bound to RUP43 receptor. In some embodiments, the present invention expressly contemplates a library of said raiiolabeled test ligands useful for detecting a hgand bound to RUP43 receptor. In certain embodiments, said library comprises at least about 10, at least about 102, at least about 103, at least about 10s, or at least ibout 106 said radiolabeled test compounds. It is a further object of this invention to develop novel RUP43 receptor assays which comprise such radioisotope-labeled test ligands.
In some embodiments, a radioisotope-labeled version of a compound is identical to the compound, but for the fact that one or more atoms are replaced or substituted by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature (i,e., naturally occurring) Suitable radionuchdeji that may be incorporated in compounds of the present invention include but are not limited to 2H (deutentm), 3H (tritium), "C, 13C, MCf 13N, 15N, 15O, 170,18O, I&F, 35S, 36C1, E2Br, 75Br, 76Br, 77Br, 123I, ml, 125I anc 131I. The radionuclide that is incorporated hi the instant radio-labeled compound will depend on the spe ;ific application of that radio-labeled compound. For example, for in viti o RUP43 receptor labeling and competition assays, compounds that incorporate 3H, 14C, B2Br, 125I, I, S or will generally be most useful, plr radio-imaging applications aC, t8F, l25I, ml, 12*L 13% 7SBr, 76Br or ?7Br will generally be most useful- In some embodiments, the radionuclide is selected from the group consisting of3H, »Cf 18F, 14C, °% l2X 131I,35S andE2Br.
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Synthetic methods for incorporating radio-isa topes into organic compounds are applicable to compounds of the invention A. Catalytic Reduction with Tritium Gas - This procedure normally yields high specific activity
products and requires halogenated or unsaturated precursors.
B. Reduction with Sodium Borohydride [3H] - This procedure is rather inexpensive and requires
precursors containing reducible functional groups such ES aldehydes, ketones, lactones, esters, and the like
C. Reduction with Lithium Aluminum Hydride [3H ] - This procedure offers products at almost
theoretical specific activities, i It also requires precursors containing reducible functional groups such as
aldehydes, ketones, lactones, esters, and the like.
D. Tritium Gas Exposure Labeling - This procedure involves exposing precursors containing
exchangeable protons to tritium gas in the presence of a suitable catalyst
E. N-Methylation using Methyl Iodide [3H] ¦ This procedure is usually employed to prepare 0-
methyl or N-methyl (3H) products by treating appropriate precursors with high specific activity methyl
iodide (3H). This method in general allows for higher specific activity, such as for example, about 70-90
Ci/mmol.
Synthetic methods for incorporating activity levels of ml into target molecules include:
A. Sandmeyer and like reactions - This procedure transforms an aryl or heteroaryl amine into a
diazonium salt, such as a tetrafluoroborate salt, and subsequently to nsl labeled compound using Na12SI A
represented procedure was reported by Zhu, D.-G. and co-workers in /, Org. Chem. 2002,67,943-948.
B. Ortho I2SIodination of phenols - Thus procedure allows for the incorporation of 125I at the ortho
position of a phenol as reported by Collier, T. L. and so-workers in J. Labeled Compd Radiophann, 1999,
42, S264-S266.
C. Aryl and heteroaryl bromide exchange with t25I - This method is generally a two step process.
The first step is the conversion of the aryl or heteroaryl bromide to the corresponding tu-alkyltin
intermediate using for example, a Pd catalyzed reacion [i.e. Pd(Ph3P)4] or through an aryl or heteroaryl
lithium, in the presence of a tri-alkyltinhalide or hexaalkylditin [e.g., (CH3)jSnSn(CH3)3]. A represented
procedure was reported by Bas, M.-D and co-workers in /. Labeled Compd Radiophann 2001, 44, S2S0-
S2S2.
In some embodiments, a radioisotope-labelec version of a compound is identical to the compound, but for the addition of one or more substituents com >rising a radionuclide. In some further embodiments, the compound is a polypeptide. In some further emb :>diments, the compound is an antibody or an antigen-binding fragment thereof. In some further embodiments, said antibody is monoclonal. Suitable said radionuclide includes but is not limited to 2H.(deuterium), 3H (tritium), nC, 13C, I4C, l3N, 15N, 15O, J7O3 l8O, 18F, 35S, 36C1, B2Br, 75Br, 76Br, "Br, 123I, 1MI, 125I and D1I. The radionuclide that is incorporated in the instant radio-labeled compound will depend on the specific application of that radio-labeled compound. For example, for in vitro RUP43 receptor labeling and competition assays, compounds that incorporate 3H, WC,
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a2Br, I2SI, 131l 35S or will generally be most useful. For radio-imaging applications "C, 18F, 12SI, mI, l2\ n\ 75Br, 76Br or 77Br will generally be r iost useful In some embodiments, the radionuclide is selected from the group consisting of 3H, "C, )EF, HCK, n% "% 35S and B2Br.
Methods for adding one or more substituents comprising a radionuchde are within the purview of the skilled artisan and include, but are not limited to, addition of radioisotopic iodine by enzymatic method [Marchalonic JJ, Biochemical Journal (1969) 113:299-305; Thorell Jl and Johansson BG, Biochimica et Biophysica Acta (1969) 251:363-9; the disclosure of each of which is hereby incorporated by reference in its entirety] and or by ChJorarnine-T/Ibdogen/Iodobead methods [Hunter WM and Greenwood FC; Nature (1962) 194:495-6, Greenwood FC et ul., Biochemical Journal (1963) 89:114-23; the disclosure of each of which is hereby incorporated by reference in its entirety].
Other uses of the disclosed receptors and methods will become apparent to those in the art based
upon, inter aha, a review of (his patent document i
EXAMPLES
The following examples are presented for purposes of elucidation, and not limitation, of the present invention. While specific nucleic acid and amino acid sequences are disclosed herein, those of ordinary skill in the art are credited with The ability to make minor modifications to these sequences while achieving fee same or substantially similar results reported below. Such modified approaches are considered within the purview of this disclosure.
The following Examples a:e provided for illustrative purposes and not as a means of limitation One of ordinary skill in the art Would be able to design equivalent assays and methods based on the disclosure herein, all of which form, part of the present invention.
Recombinant DNA techniques relating to the subject matter of the present invention and well known to those of ordinary skill in the art can be found, e.g, in Maniatis T et aL, Molecular Cloning: A Laboratory Manual (1989) Cold Spring Harbor Laboratory; U.S. Patent Number 6,399,373; and PCT Application Number PCT7IB02/0T461 published as WO 02/066505 on 29 August 2002; the disclosure of each of which is hereby incorporated by reference in its entirety.
Example 1
BULL-LENGTH CLONING OF HUMAN GPCRS
Endogenous Human RUP43 (SEQ ED NOs:l & 2)
Polynucleotide sequencfc encoding full-length 'endogenous human RUP43 (GPR131, eg GenBank® Accession No. NMj70699) can be cloned as described here. SEQ ID NO:1 is an endogenous human RUP43 (GPR131) polynucleotide coding, sequence that may be cloned as described here, SEQ ID NO:2 is the corresponding encoded endogenous human RUP43 (GPR131) polypeptide.
Full-length endogenous human RUP43 is cloned by PCR using Platinum PCR SupeiMix (Invitrogen catalog # 11306-016} and the specific primers
5'-GACAAGCATGAO3CCCAACAGCACTGGCGAG-3' (5J-primer; SEQ ID NO:3) and
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5'-CTTGAATTAGTTCAAGTCCAGGTCGACACTGC-3' (3'-primer; SEQ ED N0:4) with human DNA as template The human DNA may be genomic DNA or cDNA. The cycle condition used is 25 cycles of 95°C for 40 sec, 60°C for 50 sec, and 72°C for 1 mm. The 1 0 kb PCR product is cloned into the pCRH-TOPO™ vector (Invi xogen)
HA/Vf>His Double Tagged Endogenous Hunan RUP43 (SEQ ID NOs:5 & 6) Polynucleotide encoding mil-length endogenous human RUP43 (GPR131) polypeptide (absent the N-terminal metluonme) with N-terminal HA epitope ;ag and C-terminally disposed V5His epitope tag was cloned as described here. '"HA" epitope tag comprises ammo acid sequence MYPYDVPDYA. "V5" comprises ammo acid sequence GKPDPNPLLGLDS"; "His" comprises amino acid sequence HHHHHH. SEQ ED NO:5 is endogenous human RUP43 (GPRB1) polynucleotide coding sequence (absent the codon encoding the N-terminal methiomne) with 5'-terming 1 HA epitope tag and 3'-terminal V5His epitope tag SEQ ID NO:6 is the corresponding encoded HA/V5Eis double-tagged RLJP43 polypeptide
PCR was performed using an EST clon; (IMAGE #5221127, GenBank® Accession No BC033625) as template and pru polymerase (Stmtagene), with the buffer system provided by the manufacturer supplemented with 10% DMSO, 0.25 u M of each primer, and 0 5 mM of each 4 nucleotides The cycle condition was 25 cycles of 95°C for 40 sec, 60°Cfor 50 sec, and72°C for 1 min40sec. The 5' PCR primer incorporated a HindHI site and had the sequence:
S'-GACAAGCTTGACGCCCAACAGCACTGGCGAG-S' (SEQ ID NO:7). The 3' PCR primer incorporated an EcoRI sits and had the sequence: S'-CTTGAATTCGTTCAAGTCCAGGTCGACACTGC-3' (SEQ ID NO:8). The 1.0 kb PCR product was digested with HindlE and EcoRI and cloned into 5'HA/3'V5His double-tagged pCMV expression vector.
EXAMPLE 2
Preparation of Non-Endogenous, Constitutively Activated Human RUP43
Those skilled in the art are credited with tht; ability to select techniques for mutation of a nucleic acid sequence. Presented below are approaches that may be utilized to create non-endogenous versions of human GPCRs. The mutation disclosed here for er dogenous human RUP43 (GPR131) is based upon an algorithmic approach whereby the 16th amino acid (located in the IC3 region of the GPCR) N-terminal to a conserved proline (or an endogenous, conservative substitution therefor) residue (located m the TM6 region of the GPCR, near the TM6/IC3 interface) is mutated, preferably to a histidine, arginine or lysine ammo acid residue, most preferably to a lysine amino acid residue.
By way of illustration and not limitation, i non-endogenous, constitutively activated version of endogenous human RUP43 (GPR131) may be made by mutating alanine at ammo acid position 223 of SEQ ED NO:2, preferably to a lysine.
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1. Transformer Site-Directed ™ Mutageoesis t
Preparation of non-endogenous human GPCRs may, be accomplished on human GPCRs using, inter aha, Transformer Site-Direcued™ Mutagenesis Kit .(Clontech) according to Hie manufacturer instructions. Two mutagenesis primers are utilized, most preferably a lysine mutagenesis oligomicleotide that creates the lysine mutation, and a selection marker oligonucleotide. For convenience, the codon mutation to be incorporated into the human GPCR is also noted, in standard form.
2. QuikChange™* Site-Directed™ Mutagenesis
Preparation of non-endogerJous human GPCRs can also be accomplished by using QuikChange™ Site-Directed™ Mutagenesis Kit (ptratagene, according to manufacturer's instructions). Endogenous GPCR is preferably used as a template and two mutagenesis primers utilized, as well as, most preferably, a lysine mutagenesis ohgonucleotidb and a selection marker oligonucleotide (included in kit). For convenience, the codon imitation incorporated into the novel human GPCR and the respective ohgomicleotides are noted, in stands id form.
Example 3 Receptor Expression
Although a variety of cells are available to the art for the expression of proteins, it is most preferred that mammalian cells or melanophores be utilized. The primary reason for this is predicated upon practicalities, i.e., utilization of, eg., yeast cells for the expression of a GPCR, while possible, introduces into the protocol a non-mammalian cell which may not (indeed, in the case of yeast, does not) include the receptor-coupling, genebc-mechan ism and secretary pathways that have evolved for mammalian systems -thus, results obtained in non-mam nakan cells, while of potential use, are not as preferred as that obtained from mammalian cells or melanopaores. Of the mammalian cells, CHO, COS-7, MCB3901,293 and 293T cells aie particularly preferred, all hough the specific mammalian cell utilized can be predicated upon the particular needs of the artisan. In some embodiments, adipocytes or skeletal muscle cells obtained from a mammal may be used. See infi-a as relates to melanophores, including Example 10.
a. Transient Transfection
On day one, 6xlO6/ 10 cm dish of 293 cells are plated out. On day two, two reaction tubes are prepared (the proportions to folldw for each tube are per plate): tube A is prepared by mixing 4^g DNA (e.g., pCMV vector; pCMV vector with receptor cDNA, etc.) in 0 5 ml serum free DMEM (Gibco BRL); tube B is prepared by mixing 24u. 1 hpofectamine (Gibco BRL) in 0.5ml serum free DMEM Tubes A and B are admixed by inversions (sever il times), followed by incubation at room temperature for 30-45mrn. The admixture is referred to as the "transfection mixture". Plated 293 cells are washed with 1XPBS, followed by addition of 5 ml serum free DMEM. 1 ml of the transfection mixture is added to the cells, followed by incubation for 4hrs at 37°C/5% CO2. The transfection mixture is removed by aspiration, followed by the addition of 10ml of DMEM/10% Fetal Bovine Serum. Cells are incubated at 37°C/5% CO2. After 48hr incubation, cells are harvested add utilized for analysis.
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b. Stable Cell Lines
Approximately 12xlO6 293 cells are plated on a 15cm tissue culture plate. Grown in DME High Glucose Medium containing .ten percent fetal bovine scrum and, one percent sodium pyruvate, L-glutamine, and antibiotics. Twenty-four hours following plating of 293 cells (or to ~80% confluency), the cells are transfected using 12ug of DNA {eg., pCMV vector w th receptor cDNA) The 12u.g of DNA is combined
with 60p.L of lipofectamuie ;and 2ml of DME High Glucose ,Medium without serum. The medium is
i aspirated from the plates and the cells are wasliec once with medium without serum The DNA,
hpofectamine, and medium 'mixture aie added to the plate along with 10ml of medium without serum Following incubation at 37°C for four to five nous, the medium is aspirated and 25ml of medium containing serum is added. Twenty-four hours following transfecnon, the medium is aspirated again, and fresh medium with serum is added Forty-eight hours following transfection, the medium is aspirated and medium with serum is added containing geneticin (G418 drug) at a final concentration of approximately 12xI06 293 cells are plated on a 15cm tissue culture plate. Grown in DME High Glucose Medium containing ten percent fetal bovine serum and one pe -cent sodium pyruvate, L-glutamine, and antibiotics. Twenty-four hours following plating of 293 cells (or to ~80% confluency), the cells are transfected using 12gg of DNA (eg., pCMV vector with receptor cDNA). The 12^g of DNA is combined with 60ul of Hpofectamine and 2ml of DME High Glucose Medium without serum. The medium is aspirated from the plates and the cells are washed once with medium wi iiout serum. The DNA, Iipofectamine, and medium mixture are added to the plate along with lOmL of medium without serum. Following incubation at 37DC for four to five hours, the medium is aspirated and 2t ml of medium containing serum is added. Twenty-four hours following transfection, the medium is aspirated again, and fresh medium with serum is added. Forty-eight hours following transfection, the medium is aspirated and medium with serum is added containing geneticin (G418 drug) at a final concentration of 500fXg/ml The transfected cells now undergo selection for positively transfected cells containing the G418 resistance gene. The medium is replaced every four to five days as selection occurs. During selection, cells are grown to create stable pools, or split for stable clonal selection.
EXAMPLE 4
Assays For determination of GPCR Activation
A variety of approaches are available for assessment of activation of human GPCRs. The following are illustrative; those of ordinary skill in the art are ci edited with the ability to determine those techniques that are preferentially beneficial for the needs of the ariisan
1. Membrane Binding. Assays: [35S]GTPyS Assay
When a G protein-coupled receptor is in its active state, either as a result of Iigand binding or constitutive activation, the receptor couples to a G proi ein and stimulates the release of GDP and subsequent binding of GTP to the G protein. The alpha subunit of the G protein-receptor complex acts as a GTPase and slowly hydrolyzes the GTP to GDP, at which point the receptor normally is deactivated. Activated receptors continue to exchange GDP for GTP. The non-hydrolyzable GTP analog, [35S]GTPyS, can be
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utilized to demonstrate enhanced birding of [35S]GTPyS to membranes expressing activated receptors The advantage of using [35S]GTPyS binding to measure activation .is that: (a) it is genencally applicable to all G protein-coupled receptors; (b) it is proximal at the membrane surface making it less likely to pick-up molecules which affect the intracellular cascade
The assay utilizes the abihy of G protein coupled receptors to stimulate [35S]GTPyS binding to membranes expressing the relevant receptors. The assay can, therefore, be used in the direct identification method to screen candidate compounds to endogenous GPCRs and non-endogenous, constitutively activated GPCRs. The assay is generic and has application to drug discovery at all G protein-coupled receptors.
The [35S]GTPyS assay is in subatedin 20 mM HEPES and between 1 and about 20mM MgCl2 (this
amount can be adjusted for optimization of results, although 20mM is preferred) pH 7.4, binding buffer with
between about 0.3 and about 1.2 nM [3iS]GTPyS (this amount can be adjusted for optimization of results,
although 1.2 is preferred ) and 12 5 to 75 p.g membrane protein (e.g. 293 cells expressing the Gs Fusion
Protein, this amount can be adjusted for optimization) and 10 uM GDP (this amount can be changed for
optimization) for 1 hour. Wheatgenn aggluiinin beads (25 ^1; Amersham) are then added and the mixture
incubated for another 30 minutes kt room temperature. The tubes are then centnfuged at 1500 x g for 5
minutes at room temperature and then counted in a scintillation counter.
2. Adenylyl Cyclase
A Flash Plate™ Adenylyl Cyclase kit (New England Nuclear; Cat No. SMP004A) designed for cell-based assays can be modified for use with crude plasma membranes. The Flash Plate wells can contain . a scintillant coating which also contains a specific antibody recognizing cAMP. The cAMP generated in the wells can be quantitated by a direct competition for binding of radioactive cAMP tracer to the cAMP antibody. The following serves as a brief protocol for the measurement of changes in cAMP levels in whole cells that express the receptors.
Transfected cells are harv ;sted approximately twenty four hours after transient transfection. Media is carefully aspirated off and discarded. 10ml of PBS is gently added to each dish of cells followed by careful aspiration, lml of Sigma cell dissociation buffer and 3ml of PBS are added to each plate. Cells are pipetted off the plate and the cell suspension is collected into a 50ml conical centrifuge tube. Cells are then centnfuged at room temperature at 1,100 rpm for 5 min. The cell pellet is carefully re-suspended into an appropriate volume of PBS (about 3ml/plate). The cells are then counted using a hemocytometer and additional PBS is added to give the appropriate number of cells (with a final volume of about 50 ul/well).
cAMP standards and Defection Buffer (comprising 1 uCi of tracer [12SI] cAMP (50 ul) to 11 ml Detection Buffer) is prepared and maintained in accordance with the manufacturer's instructions. Assay Buffer is prepared fresh for screening and contains 50^1 of Stimulation Buffer, 3ul of test compound (12^M final assay concentration) and 50 al cells Assay Buffer is stored on ice until utilized. The assay, preferably carried out e.g in a 96-well plat;, is initiated by addition of 50ul of cAMP standards to appropriate wells
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followed by addition of 50ul' of PBSA to wells H-l 1 and H12 50pi of Stimulation Buffer is added to all wells. DMSO (or selected candidate compounds) is i dded to appropriate wells using a pin tool capable of dispensing 3ul of compound solution, with a final as!;ay concentration of 12^M test compound and lOOul total assay volume. The cells are then added to the veils and incubated for 60 min at room temperature. IOOul of Detection Mix containing tracer cAMP is then added to the wells. Plates are then incubated additional 2 hours followed by counting in a Wallac MicroBeta scintillation counter. Values of cAMP/well are then extrapolated from a standard cAMP curve wh ch is contained within each assay plate.
3. Cell-Based cAMP for Gi Coupled Target GPCRs
TSHR is a Gs coupled GPCR that causes the accumulation of cAMP upon activation. TSHR will be constitutivcly activated by mutatmg amino acid residue 623 (i.e., changing an alanme residue to an isoleucine residue) A Gi coupled receptor is expected to inhibit adenylyl cyclase, and, therefore, decrease the level of cAMP production, which can make assessment of cAMP levels challenging An effective technique for measuring the decrease in production of cAMP as an indication of activation of a Gi coupled receptor can be accomplished by co-transfecting, mo: t preferably, non-endogenous, constitutively activated TSHR (TSHR-A623I) (or an endogenous, constitutivcly active Gs coupled receptor) as a "signal enhancer" with a Gi linked target GPCR to establish a baselire level of cAMP. Upon creating a non-endogenous version of the Gi coupled receptor, this non-endogenous version of the target GPCR is then co-transfected with (he signal enhancer, and it is this material that cai be used for screening. We will utilize such approach to effectively generate a signal when a cAMP assiiy is used. In some embodiments, this approach is preferably used in the direct identification of candidate compounds against Gi coupled receptors. It is noted that for a Gi coupled GPCR, when this approach is used, an inverse agonist of the target GPCR will increase the cAMP, signal and an agonist will decrease the cAMP signal.
Qn day one, 2xlO4 293 cells/well will be plated out. On day two, two reaction tubes will be prepared (the proportions to follow for each tube aie per plate): tube A will be prepared by mixing 2jig DNA of each receptor transfected into the mammal an cells, for a total of 4p.g DNA (e.g., pCMV vector; pCMV vector with mutated THSR (TSHR-A623I); TSHR-A623I and GPCR, etc) in 1.2ml serum free DMEM (Irvine Scientific, Irvine, CA); tube B will be prepared by mixing 120^1 lipofectamine (Gibco BRL) in 1.2ml serum free DMEM. Tubes A and ]J will then be admixed by inversions (several times), followed by incubation at room temperature for 30-4 5min. The admixture is referred to as the "transfection mixture". Plated 293 cells will be washed with 1XFBS, followed by addition of 10ml serum free DMEM. 2.4ml of the transfection mixture will then be added t3 the cells, followed by incubation for 4hrs at 37°C/5% CO2. The transfection mixture will then be removed by aspiration, followed by the addition of 25ml of DMEM/10% Fetal Bovine Serum. Cells will then be incubated at 37°C/5% CO2. After 24hr incubation, cells will then be harvested and utilized for analysis
A Flash Plate™ Adenylyl Cyclase kit (New England Nuclear; Cat. No SMP004A) is designed for cell-based assays, ,but can be modified for use with c rude plasma membranes depending on the need of the slcilled artisan. The Flash Plate wells will contain a scinnllant coating which also contains a specific
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antibody recognizing cAMP. The cAMP generated in the wells can be quantitated by a direct competition for binding of radioactive cAMP trac sr to the cAMP antibody. The following serves as a bnef protocol for the measurement of changes in cAMP levels in whole cells that express the receptors.
Transfected cells will be harvested approximately twenty four hours after transient transfection. Media will be carefully aspirated off and discarded 10ml of PBS will be gently added to each dish of cells followed by careful aspiration, lml of Sigma cell dissociation.buffer and 3ml of PBS will be added to each plate. Cells will be pipetted off the plate and the cell suspension will be collected into a 50ml conical centrifuge tube Cells will then be centrifuged at room temperature at 1,100 rpm for 5 nun The cell pellet will be carefully re-suspended into a i appropriate volume of PBS (about 3ml/plate). The cells will then be counted using a hemocytometer and additional PBS is added to give the appropriate number of cells (with a final volume of about 50fil/well).
cAMP standards and Detecaon Buffer (comprising 1 uCi of tracer [1251] cAMP (50 p.1) to 11 ml Detection Buffer) will be prepared and maintained in accordance with the manufacturer's instructions. Assay Buffer should be prepared frosh for screening and contained 50p.i of Stimulation Buffer, 3u.l of test compound (12uM final assay concentration) and 50ul cells, Assay Buffer can be stored on ice until utilized. The assay can be initiated by addition of 50u,l ofcAMP standards to appropriate wells followed by addition of 50u.l ofPBSA to wells H-ll and HI 2. Fifty jal of Stimulation Buffer will be.added to all wells Selected compounds (e.g., TSH) will be added to appropriate wells using a pin tool capable of dispensing 3p.l of compound solution, with a final assay concentration of 12JIM test compound and 100p.l total assay volume. The cells will then be added to the wells and incubated for 60 nun at room temperature lOOul of Detection Mix containing tracer cAMP will then be added to the wells. Plates were then incubated additional 2 hours followed by counting in a "Wallac MicroBeta scintillation counter. Values of cAMP/well will then be extrapolated from a standard cAMF curve which is contained within each assay plate.
4. Reporter-Based Assays ,
CRE-Luc Reporter Assav (Gs-associated receptors)
293 and 293T cells are pis ted-out,on 96 well plates at a density of 2 x 104 cells per well and were transfected using Lipofectamine Reagent (BRL) the following day according to manufacturer instructions. A DNA/lipid mixture is prepared Jbr each 6-well transfection as follows: 260ng of plasmid DNA in 100|il of DMEM is gently mixed with 2p.l of lipid in lOOul of DMEM (the 260ng of plasmid DMA consists of 200ng of a 8xCRE-Luc reporter plasmid, 50ng of pCMV comprising endogenous receptor or non-endogenous receptor or pCMV alone, and lOng of a GPRS expression plasmid (GPRS in pcDNA3 (Invitrogen)). The 8XCRE-Luc reporter plasmid was prepared as follows: vector SRIF-p-gal was obtained by cloning the rat somatostatirt promoter (-71/+51) at BglV-HindlH site in the ppgal-Basic Vector (Clontech). Eight (8) copies of cAMP response element were obtained by PCR from an adenovirus template AdpCF126CCRE8 [see. Suzuki et al., Hum Gene Ther (1996) 7:1883-1893; the disclosure of which is hereby incorporated by reference in its entirety) and cloned into the SRTF-J5-gal vector at the Kpn-BglV site, resulting in the 8xCR3E-f5-gal reporter vector. The 8xCRE-Luc reporter plasmid was generated
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by replacing the beta-galactosidase gene in the 8xCRE-j3-gal reporter vector with the luciferase gene obtained from the pGL3-basic vector (Promega) at tin; HindHE-BaniHl site. Following 30 min. incubation at room temperature, the DNA/lrpid mixture is diluted with 400 ul of DMEM and lOOjil of the diluted mixture is added to each well. 100 ul of DMEM with 10% FCS are added to each well after a 4hr incubation in a cell culture incubator The following cay the transfected cells are changed with 200 [il/well of DMEM with 10% FCS. iBight (8) hours later, the wells are changed to 100 ul /well of DMEM without phenol red, after one wash with PBS. Luciferase acivity is measured the next day using the LucLite™ reporter gene assay kit (Packard) following 'manufacturer instructions and read on a 1450 MicroBeta™ scintillation and luminescence counter (Wallac).
b. API reporter assay (Gq-associated receptors)
A method to detect Gq stimulation depends o;i die known property of Gq-dependent phospholipase C to cause the activation of genes containing API elements in their promoter. A Pathdetect™ AP-1 cis-Reporting System (Stratagene, Catalogue # 219073) can be utilized following the protocol set forth above with respect to the CREB reporter assay, except that the components of the calcium phosphate precipitate were 410 ng pAPl-Luc, 80 ng pCMV-receptor expres sion plasnud, and 20 ng CMV-SEAP.
c. SRF-LUC Reporter Assay (Gq- asso ciated receptors)
One method to detect Gq stimulation depends on the known property of Gq-dependent phospholipase C to cause the activation of genes coitaining serum response factors in their promoter. A Pathdetect1 M SRF-Luc-Reporting System (Stratagene) can be'utilized to assay for Gq coupled activity in, e.g., COS7 cells. Cells are transfected with, the p. asmid components of the system and the indicated expression plasmid encoding endogenous or non-endogenous GPCR using a Mammalian Transfection™ Kit (Stratagene, Catalogue #200285) according to the manufacturer's instructions. Briefly, 410 ng SRF-Luc, 80 ng pCMV-receptor expression plasmid and 20 ng CMV-SEAP (secreted alkaline phosphatase expression plasmid; alkaline phosphatase activity is rr easured in the media of transfected cells to control for variations in transfection efficiency between Samples) are combined in a calcium phosphate precipitate as per the manufacturer's instructions. Half of die precipitate is equally distributed over 3 wells in a 96-well plate, kept on the cells in a serum free media for 24 hours. The last 5 hours the cells are incubated with, e.g luM, test compound. Cells are then lysed and assayed for luciferase activity using a Luclite™ Kit (Packard, Cat # 6016911) and "Trilux 1450 Micrsbeta" liquid scintillation and luminescence counter (Wallac) as per the manufacturer's instructions. Th Intracellular IP3 Accumulation Assay (Gq-associated receptors)
On day 1, cells comprising (he receptors (endogenous or non-endogenous) can be plated onto 24 well plates, usually 1x10s cells/well (although his number can be optimized. On day 2 cells can be transfected by first mixing 0.25ug DNA in 50 ul semm free DMEM/well and 2 ul lipofectamine in 50 ul serum free DMEM/well. The solutions are gently mi ted and incubated for 15-30 min at room temperature. Cells are'washed with 0.5 ml PBS and 400 ul of serum free media is mixed with the transfection media and
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added to the cells. The cells are then| incubated for 3-4 hrs at 37°C/5%CO2 and then the transfection media is removed and replaced with lml/well of regular growth media. On day 3 the cells are labeled with 3H~ myo-inositol Briefly, the media is removed and the cells are washed with 0.5 ml PBS Then 0.5 nil inositol-free/senim free media (GIBCO BRL) is added/well with 0.25 uCi of 3H-myo-inositol/ well and the cells are incubated for 16-18 hrs o/n at 37°C/5%CO2. On Day 4 the cells are washed with 0.5 ml PBS and 0.45 ml of assay medium is added containing lnositol-free/serum free media 10 uM pargyhne 10 mM lithium chloride or 0 4 ml of assay njedium and 50ul of lOx ketansenn (ket) to final concentration of 10p.M The cells are then incubated for 30 kin at 37°C. The cells are then washed with 0 5 ml PBS and 200ul of fresh/ice cold stop solution (1M KG)H; 18 mM Na-borate; 3.S mM EDTA) is added/well. The solution is kept on ice for 5-10 mm or unto cells were lysed and then neutralized by 200 ul of fresh/ice cold neutralization sol. (7.5 % HCL). The lysate is then transferred into 1.5 ml eppendorf tubes and 1 ml of chlorofornVmethanol (1:2) is addeditube. The solution is vortexed for 15 sec and the upper phase is applied to a Biorad AG1-X8™ anion exchange resin (100-200 mesh). Firstly, die resin is washed with water at 1:1 25 W/V and 0.9 ml of upper phase is loaded onto the column The column is washed with 10 mis of 5 mM myo-inositol and 10 ml of 5 mM Na-borate/60mM Na-formate The inositol tris phosphates are eluted into scintillation vials containing 10 ml of scintillation cocktail with 2 ml of 0.1 M formic acid/ 1 M ammonium formate. The columrs are regenerated by washing with 10 ml of 0.1 M formic acid73M ammonium formate and rinsed twice with dd H2O and storedjat 4°C in water,
EXAMPLES
fusion Protein Preparation
a. GPCR:Gs Fusion Constuct
The design of the GPCR-p protein fusion construct can be accomplished as follows: both the 5' and 3' ends of the rat G protein Gsa (long form; Itoh, H. et al., 83 PNAS 3776 (1986)) are engineered to include a HindHI (5'-AAGCTT-3') sequence thereon. Following confirmation of the correct sequence (including the flanking HindDI sequences), the entire sequence is shuttled into pcDNA3.1(-) (Invitrogen, cat no. V795-20) by subcloning x sing the HindlH restriction site of that vector. The correct orientation for the Gsa sequence is determined aiter subcloning into pcDNA3.1(-) The modified pcDNA3.1(-) containing the rat Gsa gene at HindDI sequence is 'then verified, this vector is now available as a "universar Gsa protein vector. The pcDNA3.1(-) vector contains a variety of well-known restriction sites upstream of the HindDI site, thus beneficially providing the ability to insert, upstream of the Gs protein, the coding sequence of an endogenous, constitutively active GPCR. This same approach can be utilized to create other "universal" G protein vectors, and, of course, other commercially available or proprietary vectors known to the artisan can be utilized—the : mportant criteria is that the sequence for the GPCR be upstream and in-frame with that of the G protein.
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Gq(6 amino acid deletion)/Gi Fusion Construct
The design of a Gq(del)/Gi fusion construct can be accomplished as follows: the N-terminai six (6) ammo acids (amino acids 2 through 7, having the sequence of TLESIM) of Gccq-subunit will be deleted and the C-terminal five (5) amino acids having the sequer.ee EYNLV will be replaced with the corresponding amino acids of the Gcri Protein, having the sequence DCGLF This fusion construct will be obtained by PCRusing'the following primers:
y-gatcAAGCTTCCATGGCGTGCTGCCTGAGCGAGGAG-3' (SEQ ID NO:9) and
5'-gatcGGATCCTTAGAACAGGCCGCAGTCCTTCAGGTTCAGCTGCAGGATGGTG-3' (SEQIDNO:10)
and Plasmid 63313 which contains the mouse Gaq-wild type version with a hemagglutinin tag as template, Nucleotides m lower caps are included as spacers.
TaqPlus Precision DNA polymerase (Stratagene) will be utilized for the amplification by the following cycles, with steps a through 4 repeated 35 times: 95°C for 2 mm; 95°C for 20 sec, 56°C for 20 sec; 72°C for 2 min; and 72°C for 7 mm. The PCR. product will be cloned into a pCRE-TOPO vector (Invitrogen) and sequenced using the ABI Big Dye Terminator kit (P.E. Biosystems). Inserts from a TOPO clone containing the sequence of the fusion construct will be shuttled into the expression vector pcDNA3 1(+) at the HindlH/BamHI site by a 2 step cloning process. Abo see, PCT Application Number PCT/US02/05625 published as WO02068600 on 6 September 2002, the disclosure of which is hereby incorporated by reference in its entirety.
EXAMPLE 6
[3SS]GTPyS ASSAY
A. Membrane Preparation j
In some embodiments membranes comprising the Target GPCR of interest and for use in the identification of candidate compounds as, e.g., inverse agonists, agonists, or antagonists, are preferably prepared as follows:
a. Materials I
"Membrane Scrape Buffer" is comprised of ::0mM HEPES and lOmM EDTA, pH 7 4; "Membrane Wash Buffer" is comprised!of 20 mM HEPES and 0.1 mM EDTA, pH 7.4; "Binding Buffer" is comprised of 20mM HEPES, 100 mM NaCI, and 10 mM MgCl;, pH 7.4.
b. Procedure
All materials will be kept on ice throughout the procedure. Firstly, the media will be aspirated from a confluent monolayer of cells, followed by rinse with 10ml cold PBS, followed by aspiration. Thereafter, 5ml of Membrane Scrape Buffer will be added to scrape cells; this will be followed by transfer of cellular extract into 50ml centrifuge tubes (centrifuged at 20,000 rpm for 17 minutes at 4°C). Thereafter, the supernatant will be aspirated and the pellet will be iesuspended in 30ml Membrane Wash Buffer followed
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by centrifuge at 20,000 rpm for 17 minutes at 4°C The supernatant will then be aspirated and the pellet resospended in Binding Buffer. This will then be homogenized using a Bnnkman Polytron™ hornogenizer (15-20 second bursts until the all Wterial1 is in suspension). This is referred to herein as "Membrane ^rotein",
Bradford Protein Assay
Following the homogenizafcon, protein concentration of the membranes will be determined using the Bradford Protein Assay (protend can be diluted to about 1,5mg/ml, aliquoted and frozen (-80°C) for later use; when frozen, protocol for use Will be as follows: on the'day of the assay, frozen Membrane Protein is thawed at room temperature, followed by vortex and then homogenized with a, Polytron at about 12 x 1,000 rpm for about 5-10 seconds, it is noted that for multiple preparations, the homogenizer should be thoroughly cleaned between homogenizationo:'different preparations)
a. Materials
Binding Buffer (as per abcive); Bradford Dye Reagent; Bradford Protein Standard will be utilized, following manufacturer instructions (Biotad, cat. no. 500-0006).
b. Procedure
Duplicate tubes will be prepared, one including the membrane, and one as a control "blank". Each contained SOOfj.1 Binding Buffer. Thereafter, 10|il of Bradford Protein Standard (lmg/ml) will be added to each tube, and lOul of membrane Protein will then be added to just one tube (not the blank) Thereafter, 200p.l of Bradford Dye Reagent will be added to each tube, followed by vortex of each. After five (5) minutes, the tubes will be re-vorteked and the material therein will be transferred to cuvettes. The cuvettes will then be read using a CECIL 3041 specttophotometer, at wavelength 595.
Identification .Assay
a. Materials
GDP Buffer consisted of 37.5 ml Binding Buffer and 2mg GDP (Sigma, cat. no. G-7127), followed by a series of dilutions in Binding Buffer to obtain 0.2 ^iM GDP (final concentration of GDP in each well was 0.1 ^iM GDF); each well comprising a candidate compound, has a final volume of 200^1 consisting of lOOul GDP Buffer (final concentJation, O.luM GDP), 50p.l Membrane Protein in Binding Buffer, and 50^1 [35S]GTP?S (0.6 nM) in Binding Buffer (2.5 nl [35S]GTPyS per 10ml Binding Buffer).
b. Procedure
Candidate compounds will be preferably screened using a 96-well plate format (these can be frozen at -80°C). Membrane Protein (or membranes with expression vector excluding the Target GPCR, as control), will be homogenized briefly until in suspension. Protein concentration will then be determined using the Bradford Protein Assa j set forth above. Membrane Protein (and control) will then be diluted to 0.25mg/ral in Binding Buffer (firjal assay concentration, 12.5|ig/well). Thereafter, 100 ul GDP Buffer was added to eacn well of a Wallac Sbintistrip™ (Wallac). A 5ul pin-tool will then be used to transfer 5 ul of a
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candidate compound into such well (1 e, 5u.l in total assay volume of 200 yd is a 1 "40 ratio such that the final screening concentration, of the candidate compomd is lOuM). Again, to avoid contamination, after each transfer step the pin tool should be rinsed in thre; reservoirs comprising water (IX), ethanol (IX) and water (2X) - excess liquid should be shaken from the tool after each nnse and dried with paper and kimwipes. Thereafter, 50 ul of Membrane Protein Will be added to each well (a control well comprising membranes without the Target GPCR was also utilized), and pre-incubated for 5-10 minutes at room temperature Thereafter, 50ul of [35S]GTPyS (0.6 iiM) in Binding Buffer will be added to each well, followed by incubation on a shaker for 60 minutes at ::oom temperatuie (again, in this example, plates were covered with foil) The assay will then be stopped by spinning of the plates at 4000 RPM for 15 minutes at 22°C The plates will then be aspirated with an S channel manifold and sealed with, plate covers. The plates will then be read on a Wallac 1450 using setting "Prot #37" (as per manufacturer's instructions).
Example? CYCLIC AMP ASSAY
Another assay approach for identifying candidate compounds as, e.g., inverse agonists, agonists, or antagonists, is accomplished by utilizing a cyclase-based assay In addition to direct identification, this assay approach can be utilized as an independent approach to provide confirmation of the results from the [35S]GTPyS approach as set forth in Example 6, supra.
A modified Flash Plate™ Adenylyl Cyclasi kit (New England Nuclear; Cat No. SMP004A) is preferably utilized for direct identification of candidate compounds as inverse agonists and agonists to endogenous or non-endogenous, constitutively actived GPCRs in accordance with the following protocol.
Transfected cells are harvested approximately three days after transfection. Membranes are prepared by homogemzation of suspended cells in suffer containing 20mM HEPES, pH 7.4 and lOmM MgCU. Homogenization is performed on ice using a Brinkman Polytron™ for approximately 10 seconds. The resulting homogenate is centnfuged at 49,000 X g for 15 minutes at 4°C. The resulting pellet is then resuspended in buffer containing 20mM HEPES, pH 7.4 and 0.1 mM EDTA, homogenized for 10 seconds, followed by ccntrifugation at 49,000 x g for 15 minu tes at 4°C. The resulting pellet is then stored at -80°C until utilized. On the day of direct identification scr sening, the membrane pellet is slowly thawed at room temperature, resuspended in buffer containing 20mM HEPES, pH 7.4 and lOmM MgCl2> to yield a final protein concentration of 0.60mg/ml (the resuspended membranes are placed on ice until use),
cAMP standards and Detection Buffer (corr prising 2 uCi of tracer {[l25I]cAMP (100 ul) to II ml Detection Buffer] are prepared and maintained in at cordance with the manufacturer's instructions. Assay Buffer was prepared fresh1 for screening and contained 20mM HEPES, pH 7.4, lOmM MgCl2, 20mM phospocreaune (Sigma), 0.1 units/ml creatine phosp hohnase (Sigma), 50 uM GTP (Sigma), and 0.2 mM ATP (Sigma); Assay Buffer was then stored on ice until utilized.
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Candidate compounds are adaed, preferably, to e,g 96-well plate wells (3ul/well; 12uM final assay concentration), together with 40 nl Membrane Protein (30ug/well) and 50^1 of Assay Buffer. This admixture was then incubated for 30 minutes at room temperature, with gentle shaking.
Following the incubation, lOOul of Detection Buffer is added to each well, followed by incubation for 2-24 hours. Plates are then counted in a Wallac MicroBeta™ plate reader using "Prot #31" (as per manufacturer's instructions).
By way of example and not limitation, an illustrative screening assay plate (96 well format) result obtained is presented in Figure 1. Each bar represents the result for a compound that differs in each well, the "Target GPCR" being a Gsa Fusion Protein construct of an endogenous, constirutively active Gs-coupled GPCR unrelated to GPR13L The results presented ;in Figure 1 also provide standard deviations based upon the mean results of each plate ("m") and the mean plus two arbitrary preference for selection of inverse agonists as "leads" from the primary screen involves selection of candidate compounds that that reduce the per cent response by at least the mean plate response, minus two standard deviations. Conversely, an arbitrary preference for selection of agonists as "leads" from the primary screen involves selection of candidate compounds that increase the per cent response by at least the mean plate response, plus the two standard deviations. Based upon these selection processes, the candidate compounds in the following wells were directly identified as putative inverse agonist (Compound A) and agonist (Compound B) to said endogenous GPCR in wells A2 and G9, respectively. See, Figure 1. It is noted for clarity: these compounds have been directly identified without any knowledge of the endogenous ligand for this GPCR. By focusing on assay techniques that are based upon receptor function, and not compound binding affinity, it is possible to ascertain compounds that are able to reduce the functional activity of this receptor (Compound A) as well as increase 1 he functional activity of the receptor (Compound B).
Example 8
Fluorometric Imaging I late Reader (FLIPR) Assay for the Measurement of Intracellular Calcium Concentration
Target Receptor (experimental) and pCMV (negative control) stably transfected cells from
respective clonal lines are seeded into poly-D-lysine pretieated 96-well plates (Becton-Dickinson, #356640)
at 5.5xlO4 cells/well with complete culture medium (DMEM with 10% FBS> 2 mM L-glutaminef 1 mM
sodium pyruvate) for assay the next day. To prepare Fluo4~AM (Molecular Probe, #F14202) incubation
buffer stock, 1 mg Fluo4-AM is dissolved in 467 ul DMSO and 467 pi Pluoronic acid (Molecular Probe,
#P3000) to give a 1 mM stock solution that can be stored at -20°C for a month. Fluo4-AM is a fluorescent
calcium indicator dye. ;
Candidate compounds ar;; prepared in wash buffer (IX HBSS/2.5 mM Probenicid/20 mM HEPES atpH7.4).
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At the time of assay, culture medium is removed from the wells and the cells are loaded with 100 ul of 4 uM Fluo4-AM/2.5 mM Probemcid (Sigma, #P3761)/20 rnM HEPES/complete medium at pH 7.4 Incubation at 37°C/5% CO2 is allowed to proceed for ¦ 50 nun.
After the 1 hr incubation, the Fluo4-AM incusation buffer is removed and the cells are washed 2X with 100 ul wash buffer. In each well is left 100 ul wash buffer The plate is returned to the incubator at 37°C/5% CO2 for 60 min.
FLIPR (Fluorometric Imaging Plate Reader, Molecular Device) is programmed to add 50 jil candidate compound on the 30th second ,and to record transient changes in intracellular calcium concentration ([Ca2+]) evoked by the candidate compound for another 150 seconds. Total fluorescence change counts are used to determine agonist activity using the FLIPR software The instrument software normalizes the fluorescent reading to give equivalent initial readings at zero.
In some embodiments, the cells comprising Target Receptor further comprise Gal5, GaI6, or the chimeric Gq/Gi alpha unit >
Although the foregoing provides a FLIPR ass ay for agonist activity using stably transfected cells, a person of ordinary skill in the art would readily b; able to modify the assay in order to characterize antagonist activity Said person of ordinary skill in tl ie art would also readily appreciate that, alternatively, transiently transfected cells could be used.
Example 9
MAP Kinase Assay
MAP kinase (mitogen activated kinase) maj[ be monitored to evaluate receptor activation. MAP lanase can be detected by several approaches. One approach is based on an evaluation of the phosphorylation state, either unphosphoryiated (inacive) or phosphorylated (active). The phosphorylated protein has a slower mobility in SDS-PAGE and car therefore be compared with the unstimulated protein using Western blotting. Alternatively, antibodies specific for the phosphorylated protein are available (New England Biolabs) which can be used to detect an increase in the phosphorylated kinase. In either method, cells are stimulated with the test compound and then > attracted with Laernmh buffer. The soluble fraction is applied to an SDS-PAGE gel and proteins are transferred electrophoreticauy to nitrocellulose or Immobilin. Immunoreactive bands are.detected by standard Western blotting technique. Visible or cherruluminescent signals are recorded on film and may be quantified b> densitometry.
Another approach is based on evaluation of the MAP kinase activity via a phosphorylation assay Cells are stimulated with the test compound and a soluble extract is prepared The extract is incubated at 30°C for 10 min with gamma-32P-ATP, an ATP regenerating system, and a specific substrate for MAP kinase such as phosphorylated heat and acid stable protein regulated by insulin, or PHAS-I. The reaction is terminated by the addition of H3PO4 and samples are transferred to ice. An aliquot is spotted onto Whatman P81 chromatography paper, which retains the phosph ^rylated protein The chromatography paper is washed and counted for 32P is a liquid scintillation counter, i alternatively, the cell extract is incubated with gamma-
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32P-ATP, an ATP regenerating systerr., and biotmylated myelin basic proem bound by streptavidin to a filter
support. The myelin basic protein is a substrate for activated MAP kinase. The phosphorylation reaction is
carried out for 10 min at 30°C TWe extract can then be aspirated through the filter, which retains, the
phosphorylated rnyelin basic protein The filter is washed and counted for 3aP by liquid scintillation
counting. |
Example 10 Melanophore Technology
Melanophores are skin cells found in lower vertebrates. They contain pigmented organelles termed melanosomcs. Melanophores are abl to redistribute these melanosomes along a microtubule network upon G-protem coupled receptor (GPCR.) activation The result of this pigment movement is an apparent lightening or darkening of the cells. In melanophores, the decreased levels of intracellular cAMP that result from activation of a Gi-coupled receptor cause melanosomes to migrate to the center of the cell, resulting in a dramatic lightening in color. If cAistP levels are- then raised, following activation of a Gs-coupled receptor, the melanosomes are re-dispersed £.nd the cells appear dark again. The increased levels of diacylglycerol that result from activation of Gq-ooupled receptors can also induce this re-dispersion. In addition, the technology is also suited to the studv of certain receptor tyrosine kinases. The response of the melanophores takes place within minutes of receptor activation and results in a simple, robust color change, The response can be easily detected using a ccnventional absorbance microplate reader or a modest video imaging system Unlike other skin cells, the melanophores derive from the neural crest and appear to express a full complement of signaling proteins. In particular, the cells express an extremely wide range of G-proteins and so are able to functionally express clmost all GPCRs.
Melanophores can be utilized to identify compounds, including natural ligands, against GPCRs. This method can be conducted by introducing test cells of a pigment cell line capable of dispersing or aggregating their pigment in response to a specific stimulus, and expressing an exogenous clone coding for the GCPR. A stimulant; e g., meliitonin, sets an initial state of pigment disposition wherein the pigment is aggregated within the test cells if abuvation of the GPCR induces pigment dispersion. However, stimulating the cell with a stimulant to set ar initial state of pigment disposition wherein the pigment is dispersed if activation of the GPCR induces pigment aggregation. The test cells are then contacted with chemical compounds, and it is determined whether the pigment disposition in the cells changed from the initial state of pigment disposition. Dispersion of pigments cells due to the candidate compound, including but not limited to a ligand, coupling to the GPCR will appear dark on a petri dish, while aggregation of pigments cells will appear light
Materials and methods will be followed according to the disclosure of U.S. Patent Number 5,462,856 and U.S. Patent Nunber 6,051,386. These patent disclosures are hereby incorporated by reference in their entirety.
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The cells are plated in e.g. 96-well plates (one receptor per plate) 48 hours post-transfection, half of the cells on each piate are treated with lOnM melatc nin. Melatonin activates an endogenous Gi-coupled receptor in the melanophores1 and causes them1 to aggr :gate their pigment. The remaining half of the cells are transferred to serum-free medium 0.7X L-15 (Gil co) After one hour,' the cells in serum-free media remain in a pigment-dispersed state while the melaton. n-treated cells are in a pigment-aggregated state At this point, the cells are treated with a dose response of 11 test/candidate compound If the plated GPCRs bind to the test/candidate compound, the melanophores woi Id be expected to undergo a color change in response to the compound. If the receptor were either a Gs or Gq coupled receptor, then the meJatonin-aggregated melanophores would undergo pigment dispersion. In contrast, if the receptor was a Gi-coupled receptor, then the pigment-dispersed cells would be expected to indergo a dose-dependent pigment aggregation.
EXAMJ'LE 11
Tissue Distribution of Human and Mouse RXJP43
The expression of RUP43 by human and mouse adipocytes and skeletal muscle cells was interrogated by RT-PCR. The expression of RUP43 by human leukocyte subsets was interrogated by TaqMan RT-PCR
a >
Human preadipocytes were purchased from Biowhittaker and either allowed to remain
undifferentiated or subjected to differentiation Humsn differentiated adipocytes were purchased from Zen
Bio. RNA was prepared from these undifferentiated or differentiated human adipocytes and converted to
cDNA. RT-PCR was then1 carried out in order to interrogate expression of RUP43 using the specific
primers •
5'-CTACCTGTACCTCGAAGTCTA-3' (ser se-primer; SEQ ID NO:11) and
5'-AGTGGCGGGCGCTGCTCAT-3' (antisense-primer; SEQ ID NO:12).
The cycle condition used was 94°C for 2 mm, 94°C for 15 sec, 55°C for 30 sec, and 72°C for 1 mm, with 35 cycles for the final three steps. RUP43 was found to be expressed endogenously by differentiated human adipocytes and to a lesser extent by human preadipocytes (Figure 2A)
hi :
Expression of RUP43 by human subcutaneous ("Sub Q") and visceral fat was interrogated by RT-PCR as in [a] above. Subcutaneous fat samples were obtained from ten individuals with BMI ranging from 19 to 35. Visceral fat samples were obtained from eight individuals with BMI ranging from 19 to 45. RT-PCR of g'lyceialdehyde-3-phosphate dehydrogenase (GAPDH) was used to show comparable loading of samples. Human RUP43 was found to be expressed sndogenously both in subcutaneous and in visceral fat (Figure 2B).
Mouse 3T3L1 cells were allowed to remair undifferentiated or were subjected to differentiation. RNA was prepared from undifferentiated 3T3L1 cells, from differentiated 3T3L1 cells, or from mouse
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skeletal muscle cells. Conversion of the RNA to cDNA was carried out either in the presence ("+") or absence ("-"; negative control) of re\ erse transcriptase. RT-PCR was then carried out in order to interrogate expression of RUP43 using the specific primers.
5'-TGAGCTGTCGGCCAlTCCCAT-3' (sense-primer;SEQIDNO:13)and S'-GATTGTCCCTCTTGGrrCTTC-S' (antisense-primer; SEQ ID N£:14). The cycle condition used was 94°C for 2 min} 94°C for 15 sec, 55°C for 30 sec, and 72°C for 1 min, with 35 cycles for the final three steps. RUP43 was found to be expressed by differentiated mouse 3T3L1 adipocytes and to a lesser exient by undifferentiated 3T3L1 adipocytes. ,RUP43 was also found to be endogenously expressed by mouse s keletal muscle cells (Figure 2C).
4
Human skeletal muscle eel is were obtained from Cambrex. RNA was prepared from the skeletal
i . muscle cells and converted to cDHA. AS a positive control, cDNA prepared as in [a] from human
adipocytes obtained from Biowhittajker wasused. RT-PCR was carried out as described in [a]. RUP43 was found to be endogenously expressed by skeletal muscle cells, and as previously shown in [a], by adipocytes (Figure 2D).
EXAMPLE 12
Adipocy te Differentiation
Differentiation of Mouse 5T3L1 Cells
3T3L1 Growth Medium 1000ml
DMEM ' 1000ml
' 10%BCS : 100ml
L-glutamine, 200mM 10ml
P/S 10ml
3T3L1 Regular Medium! 1000ml i
DMEM 1000ml
10%FBS 100ml
L-glutamine3 200mM 10ml
p/S ' 10ml
3T3L1 Inducing MediuJn 1000ml
DMEM . 1000ml'
10%FBS 100ml
L-glutamine, 200mM 10ml ,
P/S 10ml ,
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Insulin (lOmg/ml) lml
IBMax (lOmg/ml) ¦ ll.lnl
Dexaraethasone (lOmg/ml) 328^1
3T3L1 Insulin Only Medium 1000ml
DMEM 1000ml
10%FBS 100ml
L-glulamine, 200rnM lOrr I
P/S ' lOirl
Insulin (lOmg/ml) lml
DMEM- HYQ DEM/High glucose, SH300SU1,500ml. SH3OO81 02,1000ml.
BCS: Bovine Calf Serum, Hyclone SH 30073 03
FBS: Fetal Bovine Serum, Hyclone SH 30071.03
L-glutamme 200mm, lOOx. Hyclone SH4OOO>11
Penicillin-Streptomycin, 100ml Hyclone SV30010
Trypsin, HYQ, 0.05% lx,SH30236.01 100m:
HYQ DPBS/modified, lx SH30028.02,50ml
3T3L1 cells were seeded at 50% confluence such that the culture was fully confluent the next day Two days after the cells have reached 100% confluence, inducing medium was added. Two to five days later, the cells were changed to insulin only medium. Two to five days after induction, the cells were returned to regular medium for two days, completing i he process of 3T3L1 differentiation to adipocytes. b. Differentiation of Human PreacSpocytes
Human preadipocytes purchased from Camb.'ex were seeded in a 24-welI plate at ixlO6 cells/plate. After two days, when the cells reached 100% confluence, inducing medium purchased from Cambrex was added. The cells were cultured in inducing medium for ten days, thereby completing the process of
differentiation of primary human preadipocytes to adipocytes.
i
EXAMPLE 13 ,
DIFFERENTIATION OF HUMAN SKELETAL MUSCLE CELLS
Human primary undifferentiated skeletal muscle cells cultured in SKGM-2 medium purchased from Cambrex. When the skeletal myoblast culture achieved 50-70% confluence, the SKGM-2 medium was removed, and fusion medium (DMEM-F12 suppleme nted with 2% horse serum) was added.
Culture of the cells in the fusion medium Lvas continued for 4-7 days (with replacement of the fusion medium every other day) or until myotubes wi ;re observed throughout the culture.
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The resulting differentiated c ultures were observed to contain multinucleated (more than 3 nuclei) myotubes
If the myotubes were to be i sed in assays that required an extended period of time in culture, the fusion medium was removed and replaced with SKGM-2 medium. For best performance, the SKGM-2 medium was replaced every other day to maintain the culture for 2-3 weeks Myotube cultures were best used by 2 weeks post differentiation.
EXAMPLE 14
Endogenous RUP43 Couples to Gs
Gs coupling by RUP43 was interrogated by comparing the intracellular level of cAMP in HEK293 cells transfected with endogenous :auman, mouse, or rat RUP43 with mock-transfected HEK293 cells ("pCMV") Determination of intraceUular cAMP level was carried out by cyclase assay, using the Perkin Elmer Flashplate Kit (SMP004B) with 125I as the tracer (NEX130) essentially as per manufacturer's instructions.
HEK 293 cells were plated at a density of 1.2xlO7 and allowed to adhere overnight The HEK293
ceils were then transfected with pCMV alone or with pCMV containing polynucleotide encoding
endogenous human, mouse, or rat R.XJP43, using lipofectamine (120ug per 15cm dish). The transfected
cells were allowed to recover overnight. For the assay, the transfected cells were harvested and added to a
flashplate well at a final cell count of lxlO6 cells. They were.allowed to adhere, men subjected to the tracer
for two hours. All the wells were then aspirated and the plate was read using the microplate reader (Wallac
1450 microbeta counter). It was found that the intracellular level of RUP43-transfected HEK293 cells was
significantly greater than that of mock-tiansfected cells, indicating that KUP43 manifests a detectable level
of constitutive Gs coupling (Figure 3). EXAMPLE 15
Identification of Compound 1 as,an Agonist of RUP43
Materials
HEK 293 cells obtained fr )m ATCC were used for all assays. Culture media consisted of 90mls of DMEM supplemented with 10% fetal bovine serum (Gibco, BRL). Cyclic AMP measurements were determined using the Adenylyl Cyclase Activation Flashplate® Assay with direct cAMP [i25I\ Detection
system
Transient Transfection a, td JVliole-cell Cyclase Flashplate Assay
HEK 293 cells (5 x 105 ctlls/ml) were plated in 15cm dishes. The next day, cells were transfected using FuGENE 6 reagent (Roche Applied Science) as manufacturer suggested. Briefly, transfection mixture consisting of OptuMEM (Gibco, BRL) and FuGENE 6 reagent were mixed together and allow to incubate for 5 minutes at room tempera1 ure Transfection reagent was added drop-wise into a separate tube containing 2ug of endogenous human RUP43 receptor plasmid (Transfected) or 2 og of empty pCMV
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vector {Mock) and allowed to incubate for 15 minutes at room temperature The DNA/transfection mixture was added drop-wise to each perspective plate anc incubated over night in a humidified incubator maintained at 37°C, 95/5% O2I On Day 3, the cells were rinsed once with PBS and dislodged from trie plate using a nonenzymatic cell-dissociation buffer. (GibcosBRL) and resuspended in assay stimulation buffer® (Man Elmer) at a density of 2X105 cell/ml for measurement of cAMP, Compound 1 or vehicle was serially diluted in stimulation buffer at 2X the desired final concentration. Compound 1 and vehicle (50 uXVwell) were added to the perspective wells of a 96-well Flashplate® (Perkm Elmer). Transfected or Mock cells were aliquot to each well (50 uL/well) and allowed to incubate at rcW temperature on a plate form shaker for 1 hour. Detection buffer® (Perkm Elmer, 100 uL) was added to each well and incubated for 2 hours at room temperature with mild agitation. At the end of the 2 hour incubation, the plate was aspirated and cAMP levels were determined using Wallac 1450 microbeta counter.
It was found that Compound 1, in a dose dependent manner, led to an increase in lntracellular cAMP specifically in HEK293 cells transfected w;th endogenous human RUP43 and not in Mock transfected cells, identifying Compound 1 to be an agonist of RUP43 {Figure 4).
EXAMPLE 16
Identification of Compound 2 as an Agonist of RUP43
Using melanophore technology (Example 10,l supra), Compound 2 was found to be an agonist of endogenous human RUP43 {Figure 5). Briefly, melanophore cells were harvested from confluent flasks (T-185 cm3 flask) using Trypsin (0.7X), and transacted by electroporation. Polynucleotide encoding endogenous human RUP43 (30ug) was used for transfection of melanophores. After electroporation, cells were preplated in flasks approximately 3-4 hours to r.d of non-viable cells and debris Upon completion, flasks were subsequently trypsimzed and plated in triplicate onto 384 well poly-D-lysine coated plates for assay. Forty-eight hours post-transfection, assay plates were read in a spectrophotometer (absorbance To) Cells were then incubated for one hour with serially diluted Compound #2 (lOOuM-Sl^pM, 5-fold dilutions, 0.5% DMSO final) and read again (absorblnce Tfi0) Triplicate absorbance data were analyzed and depicted as percent control response as compared to positive control wells (200) and negative control wells (100). Curve height was approximately 92% of control with an EC50=0.212uM.
EXAMPLE 17
In Vitro Glucose Uptake Assay
The in vitro glucose uptake assay was carried out as described here.
Buffers and Reagents;
Starvation medium: DMEM/high glucose with 0.5% BSA.
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lmMMgS04 •lmM CaCl2
136mMNaCl 4.7mM KC1 1%BSA
2-Deoxyglucose (DOG): Sto± lOOmM: 16,4mg/ml in water (store at 4 C 1-2 weeks).
For each well, add 1 ul containing 1 uCi [3H]-2-DOG and 1 ul cold stock 2-DOG and 2ml KRPH
CytochalasinB (CytoB): StJck(10niMin95%ethanol): Keepat-20°C.
Use CytoB at lOuM final to (block carrier-mediated uptake. Also use this concentration at the end to stop the reaction. The stop buffer is PBS plus lOuM CytoB ("PBS")
l%Triton-X: This is the sohbilization buffer.
i Cells are plated in 24-well pi ites.
Procedure:
1. Starve cells for at le ist 2 hrs
2. Wash cells 2 times with KRPH buffer and add 2 mis of KRPH to the well.
3. Treat cells with insi lin and/or with test compound, or with vehicle (Control), for 20 mm.
4. After 20 min, aspirate the buffer from the well and immediately add lml of KRPH buffer
plus 2-DOG. For CytoB-treated cells, add CytoB 5 min before uptake assay. ;
5. After 4 min, aspirat; the buffer from the well and add 3 mis of cold PBS. After completing
the assay, wash fhe cells in each well 2 times with cold PBS. Aspirate the Stop PBS completely, and then
add 700ul of 1% Triton X. Place in 37°C incubator for 30 min. ;
6. Count CPM in total lysate. (Calculate CPM/well. ,
I
Subtract the CytoB value from the value obtained for each of cells treated with insulin and/or with test compound and cells treated with vehicle (Control).
EXAMPLE 18
Compound 2 Stimulates Glucose Uptake in Mouse 3T3L1 Adipocytes
Differentiated mouse 3T3IL1 adipocytes were treated with 50uM Compound 2 for various times, after which glucose uptake was determined according to Example 17. From Figure 6, it is apparent that
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Compound 2 stimulated glucose uptake in 3T3L1 adif ocytes. The results mdicate that RUP43 agonist is an attractive candidate for modulating glucose level in Kyperglycemia that insulin fads to control The rapid tune course of the stimulated glucose uptake suggt sts that RUP43 agonist may provide a more rapid therapeutic effect than do currently available drugs for lowering'blood glucose concentration
Example 19
Compound 2 Enhances Insulin-Stimulated Glucose Uptake in Mouse 3T3L1 Adipocytes
Differentiated mouse 3T3L1 adipocytes were treated with ("Compound 2") or without ("Control")
Compound 2 in serum-free medium for 3 hr. The 3T3L1 cells were then washed with fresh KRPH buffer
twice and treated with various concentrations of insulin for 20 min. After treatment with insulin, glucose
uptake was determined according to Example 17. From Figure 7, it is apparent that Compound 2 enhanced
insulin-stimulated glucose uptake in 3T3L1' adipocytes. The results indicate that RUP43 agonist can
increase insulin efficacy, thereby lowering the concen ration of insulin required to achieve maximal glucose
uptake (
EXAMPLE 20
Compound 2 Stimulates Glucose Uptake in Human Primary Human Adipocytes
Human preadipocytes (Cambrex) were differentiated into adipocytes. The differentiated primary human adipocytes were treated with or without 50(iM Compound 2 for 3 hr in serum-free medium The human adipocytes were then washed with fresh KRPH buffer twice and treated with or without lOOnM insulin for 20 min. After treatment with or without insulin, glucose uptake was determined according to Example 17. From Figure\8, it is apparent that Compound 2 stimulated glucose uptake in primary human adipocytes. From Figure 8 it is also apparent that Conpound 2 enhanced insuhn-stunulated glucose uptake in primary human adipocytes. Significantly,1 as was observed for the mouse 3T3L1 cells, RUP43 agonist can stimulate glucose uptake in primary human ad pocytes in the absence of insulin, and the level of RUP43-stunuIated glucose uptake is comparable to th; level of .insulin-stimulated glucose uptake.
EXAMPLE 21
Compound 2 Stimulates Glucose Uptake ir Rat L6 Myoblast Cells
Rat skeletal muscle L6 myoblast cells were obtained from ATCC and grown in 24-weIl plates to
confluence. !
a.
Confluent L6 cells were treated with or without various concentrations of Compound 2 in serum-free medium for 3 hr. The L6 cells were then washe d twice with KRPH buffer. L6 cells which had been treated with Compound 2 were incubated with KRPH buffer for 20 min; L6 cells which had not been treated with Compound 2 were treated with lOnM or lOOnM insulin for 20 min. After treatment with or without insulin, glucose uptake was determined according to Example 17. From Figure 9A, it is apparent that
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Compound 2 stimulated glucose uptzke in rat L6 myoblast cells. RUP43 agonist stimulated greater glucose uptake in rat L6 myoblast ¦than did insulin As skeletal muscle cells are responsible for 80% of glucose disposal in vivo, the results obtained indicate that RUP43 agonist is an attractive candidate for providing better glucose disposal in vivo than does insulin
Confluent L6 myoblast ceUd were treated with 50uM Compound 2 for various times. At the end of each treatment period, glucose uptake was determined according to Example 17. The results indicate that RUP43 agonist can stimulate glucose uptake in skeletal muscle cells within 20 min, a timeirame similar to that of insulin (Figure 9B). The results indicate that RUP43 agonist is an attractive candidate for regulating glucose level in vivo within a short pWiod of time comparable to that of insulin.
EXAMPLE 22
Compound 2 Enhances Injsulin-Stimulated Glucose Uptake in Rat L6 Myoblast Cells Confluent rat L6 myoblast j;ells were treated with or without 50uM Compound 2 for 3 hr in serum-free medium. The L6 cells were men washed twice with fresh KRPH buffer. The L6 cells were then treated with or without lOOnM insulin fan 20 mm. After treatment with or without insulin, glucose uptake was determined according to Example 17. From Figure 10, it is apparent that, analogous to what was observed for adipocytes, Compound 2 enhances insulin-stimulated glucose uptake in rat L6 myoblast cells. The results further indicate that RUP43 agonist can increase insulin efficacy, thereby lowering the concentration of insulin required to achieve maximal glucose uptake. RUP43 therefore represents an attractive therapeutic option for an individual suffering fijom hyperinsulinenna-caused problems relating to insulin resistance.
EXAMPLE 23
Compound 2 Stimulates Glucose Uptake in Primary Human Skeletal Muscle Cells
Primary human skeletal muscle cells obtained from Cambrex were grown to 50% confluence and then differentiated on culture witi inducing medium for 5 to 7 days. The differentiated primary human skeletal muscle cells were then transferred to growth medium for 7 to 10 days.
Differentiated human skeletal muscle cells were treated with or without various concentrations of Compound 2 in serum-free mediurn for 3 hr. After treatment with or without Compound 2, the cells were washed twice with fresh KRPH buffer. Cells which had been treated with Compound 2 were then incubated with KBPH buffer for 20 min; cells which had not been treated with Compound 2 were incubated with lOnM or lOOnM insulin for 20 nun. After treatment with or without insulin, glucose uptake was determined according to Example 17. From Figure 11A, it is apparent that RUP43 agonist can regulate glucose uptake in human skeletal muscle cells in the absence of insulin and more effectively man insulin. The lesu-lts obtained indicate that R.UP43 agonist is efficacious at stimulating glucose uptake in human skeletal muscle
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cells, where 80% of glucose'disposal takes place Th: results obtained indicate that RUP43 agonist is an attractive candidate for controlling glucose level in hyp irglycemia refractory to insulin action
b_
Differentiated human skeletal muscle cells WE re treated with 50uM Compound 2 for various time periods At the end of each treatment period, glucose uptake was determined according to Example 17 The results obtained are presented in Figure 11B. The rapid stimulation of glucose uptake in human skeletal
muscle cells observed for RUP43 agonist indicate RUT 43 agonist to be an attractive candidate for regulating
i
glucose level in vrvo directly and within a short period of time.
EXAMPLE 24 > Oral Bioavailability
Physicochemico analytical approaches for dir ictly assessing oral bioavailability are well known to those of ordinary skill in the art and may be used [see e.g., without limitation: Wong PC et al., Cardiovasc Drug Rev'(2002) 20-137-52; and Buchan P et al, Headache (2002) Suppl 2:SS4~62; the disclosure of each of which is hereby incorporated by reference in its entirety] By way of further illustration and not limitation, said alternative' analytical approaches may comprise liquid chromatography-tandem mass spectrometry [Chavez-Eng CM et al., J ChromatogrB Analyt Techno! Biomed Life Sci (2002) 767:117-29; Jerter A et al., Chn Pharmacol Ther (2002) 71:21-9; Zimmerman JJ et al., J Clin Pharmacol (1999) 39:1155-61; and Barrish A et al, Rapid Commun Mass Spectrum (1996) 10:1033-7; the disclosure of each of which is hereby incorporated by reference in its entirety]. Recently, positron emission tomography (PET) has been successfully used to obtain direct measurements of drug distribution, including oral bioavailability, in the mammalian body following oral administration of the drug [Gulyas et al., Eur J Nucl Med Mol Imaging (2002) 29:1031-8, the disclosure of which is hereby incorporated by reference m its entirety].
Alternatively, based upon the in vh>o data dei 'eloped, as for example by way of illustration and not limitation, through the mouse model of Example 26. The modulator is administered by oral gavage at doses ranging from 0.1 rng kg'1 to 100 mg kg"1. The effect of the modulator is shown to be dose-dependent and comparable to the effect after intraperitoneal administration, wherein the effect is reduction of blood glucose concentration (Example 26). The dose of modulator required to achieve half-maximal reduction of beneficial effect through oral administration is compared to the dose of modulator required to achieve half-maximal reduction of beneficial effect Ihrough intraperitoneal administration. By way of illustration, if said oral dose is twice said intraperitoneal dose, then the oral bioavailabilty of the modulator is taken to be 50%. More generally, if said oral dose is 9 mg kg"1 and said intraperitoneal dose is p mg kg'1, then the oral bioavailability of the modulator as a percentage is taki:n to be [(p/0) x 100].
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EXAMPLE 25
Blood Brain Barrier Model
The ability of a compound of the invention to cross the blood-brain barrier can be determined using brain-derived cells One method thai is envisioned, by way of illustration and not limitation, is to use the blood/brain barrier model of Dehouck et al [J Neurochem (1990) 54.1798-801; hereby incorporated by reference in its entirety] thatuses a co-culture of brain capillary endothelial cells and astrocytes.
Bovine capillary endothelial iBBCE) cells are isolated and characterized as described by Meresse et al. [J Neurochem (1989) 53:1363-13 71; hereby incorporated by reference in its entirety]. In brief, after isolation by mechanical homogenization from one hemisphere of bovine brain, microvessels are seeded onto dishes coated with an extracellular matrix secreted by bovine corneal endothelial cells. Five days after seeding, the first endothelial cells migrate out from the capillaries and begin to form microcolonies. "When the colonies are sufficiently large, th; five largest islands are trypsinized and seeded onto 35-mnvdiameter gelatin-coated dishes (one clone p:r dish) in the presence of Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% cjalf serum (Seromed), 3 mM glutamine, 50 ug/ml of gentamicin, 2.5 ug/ml of amphotericin B (Fungizoneh, and bovine fibroblast growth factor (1 ng/ml added every other day). Endothelial cells from one 35-rnmjdiameter dish are harvested at confluence and seeded onto 60-mm-diameter gelatin-coated dishes. After 6-8 days, confluent cells are subcultured at the split ratio of 1:20. Cells at the third passage (-100 dishus) are stored in liquid nitrogen.
Primary cultures of astrocytjs are made from newborn rat cerebral cortex. After the meninges have been cleaned off, the brain tissue is forced gently through a nylon sieve as described by Booher and Sensenbrenner [Neurobiology (197i) 2:97-105; hereby incorporated by reference in its entirety]. DMEM supplemented with 10% fetal calf serum (Seromed), 2 mM glutamine, and 50 u.g/ml of gentamicin is used for the dissociation of cerebral tissue and development of astrocytes.
Culture plate inserts (Millieell-CM; pore size, 0.4 jiM; diameter, 30 mm; Millipore) are coated on both sides with rat tail collagen prepared by a modification of the method of Bomstein [Lab Invest (1958) 7:134-139; hereby incorporated by eference in its entirety].
Astrocytes are plated at a concentration of 2 5 x 105 cells/ml on the bottom side using the filter upside down. After S days, filters ere properly positioned, and the medium is changed twice a week. Three weeks after seeding, cultures of as xocytes.become stabilized. Then, BBCE cells, frozen at passage 3, are recultured on a 60-mm-diameter gelatin-coated dish. Confluent cells are trypsinized and plated on the upper side of the filtures at a concentration of 4 x 105 cells. The medium used for the coculture is DMEM supplemented with 15% calf serum 2 mM glutamine, 50 ;ug/ml of gentamicin, and 1 ng/ml of bovine fibroblast growth factor added every other day. Under these conditions, BBCE cells form a confluent monolayer in S days.
Culture plates are set into six-well plates with 2 ml iof buffer added to me upper chamber and 2 ml added to the plate containing the inserts. The six-well plates are placed in a shaking water bath at 37°C. The compound of the invention is added to the upper chamber, and 100 u,l is removed from the lower
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chamber at various tune points. In certain embodir tents, the test compound is radiolabeled. In certain embodiments, the radiolabel is 3H or I4C. In some em bodiments, the final time point is about 20 mm, about 30 mm, about 40 min, about 50 mm, about 60 nun, about 70 nun, about 80 nun or about 90 mm The percentage of total test compound present in the lower chamber at the various tune points is determined. Leucine is used as a permeability positive control. Inn kn is used as a permeability negative control.
In certain embodiments, a determination of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90% of the compound of the invention in the lower chamber at the final time point is indicative of the compound of the invention being able to cross tie blood-brain barrier.
EXAMPLE 26
IN VIVO EFFECTS OF RUP43 AGONISTS ON I GLUCOSE HOMEOSTASIS IN RATS
A. Oral Glucose Tolerance Test (oGTT) in Rats.
Male Zucker diabetic fatty (ZDF) rats (Charles River) at age of 10 weeks are fasted for 18 hours and randomly grouped (n=ll) to receive a RUP43 Egonist at various doses, or with control rosiglitazone (RSG, lOmg/kg) known to increase insulin sensitivity The RUP43 agonist is delivered intraperitoneally. RSG is delivered intraperitoneally. A preferred dose of RUP43 agonist is 0.1-100 mg/kg. Other preferred dose is selected from the group consisting of: 0 1 mg/kg, 0 3 mg/kg, 1.0 mg/kg, 3.0 mg/kg, 10 mg/kg, 30 mg/kg and 100 mg/kg. The placebo group is administered vehicle.
Thirty minutes after administration of test co npound and control RSG, rats are administered orally with dextrose at 2 g/kg dose. Levels of blood glucose are determined at various time points using Glucometer Elite XL (Bayer). Taking the time of d ;xtrose administration to be "0 min", exemplary time points are -30 mm, 0 min, 30 rnin, 60 min, 90 mil and 120 min. The mean glucose concentration is averaged from eleven animals in each treatment group. These results can demonstrate that RUP43 agonist lowers blood glucose in a dose-dependent manner in rats after challenge with glucose.
Alternatively, the oral glucose tolerance test as described here is carried out in the rats immediately following seven daily injections of RUP43 agonist, RSG, or vehicle.
It is expressly contemplated that the oral glucose tolerance test described here may also be carried out in a different animal, for example in mouse or in r abbit.
B. Acute Response of ZDF Rats to RUP43 Agonist
Male Zucker diabetic fatty (ZDF) rats (Cha les River) at age of 10 weeks are randomly grouped (n=6) to receive vehicle (intraperitoneally), RUP43 agonist (intraperitoneally), or rosightazone (RSG, lOmg/kg, intraperitoneally).! A preferred dose of RIP43 agonist is 0.1-100 mg/kg. Other preferred dose is selected from the group consisting of: 0.1 mg/kg, 0 3 mg/kg, 1.0 mg/kg, 3.0 mg/kg, 10 mg/kg, 30 mg/kg and 100 mg/kg. After compound administration, food is removed and blood glucose levels are determined at various times. Exemplary times of glucose level determination are 0 hr, 1 hr, 2 hr, 3 hr and 4 hr, and then daily for up to a week. Reduction in blood glucose a. each time point is expressed as percentage of original
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glucose levels, averaged from six animals for each group. These animals have blood glucose levels (fed state) of 300-400 mg/dl, significantly higher than non-diabetic ,wild type animals. Treatment with R.UP43 agonist or RSG can be shown to significantly reduce glucose levels compared to vehicle control. These data can demonstrate that RUP43 agonist t as efficacy in improving glucose homeostasis in diabetic animals.
Alternatively, the rats are rrjected daily for seven days with R.UP43 agonist, RSG, or vehicle immediately before blood glucose level is determined daily for seven days.
It is expressly contemplated ihat the acute response test described here may also be carried out in a different animal, for example in mouse or in rabbit.
EXAMPLE 27
Synthesis of Compounds oji the Invention
Example 27A: 2-{l-[2-(2-i^hIoro-phenyl)-acetyl]-piperidin-4-yl}-thiazole-4-carboxylic acid
methyl (2-m ethy 1-4,5,6,7-tetrahy d ro-2H-indazol-3-yl)-amide
2-Pipendm-4'yl~thiazole-4~carboxylic acid ethyl ester dihydrobromide salts A solution of tert-butyl^(aminocarbothioyl)tetrahydropyridine-l(2H)-carboxylate (2,0 g, 8.2 mmol) and ethyl bromopyruvate (1.6 g, 8.2 mmol) in 30 mL of EtOH was stirred at 80°C for 4h. Afterwards, the mixture cooled to room temperature and then charged with. 48% HBr (1.0 mL, 14 mmol). The reaction mixture was allowed to stir an additional lh, and then concentrated to an oily solid. Tnturation with diethyl ether afforded 3.0 g (S'1%) of,a tan solid: lH NMR (400 MHz, DMSO-d*) 5 9.02 (br s, 1 H), 8.77 (br s, 1H), 8.46 (s, 1H), 7.01 fcr s, 1 H), 4.29 (q, J = 7.1 Hz, 2 H), 3.44-3.33 (m, 3 H), 3.02 (q, J = 11.7 Hz, 2 H)> 2.19 (d, J = 13.2 Hz, 2 H), 1.97-1 88 (m, 2 H), 1.29 (t, J - 7.0 Hz, 3 H). MS calculated for CnHi6N2O2S+H: 241, observed: 2^-1.
2-{l-[2-(2-ChIoro-phenylj-acetyl]-piperidin-4-yl}-thiazole-4-carbox)>lic acid ethyl ester A solution of 2-piperidin4-yl-thiazole-4-carboxylic acid ethyl ester dihydrobromide salts (1.0 g, 3.2 mmol), 2-chlorophenyl acetic acid (0.55 g, 3.2 mmol), O-(7-azabenzotna2ol-l-yl)-N,NsN'JN1-tetramefcyluromum hexafluorophosphate ,(1.4 g, 3.5 mmol), and diiospropylethylamine (3 0 mL, 17 mmol) in 30 mL of CH2CI2 was stirred at 40"C for 8h. Afterwards, the crude mixture was diluted with 30 mL of CH2C12 and washed with 1 M citric acid (3 X 50 mL)s saturated aqueous NaHCO3 (1 X 30 mL)a and saturated aqueous NaCl (1 X 30 mL). The resulting organic layer was dried over MgSO,*, filtered, and concentrated to a brown oil Purilfication on silica gel with EtOAc:hexanes (3:1) afforded 0 94 g (75%) of a light brown oil: ]H NMR (400 MHz, CDC13) 8 8.08 (s, 1 H), 7.39 (dd, J = 7.6,1.6 Hz, 1 H), 7.32 (dd, J =
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WO 2005/116C53 PCT/US2005/012447
7.2,2.0 Hz, 1 H), 7 25-7.19 (m, 2 H), 4 76 (appar d, 1 H), 4 42 (q, J = 7 2 Hz, 2 H), 3.99-3.95 (m, 1 H), 3.88 (d, ABpattern, J^ = 16.0 Hz, 1 H), 3.83 (d, ABpattern, J^ = 16.0 Hz, 1 H), 3.34 (tt, J = U.7,3.7 Hz, 1 H), 3.21-3 14 (m, 1 H), 2.83-2.76 (m, 1 H), 2 20-2 16 (m, 2 H), 1 74 (qd, J = 12 3, 4.1 Hz, I H), 1.63 (qd, J =
12 3,4.0 Hz, 1 H), 1 40 (t, J = 7.2 Hz, 3 H) MS calculated for C19H2,C1N2O3S+H: 393, observed: 393
i
2-{l-[2-(2-Chloro-p'henyl)-acetyl]-piperidin-4yl}-thiazole-4-carboxylicacid A solution of 2-{I-[2-(2-chloro-phenyI)-ac£tyi]-pipendin-4-yl}-thiazoIe-4-carboxyIic acid ethyl ester (0.94 g, 2 4 mmol) in. 20 mL of MeOH was d luted with 1 M NaOH (20 mL, 20 mmol) and was allowed to stir at 60°C for'4h Afterwards, the crude mixture was concentrated to remove the MeOH solvent The aqueous basic solution was then washed with CH2C12 (2 X 25 mL) and acidified with 5 M HC1 to pH = I. The resulting aqueous acidic solution WE.S extracted with CH2C12 (3 X 25 mL). The organic layeis were combined, dried over MgSO,j, filtered, and concentrated to afford 0.51 g (58%) of a white foamy solid: !H NMR (400'MHz, DMSO-ds) 5 12 9(i (br s, 1 H), 8.36 (s, 1 H), 7.45-7.40 (m, 1 H), 7.33-7.25 (m, 3 H), 4.43 (d, J = 13.2 Hz, 1 H), 4.06 (d, J = 13.6 Hz, 1 H), 3 87 (d, AB pattern, Jm = 16.0 Hz, 1 H), 3 82 (d, AB pattern, 3m = 16.0 Hz, 1 H), 3.38-3.31 (m, 1 H), 325 (appar t, J = 11.8 Hz, 1 H), 2.79 (appar t3 J = 11.6 Hz, 1 H), 2.08 (t, J = 10.6 Hz, 2 H), 1 67 (qd, J = 12.1,3.7 Hz, 1 H), 1.53 (qd, J = 12.2,4 0 Hz, 1 H). MS calculated for C17H,7C1N2O3S4-H: 365, observed: 365.
2-{l-[2-(2-Chloro-phenyl)-acetyl]-piperidin-^-yl}-thiazole-4-carboxylic acid methyl-(2-methyI-4,5,6]7-tetrahydro-2H-indazol-3-yl)-amide dihydrochl ?nde salts
A solution of N,l;dimethy 1-4,5,6,7-tetrahydro-lH-indazol-3-amine (23 mg, 0.14 mmol), O-(7-azaben2otriazol-l-yl)-N)N,lSl',Nt-tetramethyiuronium iiexafluorophosphate (75 rng, 0.20 mmol), and 2-{l-[2-(2-Ch]oro-phenyl)-acetyl]-pipendin-4-yI}-thiazoIe-4-carboxylic acid (50 mg, 0.14 mmol) in 10 mL of CH2C12 was stirred at 40cC for 8h Afterwards, the crude mixture was diluted with,20 mL of CH2C12 and washed with 1 M citric acid (3 X 30 mL), saturated aqueous NaHCO3 (1 X 30 mL), and saturated aqueous NaCl (1 X 30 mL). The resulting organic layer was dried over MgSO4, filtered, and concentrated to a yeflow oil. Purification by gradient HPLC (act:tonitrile-water with 0.1%TFA) and converting to dihydrochloride salts afforded 56 mg (70%) of a whte solid: lH NMR (400 MHz, CD3OD) S 8.28 (d, J = 8.8 Hz, 1 H), 7.43-7.41 (m, 1 H), 7.30-7.26 (m, 3 H) 4.50 (t,J= 11.8 Hz, 1 H), 4.09-4.03 (m, 1 H), 3.98-3.91 (m, 2 H), 3 83 (d, J = 12.8 Hz, 3 H), 3.37 (s, 3 H), 3.24-3.20 (m, 1 H), 2.89-2.82 (m, 1 H), 2 83-2 66 (m, 2 H), 2.47-2.41 (m 1 H), 2.03-1 88 (m, 4 H), 1.72-1 60 (m, 3 H), 1.46-1.26 (m, 3 H) MS calculated for C26H3oCIN502S+H: 512} observed: 512.
Example 27B: 2-(2-Chloro-phenyl)-l-{4-|4-(3,4-dihydro-2H-quinoIine-l-carbonyI)-thiazoI-2-
yll- ! ¦
piperidin-l-yl}-ethanone
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WO 2005/116653 :PCT/US2005/012447
By the same general procedure as in Example 29A, 2-(2-Chloro-phecyl)-l-{4-[4-(3,4-djhydro-2H-quinoline-l-carbonyl)-thiazol-2-yl]-piperidin-l-yl}-ethanone was obtained from 1,2,3,4-tetrahydroquinoline as a yellow solid. 'H NMR (400 MHz, CD3OD) 5 7.78 (s, 1 H), 7.42-7 40 (m, 1 H), 7.30-7.24 (m, 3 H), 7.18 ( Example 27C: 2-(l-[2-f2-Fluoro-phenvl1-acetvn-Diperidin-4-vII-thiazole-4-carbQxvlic acid methyI-(2-methyl-4,S,6,7-Ttetrahydro-2H-indazo]-3-yl)-aroide
By the same general procedure as in Example 29A, 2-{l-[2-(2-fl.uoro-phenyl)-acetyl]-piperidin-4-yl}-thiazole-4-carboxylic acid methyl-(2-methyl-4,5,6,7-tetraliydro-2H-indazol-3-yl)-aniide was obtained from 2-{l-[2-(2-fluoro-phenyl)-acetyl]-pipendin-4-yl}-thiazole-4-carboxylic acid as a white solid. JH MMR (400 MHz, CDC13) 5 7.71 (i J = 10.4 Hz, 1 H), 7.30 (t, J = 7.6 Hz, 1H), 7.26-7 22 (m, 1 H), 7.11 (t, J = 7.4 Hz, 1 H), 7.05 (t, J = 9.0 Hz: 1 H), 4 47 (appar t, J = 13.6 Hz, 1 H), 3.90-3 79 (m, 1 H), 3.74 (s, 2 H), 3.63 (d, J = 4.4 Hz, 3 H), 3.32 (s, 3 H), 3.17-3.11 (m, 1 H), 3.04-2.95 (m, 1 H), 2.91-2.79 (m, 1 H), 2.61-2 43 (m, 2 H)3 2.27 (dt, J = 15.3, 5.7 Hz, 1 H), 1.99-1.8S (ra, 3 H), 1.79-1.72 (m, 1 H), 1.64-1.38 (m, 5 H). MS calculated for C2&H3oFN5OzS+H: 496, observed: 496.
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I/We Claim:
1. An in vitro method of identifying one or more candidate compounds as a
modulator of glucose uptake in adipocytes or skeletal muscle cells in a
mammal, said method comprising the steps of:
(a) contacting the one or more candidate compounds with a RUP43 GPCR,
wherein the receptor couples to a G protein, said receptor comprising a
GPR131 amino acid sequence selected from the group consisting of:
(i) the amino acid sequence of SEQ ID NO:2;
(ii) amino acids 2-330 of SEQ ID NO:2;
(iii) amino acids 2-330 of SEQ ID NO:2, with the proviso that the
RUP43 G protein-coupled receptor does not comprise the
methionine residue at amino acid position 1 of SEQ ID NO:2;
(iv) the amino acid sequence of (i), (ii) or (iii) wherein the alanine at
amino acid position 223 of SEQ ID NO:2 is substituted with
lysine;
(v) the amino acid sequence of a G protein-coupled receptor encoded
by a polynucleotide that is amplifiable by polymerase chain
reaction (PCR) on a human DNA sample using primers SEQ ID
NO:3 and SEQ ID NO:4;
(vi) the amino acid sequence of SEQ ID NO:6;
(vii) the amino acid sequence of a G protein-coupled receptor encoded
by a polynucleotide that is amplifiable by polymerase chain
reaction (PCR) on a human DNA sample using primers SEQ ID
NO:7 and SEQ ID NO:8;
(viii) the amino acid sequence of a G protein-coupled receptor having an
amino acid sequence derived from SEQ ID NO:2 by substitution,
deletion or addition of one or several amino acids in the amino acid
sequence of SEQ ID NO:2;

(ix) the amino acid sequence of a G protein-coupled receptor encoded
by a polynucleotide that hybridizes under stringent conditions to
the complement of SEQ ID NO : 1;
(x) the amino acid sequence of a G protein-coupled receptor having an
amino acid sequence having at least 75% identity to SEQ ID NO:2;
and
(xi) the amino acid sequence of an allele or mammalian ortholog of
human RUP43 GPCR having the amino acid sequence of SEQ ID
NO:2; and
(b) determining whether the receptor functionality is inhibited or stimulated;
wherein inhibition or stimulation of said receptor functionality is indicative of the
candidate compound being a modulator of glucose uptake in adipocytes or skeletal
muscle cells in a mammal.
2. The method of claim 1, wherein an increase in receptor functionality is indicative
of the candidate compound being a compound that increases glucose uptake in adipocytes
or skeletal muscle cells in a mammal.
3. An in vitro method of identifying one or more candidate compounds as a
modulator of blood glucose concentration in a mammal, said method comprising the
steps of:
(a) contacting the one or more candidate compounds with a RUP43 GPCR,
wherein the receptor couples to a G protein, said receptor comprising a GPR131
amino acid sequence selected from the group consisting of:
(i) the amino acid sequence of SEQ ID NO:2;
(ii) amino acids 2-330 of SEQ ID NO:2;
(iii) amino acids 2-330 of SEQ ID NO:2, with the proviso that the
RUP43 G protein-coupled receptor does not comprise the
methionine residue at amino acid position 1 of SEQ ID NO:2;
(iv) the amino acid sequence of (i), (ii) or (iii) wherein the alanine at
amino acid position 223 of SEQ ID NO:2 is substituted with
lysine;

(v) the amino acid sequence of a G protein-coupled receptor encoded
by a polynucleotide that is amplifiable by polymerase chain
reaction (PCR) on a human DNA sample using primers SEQ ID
NO:3 and SEQ ID NO:4;
(vi) the amino acid sequence of SEQ ID NO:6;
(vii) the amino acid sequence of a G protein-coupled receptor encoded
by a polynucleotide that is amplifiable by polymerase chain
reaction (PCR) on a human DNA sample using primers SEQ ID
NO:7 and SEQ ID NO:8;
(viii) the amino acid sequence of a G protein-coupled receptor having an
amino acid sequence derived from SEQ ID NO:2 by substitution,
deletion or addition of one or several amino acids in the amino acid
sequence of SEQ ID NO:2;
(ix) the amino acid sequence of a G protein-coupled receptor encoded
by a polynucleotide that hybridizes under stringent conditions to
the complement of SEQ ID NO : 1;
(x) the amino acid sequence of a G protein-coupled receptor having an
amino acid sequence having at least 75% identity to SEQ ID NO:2;
and
(xi) the amino acid sequence of an allele or mammalian ortholog of
human RUP43 GPCR having the amino acid sequence of SEQ ID
NO:2; and
(b) determining whether the receptor functionality is inhibited or stimulated;
wherein inhibition or stimulation of said receptor functionality is indicative of the
candidate compound being a modulator of blood glucose concentration in a mammal.
4. The method according to claim 3, wherein an increase in receptor functionality is
indicative of the candidate compound being a compound that lowers blood glucose
concentration in a mammal.
5. The method of any one of claims 1 to 4, wherein the GPR131 amino acid
sequence is the amino acid sequence of SEQ ID NO: 2.

6. The method of any one of claims 1 to 4, wherein the GPR131 amino acid
sequence is the sequence of a G protein-coupled receptor having an amino acid sequence
having at least 75% identity to SEQ ID NO: 2.
7. The method of any one of claims 1 to 6, wherein said contacting comprises
contacting with a host cell or with membrane of a host cell that comprises recombinant
said RUP43 GPCR.
8. The method of claim 7, wherein the host cell is a mammalian cell.
9. The method of claim 8, wherein the mammalian cell is a 293 cell, a 293T cell, a
CHO cell, an MCB3901 cell, or a COS-7 cell.
10. The method of claim 7, wherein the host cell is a yeast cell.
11. The method of claim 7, wherein the host cell is a melanophore cell.
12. The method of any one of claims 1 to 11, wherein the candidate compounds are
screened as pharmaceutical agents for a metabolic disorder selected from the group
consisting of:

(a) diabetes;
(b) impaired glucose tolerance;
(c) insulin resistance; and
(d) hyperinsulinemia.
13. The method of any one of claims 1 to 11, wherein the candidate compounds are
screened as pharmaceutical agents for a complication of an elevated blood glucose
concentration, wherein the complication of an elevated blood glucose concentration is
selected from the group consisting of:
(a) Syndrome X;
(b) atherosclerosis;
(c) atheromatous disease;
(d) heart disease;
(e) hypertension;
(f) stroke;
(g) neuropathy;

(h) retinopathy;
(i) nephropathy; and
(j) peripheral vascular disease.
14. The method of any one of claims 1 to 11, wherein said contacting is carried out in
the presence of a known agonist of the RUP43 GPCR.
15. The method of claim 14, wherein said contacting is carried out in the presence of
a compound of Formula (II).
16. The method of claim 14, wherein said contacting is carried out in the presence of
2-{l-[2-(2-Chloro-phenyl)-acetyl]-piperidin-4-yl}-thiazole-4-carboxylic acid methyl-(2-
methyl-4,5,6,7-tetrahydro-2H-indazol-3-yl)-amide, 2-(2-Chloro-phenyl)-l-{4-[4-(3,4-
dihydro-2H-quinoline-l-carbonyl)-thiazol-2-yl]-piperidin-l-yl}-ethanone, or 2-{l-[2-(2-
Fluoro-phenyl)-acetyl]-piperidin-4-yl} -thiazole-4-carboxylic acid methyl-(2-methyl-
4,5,6,7-tetrahydro-2H-indazol-3-yl)-amide.
17. The method of any one of claims 1 to 11, wherein the method comprises
identifying an agonist, partial agonist, inverse agonist or antagonist of the receptor.
18. The method of claim 17, wherein the method further comprises formulating the
agonist, partial agonist, inverse agonist or antagonist as a pharmaceutical.
19. The method of any one of claims 1 to 13, wherein the method comprises
identifying an agonist or partial agonist of the receptor.
20. The method of claim 19, wherein the method further comprises formulating the
agonist or partial agonist as a pharmaceutical.
21. The method of any one of claims 1 to 20, wherein said determining is through the
measurement of the level of a second messenger selected from the group consisting of
cyclic AMP (cAMP), cyclic GMP (cGMP), isositol triphosphate (IP3), diacylglycerol
(DAG) and Ca2+.
22. The method of claim 21, wherein the intracellular level of cAMP is increased.
23. The method of any one of claims 1 to 20, wherein said determining comprises
detecting cAMP.

24. The method of any one of claims 1 to 21, wherein said determining is through the
use of a Melanophore assay, or through the measurement of GTPyS binding to a
membrane comprising the RUP43 GPCR.
25. The method of any one of claims 1 to 20, wherein the method further comprises
the step of comparing the modulation of the receptor caused by the candidate compound
to a second modulation of the receptor caused by contacting the receptor with a known
modulator of the receptor.
26. The method of any one of claims 1 to 25, wherein the candidate compound is not
a bile acid.
27. A process for making a modulator of a RUP43 GPCR, comprising the steps of:

(a) identifying said modulator according to the in vitro method of any one of
claims 1 to 26; and
(b) synthesizing the modulator identified in (a).
28. An in vitro method for identifying agonists and partial agonists of a G protein-
coupled receptor for use as pharmaceutical agents for a metabolic disorder selected from
the group consisting of diabetes, impaired glucose tolerance, insulin resistance, and
hyperinsulinemia, the method comprising contacting a candidate compound with a G
protein-coupled receptor comprising an amino acid sequence selected from the group
consisting of:
(a) the amino acid sequence of SEQ ID NO:2;
(b) amino acids 2-330 of SEQ ID NO:2;
(c) amino acids 2-330 of SEQ ID NO:2, with the proviso that the
RUP43 G protein-coupled receptor does not comprise the
methionine residue at amino acid position 1 of SEQ ID NO:2;
(d) the amino acid sequence of (i), (ii) or (iii) wherein the alanine at
amino acid position 223 of SEQ ID NO:2 is substituted with
lysine;
(e) the amino acid sequence of a G protein-coupled receptor encoded
by a polynucleotide that is amplifiable by polymerase chain

reaction (PCR) on a human DNA sample using primers SEQ ID
NO:3 and SEQ ID NO:4;
(f) the amino acid sequence of SEQ ID NO:6;
(g) the amino acid sequence of a G protein-coupled receptor encoded
by a polynucleotide that is amplifiable by polymerase chain
reaction (PCR) on a human DNA sample using primers SEQ ID
NO:7 and SEQ ID NO:8;
(h) the amino acid sequence of a G protein-coupled receptor having an
amino acid sequence derived from SEQ ID NO:2 by substitution,
deletion or addition of one or several amino acids in the amino acid
sequence of SEQ ID NO:2;
(i) the amino acid sequence of a G protein-coupled receptor encoded
by a polynucleotide that hybridizes under stringent conditions to
the complement of SEQ ID NO : 1;
(j) the amino acid sequence of a G protein-coupled receptor having an
amino acid sequence having at least 75% identity to SEQ ID NO:2;
and
(k) the amino acid sequence of an allele or mammalian ortholog of
human RUP43 GPCR having the amino acid sequence of SEQ ID
NO:2, and
determining whether receptor functionality is stimulated; wherein stimulation of said
receptor functionality is indicative of the candidate compound being an agonist or partial
agonist of said receptor.
29. A modulator identified according to the method of any one of claims 1 to 26 or
according to the method of claim 28.
30. A compound, wherein said compound is: 2-(2-Chloro-phenyl)-l-{4-[4-(3,4-
dihydro-2H-quinoline-l-carbonyl)-thiazol-2-yl]-piperidin-l-yl}-ethanone.
31. A compound of Formula (II):


or a pharmaceutically acceptable salt thereof,
wherein:
R1 is H or C1-6 alkyl;
R2 is a 2-methyl-4,5,6,7-tetrahydro-2H-indazol-3-yl group; or
R1 and R2 together with the nitrogen to which they are bonded form a 3,4-
dihydro-2H-quinoline-l-yl group;
and R10 and R11 are each independently H or halogen.
32. A compound according to claim 31, wherein said compound is 2-{ l-[2-(2-Chloro-
phenyl)-acetyl]-piperidin-4-yl}-thiazole-4-carboxylic acid methyl-(2-methyl-4,5,6,7-
tetrahydro-2H-indazol-3-yl)-amide, 2-(2-Chloro-phenyl)-l-{4-[4-(3,4-dihydro-2H-
quinoline-1 -carbonyl)-thiazol-2-yl]-piperidin-1 -yl} -ethanone, or 2-{ 1 -[2-(2-Fluoro-
phenyl)-acetyl]-piperidin-4-yl}-thiazole-4-carboxylic acid methyl-(2-methyl-4,5,6,7-
tetrahydro-2H-indazol-3-yl)-amide.
33. A method of preparing a pharmaceutical composition comprising admixing a
compound according to claim 31 or claim 32 and a pharmaceutically acceptable carrier.
34. A pharmaceutical composition comprising a compound according to claim 31 or
claim 32 and a pharmaceutically acceptable carrier.
35. An agonist of a mammalian RUP43 GPCR or a pharmaceutical composition
comprising the agonist and a pharmaceutically acceptable carrier for use in a method of
treatment of the human or animal body by therapy.

36. An agonist of a mammalian RUP43 GPCR or a pharmaceutical composition
comprising the agonist and a pharmaceutically acceptable carrier for use in a method of
lowering blood glucose concentration in the human or animal body by therapy.
37. An agonist of a mammalian RUP43 GPCR or a pharmaceutical composition
comprising the agonist and a pharmaceutically acceptable carrier for use in a method of
increasing glucose uptake in adipocytes or skeletal muscle cells in the human or animal
body by therapy.
38. An agonist of a mammalian RUP43 GPCR or a pharmaceutical composition
comprising the agonist and a pharmaceutically acceptable carrier for use in a method of
preventing or treating a metabolic disorder in a human or animal body by therapy,
wherein said metabolic disorder is selected from the group consisting of:

(a) diabetes;
(b) impaired glucose tolerance;
(c) insulin resistance; and
(d) hyperinsulinemia.
39. An agonist of a mammalian RUP43 GPCR or a pharmaceutical composition
comprising the agonist and a pharmaceutically acceptable carrier for use in a method of
preventing or treating a complication of an elevated blood glucose concentration in the
human or animal body by therapy, wherein the complication is selected from the group
consisting of:
(a) Syndrome X;
(b) atherosclerosis;
(c) atheromatous disease;
(d) heart disease;
(e) hypertension;
(f) stroke;
(g) neuropathy;
(h) retinopathy;
(i) nephropathy; and
(j) peripheral vascular disease.

40. The agonist or pharmaceutical composition of any one of claims 35 to 39 wherein
the agonist is a compound that stimulates glucose uptake in adipocytes or skeletal muscle
cells obtained from the mammal.
41. The agonist or pharmaceutical composition of any one of claims 35 to 40, wherein
the agonist is a partial agonist.
42. The agonist or pharmaceutical composition of any one of claims 35 to 41, wherein
the agonist is a compound according to claim 31 or claim 32, an agonist identified
according to any one of claims 19 to 26, or an agonist identified according to the method
of claim 28.
43. The agonist or pharmaceutical composition of any one of claims 35 to 42 wherein
the mammalian RUP43 GPCR is human RUP43 GPCR.
44. A process for manufacture of a medicament comprising an agonist of a
mammalian RUP43 GPCR for lowering blood glucose concentration, said process
comprising the steps of identifying said agonist according to the method of any one of
claims 1 to 26, and formulating said agonist as a medicament.
45. A process for manufacture of a medicament comprising an agonist of a
mammalian RUP43 GPCR for increasing glucose uptake in adipocytes or skeletal muscle
cells, said process comprising the steps of identifying said agonist according to the
method of any one of claims 1 to 26, and formulating said agonist as a medicament.
46. A process for manufacture of a medicament comprising an agonist of a
mammalian RUP43 GPCR for preventing or treating a metabolic disorder, wherein the
metabolic disorder is selected from the group consisting of:

(a) diabetes;
(b) impaired glucose tolerance;
(c) insulin resistance; and
(d) hyperinsulinemia,
said process comprising the steps of identifying said agonist according to the method of
any one of claims 1 to 26, and formulating said agonist as a medicament.

47. A process for manufacture of a medicament comprising an agonist of a
mammalian RUP43 GPCR for preventing or treating a complication of an elevated blood
glucose concentration, wherein the complication is selected from the group consisting of:
(a) Syndrome X;
(b) atherosclerosis;
(c) atheromatous disease;
(d) heart disease;
(e) hypertension;
(f) stroke;
(g) neuropathy;
(h) retinopathy;
(i) nephropathy; and
(j) peripheral vascular disease,
said process comprising the steps of identifying said agonist according to the method of
any one of claims 1 to 26, and formulating said agonist as a medicament.
48. The process of any one of claims 44 to 47 wherein the agonist is a compound that
stimulates glucose uptake in adipocytes or skeletal muscle cells obtained from the
mammal.
49. The process of any one of claims 44 to 48, wherein the agonist is a partial agonist.
50. The process of any one of claims 44 to 49, wherein the agonist is a compound
according to claim 31 or claim 32, an agonist identified according to any one of claims 19
to 26, or an agonist identified according to the use of claim 28.
51. The process of any one of claims 44 to 50 wherein the mammalian RUP43 GPCR
is human RUP43 GPCR.
52. An in vitro method of identifying one or more candidate compounds as a
compound that binds to a RUP43 GPCR, said receptor comprising a GPR131 amino acid
sequence selected from the group consisting of:

(a) the amino acid sequence of SEQ ID NO:2;
(b) amino acids 2-330 of SEQ ID NO:2;

(c) amino acids 2-330 of SEQ ID NO:2, with the proviso that the RUP43 G
protein-coupled receptor does not comprise the methionine residue at
amino acid position 1 of SEQ ID NO:2;
(d) the amino acid sequence of (i), (ii) or (iii) wherein the alanine at amino
acid position 223 of SEQ ID NO:2 is substituted with lysine;
(e) the amino acid sequence of a G protein-coupled receptor encoded by a
polynucleotide that is amplifiable by polymerase chain reaction (PCR) on
a human DNA sample using primers SEQ ID NO:3 and SEQ ID NO:4;
(f) the amino acid sequence of SEQ ID NO:6;
(g) the amino acid sequence of a G protein-coupled receptor encoded by a
polynucleotide that is amplifiable by polymerase chain reaction (PCR) on
a human DNA sample using primers SEQ ID NO:7 and SEQ ID NO:8;
(h) the amino acid sequence of a G protein-coupled receptor having an amino
acid sequence derived from SEQ ID NO:2 by substitution, deletion or
addition of one or several amino acids in the amino acid sequence of SEQ
IDNO:2;
(i) the amino acid sequence of a G protein-coupled receptor encoded by a
polynucleotide that hybridizes under stringent conditions to the
complement of SEQ ID NO : 1;
(j) the amino acid sequence of a G protein-coupled receptor having an amino
acid sequence having at least 75% identity to SEQ ID NO:2; and
(k) the amino acid sequence of an allele or mammalian ortholog of human
RUP43 GPCR having the amino acid sequence of SEQ ID NO:2;
comprising the steps of:
(a') contacting the receptor with a detectably labeled known ligand of the
receptor in the presence or absence of the candidate compound; and
(b') determining whether the binding of said labeled known ligand to the
receptor is inhibited in the presence of the candidate compound;
wherein said inhibition is indicative of the candidate compound being a compound that
binds to the RUP43 GPCR.

53. The method of claim 52, wherein said contacting comprises contacting with a host
cell or with membrane of a host cell that comprises recombinant said RUP43 GPCR.
54. The method of claim 52 or claim 53, wherein the known ligand is a compound
according to claim 31 or claim 32.
55. An in vitro method for detecting ligands that bind to a RUP43 GPCR, said
receptor comprising a GPR131 amino acid sequence selected from the group consisting
of:
(a) the amino acid sequence of SEQ ID NO:2;
(b) amino acids 2-330 of SEQ ID NO:2;
(c) amino acids 2-330 of SEQ ID NO:2, with the proviso that the RUP43 G
protein-coupled receptor does not comprise the methionine residue at
amino acid position 1 of SEQ ID NO:2;
(d) the amino acid sequence of (i), (ii) or (iii) wherein the alanine at amino
acid position 223 of SEQ ID NO:2 is substituted with lysine;
(e) the amino acid sequence of a G protein-coupled receptor encoded by a
polynucleotide that is amplifiable by polymerase chain reaction (PCR) on
a human DNA sample using primers SEQ ID NO:3 and SEQ ID NO:4;
(f) the amino acid sequence of SEQ ID NO:6;
(g) the amino acid sequence of a G protein-coupled receptor encoded by a
polynucleotide that is amplifiable by polymerase chain reaction (PCR) on
a human DNA sample using primers SEQ ID NO:7 and SEQ ID NO:8;
(h) the amino acid sequence of a G protein-coupled receptor having an amino
acid sequence derived from SEQ ID NO:2 by substitution, deletion or
addition of one or several amino acids in the amino acid sequence of SEQ
IDNO:2;
(i) the amino acid sequence of a G protein-coupled receptor encoded by a
polynucleotide that hybridizes under stringent conditions to the
complement of SEQ ID NO : 1;
(j) the amino acid sequence of a G protein-coupled receptor having an amino
acid sequence having at least 75% identity to SEQ ID NO:2; and
(k) the amino acid sequence of an allele or mammalian ortholog of human
RUP43 GPCR having the amino acid sequence of SEQ ID NO:2;
comprising the steps of:
(a') contacting a test ligand with a host cell or with membrane of a host cell
that expresses said receptor, under conditions which permit interaction
between said receptor and said test ligand; and
(b') detecting a ligand bound to said receptor.
56. The method of claim 55, wherein said contacting comprises contacting with a host
cell or with membrane of a host cell that comprises recombinant said RUP43 GPCR.


The present invention relates lo methods of identifying whether one or more candidate compounds is> a modulator of a G protein-coupled receptor (GPCR) or a modulator of blood glucose concentration In certain embodiments, the GPCR is human the present invention also relates to methods of using a modulator of the GPCR A preferred modulator is agonist. Agonists of the invention are useful as therapeutic agents for lowering blood glucose concentration for preventing or treating certain metabolic disorders, such a& insulin resistance, impaired glucose tolerance, and diabetes, and for preventing or treating a complication of an clevated blood glucose concentration, such as atherosclerosis heart disease, stroke, hypertension and peripheral vascular disease.

Documents:

03236-kolnp-2006 abstract.pdf

03236-kolnp-2006 claims.pdf

03236-kolnp-2006 correspondence others.pdf

03236-kolnp-2006 description(complete).pdf

03236-kolnp-2006 drawings.pdf

03236-kolnp-2006 form-1.pdf

03236-kolnp-2006 form-13.pdf

03236-kolnp-2006 form-3.pdf

03236-kolnp-2006 form-5.pdf

03236-kolnp-2006 gpa.pdf

03236-kolnp-2006 international publication.pdf

03236-kolnp-2006 international search authority report.pdf

03236-kolnp-2006 others.pdf

03236-kolnp-2006 pct request form.pdf

03236-kolnp-2006 priority document.pdf

3236-KOLNP-2006-ABSTRACT 1.1.pdf

3236-kolnp-2006-amanded claims.pdf

3236-KOLNP-2006-AMANDED PAGES OF SPECIFICATION.pdf

3236-KOLNP-2006-ASSIGNMENT-1.1.pdf

3236-KOLNP-2006-CLAIMS.pdf

3236-KOLNP-2006-CORRESPONDENCE 1.2.pdf

3236-KOLNP-2006-CORRESPONDENCE 1.3.pdf

3236-KOLNP-2006-CORRESPONDENCE-1.1.pdf

3236-KOLNP-2006-DESCRIPTION (COMPLETE) 1.1.pdf

3236-KOLNP-2006-EXAMINATION REPORT REPLY RECIEVED.pdf

3236-KOLNP-2006-FORM 1 1.1.pdf

3236-KOLNP-2006-FORM 13.1.1.pdf

3236-kolnp-2006-form 18.pdf

3236-KOLNP-2006-FORM 2.pdf

3236-kolnp-2006-form 3 1.1.pdf

3236-KOLNP-2006-FORM 3.pdf

3236-KOLNP-2006-IPRB.pdf

3236-KOLNP-2006-OTHERS 1.1.pdf

3236-KOLNP-2006-OTHERS DOCUMENTS.pdf

3236-KOLNP-2006-PA.pdf

3236-KOLNP-2006-PCT SEARCH REPORT.pdf

abstract-03236-kolnp-2006.jpg


Patent Number 246293
Indian Patent Application Number 3236/KOLNP/2006
PG Journal Number 08/2011
Publication Date 25-Feb-2011
Grant Date 23-Feb-2011
Date of Filing 06-Nov-2006
Name of Patentee ARENA PHARMACEUTICALS, INC.
Applicant Address 6166 NANCY RIDGE DRIVE, SAN DIEGO, CA 92121-3223,
Inventors:
# Inventor's Name Inventor's Address
1 QIU JUN 10558 Caminito Alvarez, San Diego, CA 92126,
2 UNETT DAVID J 11422 Cypress Terrace Place, San Diego, CA 92131
3 CONNOLLY DANIEL T 239 S. Helix Avenue, Solana Beach CA 92075
4 GATLIN JOEL E 9324 HILLERY DRIVE, APT. 5205, SAN DIEGO, CA 92126
5 WEBB ROBERT R 7625 Salix Road, San Diego, CA 92129,
PCT International Classification Number A61K31/427; A61K31/4
PCT International Application Number PCT/US2005/012447
PCT International Filing date 2005-04-12
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/561,954 2004-04-13 U.S.A.