Title of Invention

"POLYPEPTIDE FUSION CONSTRUCTS THAT INHIBIT NF-KB PATHWAY"

Abstract The present invention relates to polypeptides that inhibit the NF-KB signaling pathway and polynucleotides encoding the same. The present invention further provides methods for the modulation of and/or treatment of inflammatory responses, oncogenesis, viral infection; the regulation of cell proliferation and apoptosis; and regulation of B or T lymphocytes in antigenic stimulation, by administering the polypeptides of the present invention to a subject in need thereof. Finally, the present invention provides a method of identifying polypeptides that modulate oligomerization of NEMO.
Full Text SELECTIVE INHIBITION OF NF-kB ACTIVATION BY PEPTIDES
DESIGNED TO DISRUPT NEMO OLIGOMERIZATION
The present invention relates to polypeptides that inhibit the NF-KB
signaling pathway and polynucleotides encoding the same. The present invention
further provides methods for the modulation of and/or treatment of inflammatory
responses, oncogenesis, viral infection; the regulation of cell proliferation and
apoptosis; and regulation of B or T lymphocytes in antigenic stimulation, by
administering the polypeptides of the present invention to a subject in need thereof.
Finally, the present invention provides a method of identifying polypeptides that
modulate oligomerization of NEMO.
Nuclear factor-KB (NF-KB) signaling is an essential signal transduction
pathway involved in inflammatory responses, oncogenesis, viral infection, the
regulation of cell proliferation and apoptosis and in the case of B and T lymphocytes
in antigenic stimulation (Ghosh, 1998, Annu. Rev. Immunol.; Karin, 1999, J. Biol.
Chem.; Israel, 2000, Trends Cell Biol.; Santoro, 2003, EMBO J.). In mammalian cells,
there are five NF-KB family members mat dimerize: RelA, RelB, c-Rel, NFKB2/
plOO/p52 and NF-icBl/pl05/p50. NF-KB whose predominant form is a
heterodimeric transcription factor composed of p50 and RelA subunits, remains
sequestered in the cytoplasm through association with members of an inhibitory
family of proteins known as 1KB. Upon stimulation by the cytokines TNF-a and
interleukin-1, endotoxin (LPS), microbial and viral infections, pro-inflammatory
signals converge on the canonical IkB kinase complex (IKK), a protein complex that
is composed of two kinases subunits, IKKct/IKK-1 and IKKp/IKK-2 and a
structural/regulatory subunit NEMO/IKK-y. Once activated IKK complex
phosphorylates IkB proteins, triggering their ubiquitination and subsequent degradation
by proteasome. Free NF-KB can then move into nucleus to initiate or up-regulate
gene expression. Although IKKa and IKKp exhibit striking structural similarity
(52%), exquisite genetic studies have shown that they are involved in two pathways
for the activation of NF-KB (Pomerantz, 2002, Mol Cell). IKKp is the proinflammatory
kinase that is responsible of activation of classical NF-KB complexes
whereas IKKa in association with NF-icB inducing kinase (NIK) plays essential roles
in the non-canonical NF-KB signaling pathway (Senftleben, 2001, Science).
IKKct plays also a role in keratinocyte differentiation but this process is independent
of its kinase activity (Hu, 2001, Nature).
The NEMO protein (NF-KB essential modulator) plays a key role in
the NF-KB pathway activation. The NEMO protein is associated with IKKa and IKK0
protein kinases in a high molecular weight complex called the IKK complex. The IKK
kinases are activated by phosphorylation upon an unknown mechanism, which is
believed to be a result of NEMO oligomerization (Agou et al., 2004, J. Biol. Chem.).
The presence of the NEMO protein underlies IKK activation since NEMO-deficient
cells are unable to activate NF-icB in response to many stimuli. NEMO is composed
of an N-terminal IKK-binding domain including a large coiled-coil (CC1). The Cterminal
domain functions as the regulatory part of the protein, which has often been
reported as a binding template to link many upstream signaling molecules or viral
proteins (Ghosh, 1998, Annu. Rev. Immunol.; Santoro, 2003, EMBO J.) Interestingly,
mutations responsible for IP and EDA-ID pathologies were mainly found in this part
of the molecules (Doffinger, 2001, Nature Gen.; Zonana, 2000, Am. J. Hum. Genet.).
The C-terminal domain is composed of the minimal oligomerization domain including
two sucessives coiled-coil motifs, CC2 (residues 246-286) and LZ (residues 390-412)
(Tegethoff, 2003, Mol. Cell Biol.; Agou et al., 2004, J. Biol. Chem.), and a zinc finger
motif at the extremity of the C-terminus.
The biochemical mechanisms triggering the activation of IKK in
response to pro-inflammatory stimuli remain unclear. It has been demonstrated that
phosphorylation on two serine residues in the activation T-loop induces activation of
the IKKp. However, the mechanism that leads to this phosphorylation event is still
unknown. One possible mechanism consists of the conformation change of the kinase
induced by NEMO oligomerization (Agou et al., 2004, J. Biol. Chem.). This change of
the oligomeric state may induce the T-loop activation by a mechanism of trans-autophosphorylation
(Zandi, 1997, Cell; Tang, 2003, J. Biol. Chem.). Consistent with the
role of NEMO oligomerization in IKK activation, mutations in the minimal
oligomerization domain failed to rescue NF-tcB by genetic complementation in
NEMO-deficient cells activation in responses to many stimuli. Moreover, enforced
oligomerization of NEMO lead to full activation of IKK complex. (Inohara, 2000, J.
Biol. Chem.; Poyet, 2000, J. Biol. Chem.; Poyet, 2001, J. Biol. Chem.). Recently, the
phosphorylation and the ubiquitination of NEMO in response to TNF-a have been
reported, (Carter, 2001, J. Biol. Chem.; Trompouky, 2003, Nature; Kovalenko, 2003,
Nature). However, these NEMO modifications have not been demonstrated yet as a
crucial step to activate IKK complex in response to several pro-inflammatory stimuli.
Inhibition of NF-KB activation constitutes a privileged target for
development of new anti-inflammatory and anti-cancer drugs (May, 2000, Science;
Poulaki, 2002, Am J Pathol..). Among many protein actors in NF-KB signaling
pathway, IKK complex represents one of the most promising molecular targets for
discoveries of the new specific NF-KB inhibitors. To minimize the potential toxicity
effects in vivo, therapeutical success will greatly depend on the abilities of the NF-KB
inhibitors to block activating signals without modifying the basal level of NF-KB
activity. May et al. described a cell-permeable peptidic inhibitor that block specifically
the pro-inflammatory NF-KB activation by disrupting the constitutive NEMO
interaction with IKK kinases (May, 2000, Science; May, 2002, J. Biol. Chem.).
Modulating protein-protein interactions by the rational design of peptide that alter
protein's function provides an important tool for both basic research and development
of new classes of therapeutic drugs (Souroujon, 1998, Nat Biotechnol.), especially
with signaling proteins that exhibit flexible and dynamic binding properties (Pawson,
2003, Science). Numerous studies of peptide modulators have been described in the
literature where peptides mediate protein's function by interfering with localization
(translocation) (Lin, 1995, J. Biol. Chem.), recruitment to receptor (Chang, 2000, J.
Biol. Chem.), intramolecular interactions (Souroujon, 1998, Nat Biotechnol.) and
oligomerization (Judice, 1997, P.N.A.S.). In the latter, inhibition of HIV-1 gp41
fusion protein with various peptides provides a clear proof-of concept (for a review
see Chan, 1998, Cell and Eckert, 2001, Ann. Rev. Biochem.).
Under this theory that inhibition of NF-icB activation provides a
desirable target for the development of new anti-inflammatory and anti-cancer drugs,
the present inventors have set forth to discover candidate anti-inflammatory and anticancer
drugs, as well as to provide a method of screening for the same.
It is an object of the present invention to provide polypeptides
derived from NEMO that are useful for the regulation and/or inhibition of the NF-icB
signaling pathway.
To this end, the present invention provides NEMO-derived polypeptides
that inhibit the NF-icB signaling pathway.
In one embodiment of the present invention, the NEMO-derived
polypeptide is the CC2 domain (murine: SEQ ID NO: 3 or human: SEQ ID NO: 14).
In another embodiment of the present invention, the NEMO-derived
polypeptide is the LZ domain (murine: SEQ ID NO: 7 or human: SEQ ID NO: 16).
In a preferred embodiment of the present invention, the NEMOderived
polypeptides are fused via a spacer sequence to a polypeptide having a high
transduction potential.
Further, in another embodiment of the present invention are
polynucleotides that encode for the NEMO-derived polypeptides either with or
without the spacer sequence and the polypeptide having a high transduction potential.
In yet another embodiment of the present invention is methods of
modulating or treating disorders regulated by the NF-KB signaling pathway by
administering the NEMO-derived polypeptides to a subject in need thereof. The
disorders regulated by the NF-KB signaling pathway include: inflammatory responses,
oncogenesis, and viral infection.
The present invention also provides a method of regulating cell
proliferation or apoptosis by administering the NEMO-derived polypeptides to a
subject in need thereof.
In still another embodiment of the present invention is a method of
regulating B or T lymphocytes in antigenic stimulation by administering the NEMOderived
polypeptides .to a subject in need thereof.
In yet another embodiment, the present invention further provides a
method of identifying polypeptides that modulate oligomerization of NEMO by
a) identifying a candidate polypeptide sequence;
b) creating a polypeptide fusion construct by linking said candidate
polypeptide sequence to a polypeptide having a high transduction potential via a
spacer sequence;
c) contacting a cell culture with the polypeptide fusion construct;
and
d) monitoring the activity of the NF-icB signaling pathway;
e) comparing the activity of the NF-icB signaling pathway in the
presence of said polypeptide fusion construct to the activity of the NF-icB signaling
pathway in the absence of said polypeptide fusion construct to determine the relative
inhibition by said polypeptide fusion construct; and
f) correlating relative inhibition by said polypeptide fusion construct
to NEMO oligomerization.
The above objects highlight certain aspects of the invention.
Additional objects, aspects and embodiments of the invention are found in the
following detailed description of the invention.
A more complete appreciation of the invention and many of the
attendant advantages thereof will be readily obtained as the same becomes better
understood by reference to the following Figures in conjunction with the detailed
description below.
Figure 1 : Functional domains of the NEMO protein.
(A) The murine NEMO protein contains 412 amino acids and
multiple domains including the N-terminal IKK binding domain and the oligomerization
domain, the proline rich motif (PPP) and the zinc finger motif (ZF) at the Cterminus.
The coiled-coil predictions (open boxes) using the algorithm developed by
Wolf et al. (1997, Protein Sci.) and the NLM conserved motif (black bar) is shown.
The sequence of NEMOass-as? (residues 253-337 of SEQ ID NO: 12) corresponding to
the second coiled-coil (CC2) and leucine zipper (LZ) motifs, which contains all
determinants required for NEMO oligomerization (Agou et al., 2004, J. Biol. Chem.),
is indicated with the NLM conserved motif (residues 293-322 of SEQ ID NO: 12)
underlined and with the coiled-coil sequences showed as cylinders below the
sequence. Letters immediately above the sequence indicate the heptad repeat 'a1 and 'd1
positions which is a key feature of coiled-coil sequences (Vinson, 2002, Mol. Cell.
Biol.). (B) Multiple sequence alignment of NEMO proteins from Mus musculus (Mm),
Homo sapiens (Hs), Bos taurus (Bt) and Drosophila melanogaster (Dm), showing the
NEMO like motif (NLM) shared with NRP/optineurin, ABIN-1/Naf 1, ABIN-2 and
ABIN-3/LIND of different species (SEQ ID NOs: 19-29, respectively). The multiple
sequence alignment was constructed by parsing PSI-BLAST-generated highestscoring
pairs of sequence segments and realigning the same using with CLUSTAL W
(Thompson, 1994, N.A.R.). Identical and similar amino acid residues (shaded) are
indicated by, respectively.
Figure 2: Flow cytometry analysis of NEMO peptide uptake
(A) Cellular delivery of NEMO peptides mediated by conjugation
with the Antennapedia peptide. 70Z/3 cells were incubated for 2 h at 37°C in the
absence (W/O) or in the presence of 2 uM BODIPY-tagged Ant-CC2 wild type (WT),
or Ant-CC2 mutant (Mu), or Ant-LZ wild type (WT) or Ant-LZ mutant (Mu) peptide
as indicated, or with controls corresponding to 2 uM BODIPY-conjugated BSA
(BODIPY-BSA) or BODIPY-FL alone. (B) Concentration dependence of
antennapedia-mediated uptake of 0, 0.2, 2 and 20 uM Ant-CC2 at 37°C for 5 h in
70Z/3-C3 cells (left panel) and FACS kinetic analysis of BODIPY-conjugated Ant-
CC2 at 0, 0.5, 1,2 or 5 h after addition of 20 uM Ant-CC2 at 37°C.
Figure 3: Inhibition of LPS-induced NF-icB activation by cell-permeable Ant-
CC2 and Ant-LZ peptide
(A) 70Z3 lymphocyte B were stably transfected with pILl-pgalactosidase,
which bears the (3-galactosidase gene under the control of the NF-KB
(see "Materials and methods"). The resulting cell line, 70Z3-C3, was incubated for 2
hours in the absence or in the presence of 20 uM of antennapedia peptide (Ant), or
BODIPY-labeled antennapedia peptide (BODIPY-Ant), or BODIPY-labeled
antennapedia peptide coupled to CC2 (BODIPY-Ant-CC2) or LZ (BODIPY-Ant-LZ)
peptides, After peptide internalization, cells were treated for 5 hours with LPS (3
ug/ml, (+) in left panel) or untreated (right panel and (-) in left panel) and the NF-KB
activity was measured by p-galactosidase assay. Error bars represent the standard
deviation of three separate experiments. (B) Concentration dependence of inhibition
of LPS-induced NF-KB activation by BODIPY-Ant-CC2 peptide (left panel) or
BODIPY-Ant-LZ peptide (right panel). Cells were treated as in (A) but with different
concentration of peptide as indicated. The potential of each peptide to inhibit LPSinduced
NF-KB activation was measured by determining the ICso value that
correspond to 50% inhibition of LPS-induced NF-icB activation as compared to the
control (no peptide) (C). (D) Effect of the N-fusion sequence of antennapedia on the
inhibition of NF-KB activation. Control (no peptide) or CC2, or LZ peptides, with
(BODIPY-ANT-CC2, BODIPY-ANT-LZ) or without the antennapedia sequence at
the N-terminus (CC2, LZ) were incubated for 2 hours with 70Z3-C3 cells followed
with (+) or without (-) the LPS-treatment for 3 hours. NF-icB activity was then
measured by P-galactosidase assay.
Figure 4: Specific inhibition of NF-KB activation in response to LPS depends on a
few mutations in the hydrophobic core of CC2 and LZ coiled-coils
Left panels show a helical wheel diagram of CC2 (A) and LZ (B)
peptides. The view is from the top the molecule. The (a) through (g) positions, which
are an essential feature of coiled coil sequence (Vinson, 2002, Mol. Cell. Biol.)
represent sequential positions in each peptide sequence. The first (a) and fourth (d)
positions that are generally occupied by hydrophobic amino acids constitute the
hydrophobic core for parallel as well as antiparallel-coiled coils. Mutations that were
introduced in (a) positions of the CC2 variant (BODIPY-Ant-CC2 (Mu); shown as
residues 6-40 of SEQ ID NO:3, with glycine mutations corresponding to residues 6-40
of SEQ ID NO:5) or in (d) positions of the LZ variant (BODIPY-Ant-(Mu) are shown.
In the right panels, 70Z3-C3 cells were incubated for 2 hours in the absence (control)
or in the presence of 10 uM of cell permeable wild type (BODIPY-Ant-CC2 (WT)) or
mutant (BODIPY-Ant-CC2 (Mu)) CC2 peptides (A) or wild type (BODIPY-Ant-LZ
(WT)) or mutant (BODIPY-Ant-(Mu)) LZ peptides (B). The cells were then extensively
washed to remove the excess peptide which had not been internalized and the
cells were then diluted three times and allowed to grow for for 24 hours before treatment
for 5 hours with (+) or without LPS (-). NF-KB activity was measured using the
P-galactosidase assay. Error bars represent the standard deviation of two independent
experiments.
Figure 5: Inhibition of NF-KB activation by the LZ peptide occurs through the
formation of specific coiled-coil strands.
(A) Sequence alignement of the NEMO-derived LZ (residues 301-
336 of SEQ ID NO:12) and the GCN4 peptides (residues 23-55 of SEQ ID N0:8).
Both coiled-coil motifs were aligned using clustalX. Identical and similar amino acid
residues (shaded) are indicated by, respectively. (B) Overview and helical
wheel diagram of the GCN4 coiled-coil (top view). The amino-acid sequence of
GCN4 is shown with its corresponding [a - g] positions and residues that differ from
the corresponding NEMO-derived LZ sequence are boxed according to their degree of
conservation. Identical (open square) and similar residues (open triangle) are
indicated, (C) Comparison of the cell permeable NEMO-derived LZ and GCN4
peptide on the inhibition of LPS-induced NF-icB activation. 70Z3-C3 cells were incubated
for 2 hours in the absence (no peptide) or in the presence of 10 uM of the
antennapedia fusion LZ (BODIPY-Ant-LZ) or GCN4 (BODIPY-Ant-GCN4) peptide.
Cells were then extensively washed to remove any peptide excess which was not
internalized, and diluted three times to facilitate 24 hours of growth before treatment
for 5 hours with (+) or without LPS (-). NF-icB activity was measured using the Pgalactosidase
assay. Error bars represent the standard deviation of two independant
experiments.
Figure 6: Oligomerization properties of NEMO-derived polypeptides with or
without the antennapedia sequence
All peptides were loaded at a 10 uM concentration on a superdex 75
HR10/30 column equilibrated in a buffer containing 0.1 mM DDM to improve
recovery (see "Materials and methods"). Chromatographic profiles of the CC2 mutant
(dashed line) and the CC2 wild type (solid line) fused (BODIPY-Ant-CC2 (WT),
BODIPY-Ant-CC2 (Mu), or not fused to the antennapedia sequence (CC2 (WT), CC2
(Mu)) are shown in left panels, and elution profiles of the LZ mutant (dashed line) and
the wild type (solid line) fused (BODIPY-Ant-LZ (WT), BODIPY-Ant-LZ (Mu), or
not fused to the antennapedia sequence (LZ (WT), LZ (Mu)) are represented in right
panels. Elution volumes of globular protein markers are indicated by arrows : Oval,
ovalbumin (43 kDa); Chym, chymotrypsinogene A (25 kDa); Ribo, Ribonuclease
(13.4 kDa) and Apro, aprotinin (6.5 kDa).
Figure 7: Association of Ant-CC2 and Ant-LZ peptides to the CC2 peptide
(A) Direct titration of BODIPY-Ant-CC2 (I uM) with CC2 by
fluorescence anitropy. The concentration of CC2 was determined by amino acid
analysis. Anisotropy values of BODIPY-Ant-CC2 in milliunits (mA) were plotted
against an increasing concentration of the CC2 peptide. Data points were fitted (solid
line) to the binding isotherm equation with a KD of 15.2 u,M (Materials and Methods).
The two dashed lines represent a stoichiometric titration and intersect at an CC2
concentration of 16 uM. Given the 1 uM concentration of the BODIPY-Ant-CC2, this
gives a complex stoechiometrie of 0.8. (B) Direct titration of BODIPY-Ant-LZ (0.1
uM) with CC2 by fluorescence anitropy. The anisotropy values of the BODIPY-Ant-
LZ alone (white bar) or in the presence of the CC2 peptide (30uM, grey bar; 100 uM,
black bar) are given in milliunits (mA).
Figure 8: Cell death induced in the retinoblastoma cell line Y79 by Ant-CC2 and
Ant-LZ peptides
Rb cell line Y79 were treated with various concentration of the Ant-
CC2 (WT) (filled squares) or Ant-CC2 (Mu) (open squares) (A), or Ant-LZ (WT)
(filled circles), or Ant-LZ (Mu) (open circles) (B), or Ant peptide (open triangle) (C)
for 3 hours (A, B) or 16 hours (C). Cell survival was then evaluated using the MTS
assay as described in "Materials and methods"
Unless specifically defined, all technical and scientific terms used
herein have the same meaning as commonly understood by a skilled artisan in
enzymology, biochemistry, cellular biology, molecular biology, and the medical
sciences.
All methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present invention, with suitable
methods and materials being described herein. All publications, patent applications,
patents, and other references mentioned herein are incorporated by reference in their
entirety. In case of conflict, the present specification, including definitions, will
control. Further, the materials, methods, and examples are illustrative only and are
not intended to be limiting, unless otherwise specified.
In this application, the present inventors studied the inhibition of
NF-KB activation by peptides designed to disrupt NEMO oligomerization. The present
inventors have previously shown that the minimal trimerization domain comprises the
CC2-LZ coiled-coil subdomain and that the isolated and/or purified CC2 and LZ
domains bind to each other to form a stable trimer of heterodimers. This structural
model is reminiscent of the fold of the gp41 ectodomain from HIV-1 (Agou et al.,
2004, J. Biol. Chem.). It consist of a central three-stranded coiled coil (formed by the
CC2 coiled coil motif of NEMO) which is surrounded by the LZ helical motif derived
from the C-terminal end of NEMO, packed in an antiparallel manner around the
outside of the CC2 coiled-coil. On the basis of this model, the present inventors
rationally designed two cell-permeable peptides corresponding to optimal portions of
CC2 or LZ subdomains that mimic the contact area between NEMO subunits. Peptide
transduction was monitored by FACS and their effect on LPS-induced NF-KB activation
was quantified using a NF-icB dependent p-galactosidase assay in stably
transfected pre-B 70Z/3 lymphocytes. The present inventors have also demonstrated
that the LZ peptide and, to a lesser extent the CC2 peptide, inhibit specifically NF-KB
activation with ICso values in the uM range. The effects were specific because control
peptides including mutated CC2 and LZ peptides as well as heterologous coiled-coil
peptides (GCN4), had no inhibitory effect on NF-KB activation. Furthermore, the
present inventors have shown that these NF-KB peptidic inhibitors induced the cell
death in the human retinoblastoma cell lines Y79 that exhibit constitutive NF-KB
activity. Collectively, the present inventors have provided a new and promising
strategy to inhibit the NF-xB pathway by targeting NEMO's oligomerization.
The present inventors have proven that NEMO constitutes a
preferential target for the search for drugs inhibiting the NF-KB signaling path,
because this protein acts upstream from the NF-KB activation path. The role of
NEMO and its various domains was partially studied and published in the following
article, "NEMO trimerizes through its coiled-coil C-terminal domain. " J Biol Chem,
2002 May 17;277(20): 17464-75. Agou F. et al., a copy of which is incorporated by
reference.
In the present invention, the inventors have synthesized peptides that
mimic either the oligomerization domain (CC2 domain = approx. 40 residues), or the
LZ motif (LZ domain = approx. 40 residues). The combination of these peptides
alters either the oligomerization of NEMO or the combining thereof with the proteinic
effector, in both cases inhibiting the NF-icB pathway.
In an aspect of the present invention, peptide drugs have been
chemically combined with a peptide of 16 amino acids in length
(penetratin/antennapedia), thereby enabling intracellular transport thereof possible.
The resulting peptides also may be chemically coupled with a fluorescent tracer in
order to monitor internalization into B lymphocyte cell lines through FACS.
The action of these peptides was tested directly on B lymphocytes
having stably integrated the beta-galactosidase carrier gene also bearing upstream
from its promoter several NF-KB transcription factor (Clone C3) activation sites (see
Examples herein below).
The present inventors have successfully been able to monitor the
inhibitory effect of these peptides by measuring the same following stimulation of the
B lymphocytes by LPS.
The results as a whole reveal that the presence of the peptide
mimicking the "CC2" motif reduces the NF-KB activity by 70% as compared with a
control peptide at a relatively low dose of 20 uM. At this concentration, the effect of
the "Leucine zipper" peptide is still more significant, since its presence in the medium
completely eliminates cell response.
These new inhibitors of the NF-KB cellular signalling path offer a
major advantage as anti-inflammatory compounds and also as anti-tumor compounds,
which may be used for the treatment and/or prevention of cancers and other disorders.
The present invention relates to compounds, peptides, or compositions
that are used for modulating the oligomerization of NEMO. In particular, the
peptide compounds described herein below may be in an isolated and/or purified or
coupled form with or without a vectorizing agent. It is to be understood that the
present invention also embraces peptides having at least 70% homology with the
NEMO-derived polypeptides, so long as the homologs possess said inhibitory activity.
Methods for assessing inhibitory activity of the NEMO-derived polypeptides, and
homologs thereof, are provided below and exemplified in the Examples of the present
application. The peptides of the present invention and the doses thereof are deemed to
possess inhibitory activity when the NF-KB activity is reduced by at least 50% as
compared with a control peptide.
The present invention also relates to pharmaceutical compositions
containing said peptides, especially for the preparation of medicines used for the
treatment of inflammatory responses, oncogenesis, viral infection, the regulation of
cell proliferation and apoptosis and antigenic stimulation. In a preferred embodiment,
the pharmaceutical compositions containing said peptides are useful for the treatment
of cancer.
Also embraced by the present invention are methods of obtaining,
making, and identifying peptides and compounds that inhibit the NF-icB signaling
pathway, in particular by means of the 70Z/3-C3 cell line filed with the CNCM
(Collection Nationale de Cultures de Microorganismes), 28 rue du Docteur Roux,
75724 PARIS Cedex 15, France, on April 1st, 2003 under number 1-3004.
As used herein, the term "reduced" or "inhibited" means decreasing
the intracellular activity of one or more enzymes in the NF-KB pathway either directly
or indirectly. The phrase "inhibiting the NF-icB pathway" preferably means that the
NF-KB pathway is inhibited by disruption of NEMO oligomerization.
The term "enhanced" as used herein means increasing the intracellular
activity or concentration of the NEMO derived peptides, which are encoded
by the corresponding DNA. Enhancement can be achieved with the aid of various
manipulations of the bacterial cell. In order to achieve enhancement, particularly
over-expression, the number of copies of the corresponding gene can be increased, a
strong promoter can be used, or the promoter- and regulation region or the ribosome
binding site which is situated upstream of the structural gene can be mutated.
Expression cassettes which are incorporated upstream of the structural gene act in the
same manner. In addition, it is possible to increase expression by employing inducible
promoters. A gene can also be used which encodes a corresponding enzyme with a
high activity. Expression can also be improved by measures for extending the life of
the mRNA. Furthermore, preventing the degradation of the enzyme increases enzyme
activity as a whole. Moreover, these measures can optionally be combined in any
desired manner. These and other methods for altering gene activity in a plant are
known as described, for example, in Methods in Plant Molecular Biology, Maliga et
al, Eds., Cold Spring Harbor Laboratory Press, New York (1995).
A gene can also be used which encodes a corresponding or variant
NEMO derived peptide with a high activity of inhibiting the NF-KB pathway. Preferably
the corresponding enzyme has a greater ability than the native form of the NEMO
protein to inhibit the NF-xB pathway, more preferably at least in the range of 5, 10,
25% or 50% more inhibition. Most preferably the NEMO derived peptides of the
present invention reduce the NF-KB pathway by at least 50%, at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, as compared to the pathway in the
presence of the native NEMO protein.
In the context of the present Application, a polynucleotide sequence
is "homologous" with the sequence according to the invention if at least 70%, preferably
at least 80%, most preferably at least 90% of its base composition and base
sequence corresponds to the sequence according to the invention. According to the
invention, a "homologous protein" or "homologous peptide" is to be understood to
comprise proteins (peptides) which contain an amino acid sequence at least 70 % of
which, preferably at least 80 % of which, most preferably at least 90 % of which,
corresponds to the amino acid sequence of the CC2 region of NEMO (SEQ ID NO: 3)
or the LZ region of NEMO (SEQ ID NO: 7) in the case of murine-derived NEMO and
the CC2 region of NEMO (SEQ ID NO: 14) or the LZ region of NEMO (SEQ ID NO:
16) in the case of human-derived NEMO, wherein corresponds is to be understood to
mean that the corresponding amino acids are either identical or are mutually homologous
amino acids. It is further to be understood that, as evinced by the Examples of
the present invention, the homologous peptide of CC2 preferably retains the coiledcoil
motif structure and the homologous peptide of LZ preferably retains the helical
motif structure. With the guidance proffered by the identification of SEQ ID NO: 3,
SEQ ID NO: 7, SEQ ID NO: 14, and SEQ ID NO: 16 and the detailed description in
the Examples below, screening of theoretical mutations within the scope of the present
invention would require nothing more than a technicians level of skill in the art. More
specifically, as is routine in the art, with the identification of a candidate sequence
(i.e., the regions corresponding to SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 14,
and SEQ ID NO: 16) the artisan would assay and screen one or all possible permutations
of the said sequence to identify mutants possessing the same or better therapeutic
efficacy.
The expression "homologous amino acids" denotes those that have
corresponding properties, particularly with regard to their charge, hydrophobic
character, steric properties, etc.
Homology, sequence similarity or sequence identity of nucleotide or
amino acid sequences may be determined conventionally by using known software or
computer programs such as the BestFit or Gap pairwise comparison programs (GCG
Wisconsin Package, Genetics Computer Group, 575 Science Drive, Madison,
Wisconsin 53711). BestFit uses the local homology algorithm of Smith and
Waterman, Advances in Applied Mathematics 2: 482-489 (1981), to find the best
segment of identity or similarity between two sequences. Gap performs global
alignments: all of one sequence with all of another similar sequence using the method
of Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970). When using a sequence
alignment program such as BestFit, to determine the degree of sequence homology,
similarity or identity, the default setting may be used, or an appropriate scoring matrix
may be selected to optimize identity, similarity or homology scores. Similarly, when
using a program such as BestFit to determine sequence identity, similarity or
homology between two different amino acid sequences, the default settings may be
used, or an appropriate scoring matrix, such as blosum.45 or blosumSO, may be
selected to optimize identity, similarity or homology scores.
The present invention also relates to polynucleotides which encode
the CC2 region of NEMO (SEQ ID NO: 3) or the LZ region of NEMO (SEQ ID NO:
7) in the case of murine-derived NEMO and the CC2 region of NEMO (SEQ ID NO:
14) or the LZ region of NEMO (SEQ ID NO: 16) in the case of human-derived
NEMO, or fragments thereof, and which can be obtained by screening by means of the
hybridization of a corresponding gene bank with a probe which contains the sequence
of said polynucleotide that encodes the CC2 region of NEMO (SEQ ID NO: 3) or the
LZ region of NEMO (SEQ ID NO: 7) in the case of murine-derived NEMO and the
CC2 region of NEMO (SEQ ID NO: 14) or the LZ region of NEMO (SEQ ID NO: 16)
in the case of human-derived NEMO, or fragments thereof, and isolation of said DNA
sequence.
Polynucleotide sequences according to the invention are suitable as
hybridization probes for RNA, cDNA and DNA, in order to isolate those cDNAs or
genes that exhibit a high degree of similarity to the sequence that encodes the CC2
region of NEMO (SEQ ID NO: 3) or the LZ region of NEMO (SEQ ID NO: 7) in the
case of murine-derived NEMO and the CC2 region of NEMO (SEQ ID NO: 14) or the
LZ region of NEMO (SEQ ID NO: 16) in the case of human-derived NEMO, or
fragments thereof.
Polynucleotide sequences according to the invention are also
suitable as primers for polymerase chain reaction (PCR) for the production of DNA,
which encodes a NEMO-derived polypeptide having an ability to inhibit the NF-KB
pathway.
Oligonucleotides such as these, which serve as probes or primers,
can contain more than 30, preferably up to 30, more preferably up to 20, most preferably
at least 15 successive nucleotides. Oligonucleotides with a length of at least 40 or
50 nucleotides are also suitable.
The term "isolated and/or purified" means separated from its natural
environment.
The term "polynucleotide" refers in general to polyribonucleotides
and polydeoxyribonucleotides, and can denote an unmodified RNA or DNA or a
modified RNA or DNA.
The term "polypeptides" is to be understood to mean peptides or
proteins that contain two or more amino acids that are bound via peptide bonds.
The polypeptides according to invention include polypeptides
corresponding to the CC2 region of NEMO (SEQ ID NO: 3) or the LZ region of
NEMO (SEQ ID NO: 7) in the case of murine-derived NEMO and the CC2 region of
NEMO (SEQ ID NO: 14) or the LZ region of NEMO (SEQ ID NO: 16) in the case of
human-derived NEMO, or fragments thereof, particularly those with the biological
activity of inhibition the NF-icB pathway, and also includes those, at least 70 % of
which, preferably at least 80% of which, are homologous with the polypeptide
corresponding to the CC2 region of NEMO (SEQ ID NO: 3) or the LZ region of
NEMO (SEQ ID NO: 7) in the case of murine-derived NEMO and the CC2 region of
NEMO (SEQ ID NO: 14) or the LZ region of NEMO (SEQ ID NO: 16) in the case of
human-derived NEMO, or fragments thereof, and most preferably those which exhibit
a homology of at least 90 % to 95 % with the polypeptide corresponding to the CC2
region of NEMO (SEQ ID NO: 3) or the LZ region of NEMO (SEQ ID NO: 7) in the
case of murine-derived NEMO and the CC2 region of NEMO (SEQ ID NO: 14) or the
LZ region of NEMO (SEQ ID NO: 16) in the case of human-derived NEMO, or
fragments thereof, and which have the cited activity.
The invention also relates to coding DNA sequences that encode the
CC2 region of NEMO (SEQ ID NO: 3) or the LZ region of NEMO (SEQ ID NO: 7) in
the case of murine-derived NEMO and the CC2 region of NEMO (SEQ ID NO: 14) or
the LZ region of NEMO (SEQ ID NO: 16) in the case of human-derived NEMO, or
fragments thereof, by degeneration of the genetic code. One of skill in the art would
appreciate that the aforementioned DNA sequences may be based on the full-length
DNA sequences for the murine-derived NEMO (SEQ ID NO: 11) and the humanderived
NEMO (SEQ ID NO: 17) and thereby these sequences may be used to
ascertain the scope of these sequences in accordance with the present invention.
In the same manner, the invention further relates to DNA sequences
that hybridize with DNA sequences that encode the CC2 region of NEMO (SEQ ID
NO: 3) or the LZ region of NEMO (SEQ ID NO: 7) in the case of murine-derived
NEMO and the CC2 region of NEMO (SEQ ID NO: 14) or the LZ region of NEMO
(SEQ ID NO: 16) in the case of human-derived NEMO, or fragments thereof.
Moreover, one skilled in the art is also aware of conservative amino
acid replacements such as the replacement of glycine by alanine or of aspartic acid by
glutamic acid in proteins as "sense mutations" which do not result in any fundamental
change in the activity of the protein, i.e. which are functionally neutral. It is also
known that changes at the N- and/or C-terminus of a protein do not substantially
impair the function thereof, and may even stabilize said function.
In the same manner, the present invention also relates to DNA
sequences that hybridize with the DNA sequence that encodes the CC2 region of
NEMO (SEQ ID NO: 3) or the LZ region of NEMO (SEQ ID NO: 7) in the case of
murine-derived NEMO and the CC2 region of NEMO (SEQ ID NO: 14) or the LZ
region of NEMO (SEQ ID NO: 16) in the case of human-derived NEMO, or
fragments thereof. The present invention also relates to DNA sequences that are
produced by polymerase chain reaction (PCR) using oligonucleotide primers that
result from the DNA sequence that encodes the CC2 region of NEMO (SEQ ID NO:
3) or the LZ region of NEMO (SEQ ID NO: 7) in the case of murine-derived NEMO
and the CC2 region of NEMO (SEQ ID NO: 14) or the LZ region of NEMO (SEQ ID
NO: 16) in the case of human-derived NEMO, or fragments thereof. Oligonucleotides
of this type typically have a length of at least 15 nucleotides.
The terms "stringent conditions" or "stringent hybridization conditions"
includes reference to conditions under which a polynucleotide will hybridize to
its target sequence, to a detectably greater degree than other sequences (e.g., at least 2-
fold over background). Stringent conditions are sequence-dependent and will be
different in different circumstances. By controlling the stringency of the hybridization
and/or washing conditions, target sequences can be identified which are 100%
complementary to the probe (homologous probing). Alternatively, stringency conditions
can be adjusted to allow some mismatching in sequences so that lower degrees
of similarity are detected (heterologous probing).
Typically, stringent conditions will be those in which the salt
concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion
concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about
30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long
probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved
with the addition of destabilizing agents such as formamide. Exemplary low stringency
conditions include hybridization with a buffer solution of 30 to 35%
formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37°C, and a wash in IX
to 2X SSC (20X SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55°C Exemplary
moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M
NaCl, 1% SDS at 37°C, and a wash in 0.5X to IX SSC at 55 to 60°C Exemplary high
stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at
37°C, and a wash in 0.1X SSC at 60 to 65°C.
Specificity is typically the function of post-hybridization washes, the
critical factors being the ionic strength and temperature of the final wash solution. For
DNA—DNA hybrids, the Tm can be approximated from the equation of Meinkoth and
Wahl, Anal. Biochem., 138:267-284 (1984): Tm =81.5°C +16.6 (log M)+0.41 (%GC)-
0.61 (% form)-500/L; where M is the molarity of monovalent cations, %GC is the
percentage of guanosine and cytosine nucleotides in the DNA, % form is the
percentage of formamide in the hybridization solution, and L is the length of the
hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH)
at which 50% of a complementary target sequence hybridizes to a perfectly, matched
probe. Tm is reduced by about 1°C for each 1% of mismatching; thus, Tm, hybridization
and/or wash conditions can be adjusted to hybridize to sequences of the desired
identity. For example, if sequences with approximately 90% identity are sought, the
Tm can be decreased 10°C. Generally, stringent conditions are selected to be about
5°C lower than the thermal melting point (Tm) for the specific sequence and its
complement at a defined ionic strength and pH. However, severely stringent conditions
can utilize a hybridization and/or wash at 1,2, 3, or 4°C lower than the thermal
melting point (Tm); moderately stringent conditions can utilize a hybridization and/or
wash at 6, 7, 8, 9, or 10°C lower than the thermal melting point (Tm); low stringency
conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20°C lower
than the thermal melting point (Tm). Using the equation, hybridization and wash
compositions, and desired Tm, those of ordinary skill will understand that variations
in the stringency of hybridization and/or wash solutions are inherently described. If
the desired degree of mismatching results in a Tm of less than 45°C (aqueous solution)
or 32°C (formamide solution) it is preferred to increase the SSC concentration so that
a higher temperature can be used. An extensive guide to the hybridization of nucleic
acids is found in Current Protocols in Molecular Biology, Chapter 2, Ausubel, et al.,
Eds., Greene Publishing and Wiley-Interscience, New York (2000).
Thus, with the foregoing information, the skilled artisan can identify
and isolated and/or purified polynucleotides, which are substantially similar to the
present polynucleotides. In so isolating such a polynucleotide, the polynucleotide can
be used as the present polynucleotide in, for example, inhibiting the NF-KB pathway.
One embodiment of the present invention is methods of screening
for polynucleotides, which have substantial homology to the polynucleotides of the
present invention, preferably those polynucleotides encoding a protein having an
ability of inhibiting the NF-icB pathway.
The polynucleotide sequences of the present invention can be carried
on one or more suitable plasmid vectors, as known in the art for plants or the like.
In one embodiment, it may be advantageous for propagating the
polynucleotide to carry it in a bacterial or fungal strain with the appropriate vector
suitable for the cell type. Common methods of propagating polynucleotides and
producing proteins in these cell types are known in the art and are described, for
example, in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Laboratory Press, New York (1982) and Sambrook et al., Molecular Cloning:
A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York (1989).
The aforementioned embodiments are described in the context of
SEQ ID NO: 3 (CC2 region of the NEMO protein; i.e., amino acids 246-286 of SEQ
ID NO: 12) and SEQ ID NO: 7 (LZ region of the NEMO protein; i.e., amino acids
390-412 of SEQ ID NO: 12), where SEQ ID NO: 12 is murine-derived NEMO. The
aforementioned embodiments have been further described based on SEQ ID NO: 14
and SEQ ID NO: 16, which are derived from SEQ ID NO: 18 (human-derived
NEMO). However, it is understood that the present invention preferably provides
peptide derivatives of the NEMO protein that may be internalized into eukaryotic
cells. Internalization of the NEMO peptide derivatives may be imparted by fusing the
NEMO peptide derivative(s), or homologues thereof, to a polypeptide having a high
transduction potential. The skilled artisan would readily appreciate that the term
"high transduction potential" as used herein means that the polypeptide, and the fusion
protein thereof, readily transverses the cellular membrane resulting in the intemalization
of fusion peptide into the cellular milieu. Examples of peptides having a high
transduction potential include: the third helix of the Antennapedia/penetratin protein
(Ant) (Prochiantz, 2000,Curr. Opin. Cell Biol.), TAT derived peptides (Fawel, 1994,
P.N.A.S.), VP22 from HSV-1 (Stroh C. 2003, Oncogene), Pep. 1 (Morris, 2001,
Nature Biotech.).
To exemplify the present invention and the utility thereof, the
present inventors have fused SEQ ID NO: 3 and SEQ ID NO: 7 to the internalization
peptide Ant (SEQ ID NO: 1) separated by a short SKGMQ linker (SEQ ID NO: 9) or
by a LKAQADI linker (SEQ ID NO: 10). The resultant Ant-CC2 construct has the
sequence: CRQIKIWFQNRRMKWKKSKGMQLEDLRQQLQQAEEALVAKQELI
DKLKEEAEQHKIV (SEQ ID NO: 2), where the N-terminal cysteine has been added
for coupling to a flurophore to facilitate detection of internalization and/or inhibition.
The resultant Ant-LZ construct has the sequence:
CRQIIGWFQNRI^KWKKLKAQADIYKADFQAERHAREKLVEKKEYLQEQLE
QLQREFNKL (SEQ ID NO: 6), where the N-terminal cysteine has been added for
coupling to a flurophore to facilitate detection of internalization and/or inhibition.
In the present invention the N-terminal cysteine is an optional addition
and, as such, this residue may be omitted from the final inhibitory peptide.
Further, in the present invention the linker between the peptide having a high
transduction potential (e.g., Ant) and the CC2 or LZ peptide can be of a variable
sequence and/or length, so long as the linker sequence does not significantly diminish
the inhibitory property of CC2 or LZ peptide. To this end, the linker may be of a
length ranging from 1-35 amino acids, preferably 2-25 amino acids, more preferably
3-15 amino acids, most preferably 4-10 amino acids. In a particularly preferred
embodiment, the linker sequence is that of SEQ ID NO: 9 or SEQ ID NO: 10.
As set forth hereinabove, it is to be understood that the homologous
peptide of CC2 preferably retains the coiled-coil motif structure and the homologous
peptide of LZ preferably retains the helical motif structure, even when in the fusion
construct set forth above. As such, the present invention embraces homologous
peptides, within the homology constraints above, of SEQ ID NO: 2 and SEQ ID NO:
6 (murine-derived) and SEQ ID NO: 13 and SEQ ID NO: 15 (human-derived) with
the caveat that said homologous peptides retain the structure of CC2 and LZ respectively,
as well as the ability to inhibit the NF-icB pathway.
In an embodiment of the present invention, the inventors explored
the N-terminal region of the wild-type NEMO, in particular the NLM conserved motif
(residues 293-322 of SEQ ID NO: 12) appearing in Figure 1A. To this end, the
following sequences were produced (see Table 1 for the corresponding sequence):
NLM-DR (SEQ ID NO : 30)
Ant.NLM-DR (SEQ ID NO : 31)
Tat NLM-DR (SEQ ID NO : 32)
R7-NLM-DR (SEQ ID NO : 33)
R9-NLM-DR (SEQ ID NO : 34)
NLM-DR is a 21 amino acid "motif (and the corresponding wild
type NLM covering the same amino acid range) derived from the larger 30 amino acid
conserved NLM motif set forth in Figure 1 A. The NLM-DR has been mutated from
the wild type NLM sequence in that the aspartic acid at residue 11 in the wild type
sequence has been replaced by an arginine (see Table 1 and SEQ ID NO: 30). This
mutation was selected because, as confirmed by structural studies, the resulting
polypeptide would facilitate an intramolecular salt bridge allowing the stabilization of
the peptide in its helicoidal form.
From circular dichroism studies (CD), it appears that CC2 and LZ
peptides adopt a helicoidal structure, depending on their concentration. CC2 creates a
helix more stable than LZ. RMN and Rayon X Diffraction studies have confirmed the
structure of CC2. Always, by CD studies, the NLM-DR peptide is structured as a
helix more stable than the wild-type peptide.
Although, the polypeptides utilized in this example are 21 amino
acids long, it is contemplated in the present invention that the operable size of the
NLM fragment may be as short as 15 amino acids. In addition, any mutation in the
sequence of the NLM polypeptides that are able to reinforce the helicity and the
intermolecular interactions between the peptides and their molecular target would be
of particular interest and is within the scope of the present invention.
In the present invention it is speculated that Antennapedia mediated
monomerization of peptides may be crucial. Specifically, it is speculated that
monomerization allows them to interfer with NEMO oligomerization.
Nuclear factor-kB (NF-icB) signaling is an essential signal transduction
pathway involved in inflammatory responses, oncogenesis, viral infection, the
regulation of cell proliferation and apoptosis; and in the case of B and T lymphocytes
in antigenic stimulation (Ghosh, 1998, Annu. Rev. Immunol.; Karin, 1999, J. Biol.
Chem.; Israel, 2000, Trends Cell Biol.; Santoro, 2003, EMBO J..). As such, the
inventive peptides are useful for the modulation of and/or treatment of inflammatory
responses, oncogenesis, viral infection; the regulation of cell proliferation and
apoptosis; and regulation of B or T lymphocytes in antigenic stimulation. Therefore,
the present invention provides for a method of treating the same by administering to a
subject in need thereof a peptide in accordance with the present invention.
In the present invention, the "subject in need thereof may be a
human, a domestic animal, a farm animal, or an animal that is generally found in the
wild. For example, the subject may be selected from a human, a dog, a cat, a horse, a
cow, a mouse, a guinea pig, a sheep, a pig, etc.
Clearly, the amount of the peptide to be administered will depend on
the subject to which it is to be administered. In the case where the subject is a human,
the amount of the peptide to be administered will depend on a number of factors
including the age of the patient, the severity of the condition and the past medical
history of the patient and always lies within the sound discretion of the administering
physician. Generally, the total daily dose of the compounds of this invention
administered to a human or other mammal in single or in divided doses can be in
amounts, for example, of from 0.1 mg/Kg/day to 30 mg/Kg/day of the peptide,
preferably from 0.1 mg/Kg/day to 20 mg/Kg/day of the peptide, more preferably from
2 mg/Kg/day to 10 mg/Kg/day of the peptide, in single or multiple doses. Single dose
compositions may contain such amounts or submultiples thereof to make up the daily
dose. In a preferred embodiment, the preferred dose is 10 mg/patient to be
administered twice a day. In a particularly preferred embodiment, the administration
route is intravenous.
The peptides of the present invention may also be administered as a
component of a pharmaceutically administrate composition. In other words, the
peptide may be present in a formulation for administration to a subject in need thereof.
The inventive peptide may be the sole active ingredient for NF-icB pathway inhibition
or for treatment of inflammatory responses, oncogenesis, viral infection, the regulation
of cell proliferation and apoptosis and antigenic stimulation. Alternatively, the
composition may also contain one or more additional compounds that may be used to
treat the same. In addition, the peptide of the present invention may be in a composition
that contains one or more compounds that are useful for treatment of a disorder
not caused by the NF-KB pathway.
A therapeutically effective amount of the peptides suitable for
administration in the present invention may be administered alone or in combination
with one or more pharmaceutically acceptable carriers. As used herein, the term
"pharmaceutically acceptable carrier" means a non-toxic, inert solid, semi-solid or
liquid filer, diluent, encapsulating material or formulation auxiliary of any type. Some
examples of materials which can serve as pharmaceutically acceptable carriers are
sugars such as lactose, glucose and sucrose; starches such as corn starch and potato
starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl
cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients
such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil;
safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols; such a propylene
glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as
magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water;
isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as
well as other non-toxic compatible lubricants such as sodium lauryl sulfate and
magnesium stearate, as well as coloring agents, releasing agents, coating agents,
sweetening, flavoring and perfuming agents, preservatives and antioxidants can also
be present in the composition, according to the judgment of the formulator.
The pharmaceutical compositions suitable for administration in the
invention can be administered to humans and other animals orally, rectally, nasally,
parenterally (e.g., intramuscular, intraperitoneal, intravenous or subcutaneous injection,
or implant), intracisternally, intravaginally, intraperitoneally, sublingually, topically
(e.g., as a powder, ointment, or drop), bucally, as an oral spray, or a nasal spray.
The pharmaceutical compositions can be formulated in dosage forms appropriate for
each route of administration.
Liquid dosage forms for oral administration include pharmaceutically
acceptable emulsions, microemulsions, solutions, suspensions, syrups and
elixirs. In addition to the active NEMO-derived polypeptides, the liquid dosage forms
may contain inert diluents commonly used in the art. The inert diluents may include,
water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol,
isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,
cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl
alcohol, polyethylene glycols and fatty acid esters of sorbitan, and
mixtures thereof. The liquid dosage form for oral administration may also contain
adjuvants, which include wetting agents, emulsifying and suspending agents,
sweetening, flavoring, and perfuming agents. Other dosage forms for oral
administration include, for example, aqueous suspensions containing the active
NEMO-derived polypeptides in an aqueous medium in the presence of a non-toxic
suspending agent such as sodium carboxy-methylcellulose, and oily suspensions
containing a NEMO-derived polypeptides of the present invention in a suitable
vegetable oil, for example arachis oil.
Injectable preparations, for example, sterile injectable aqueous or
oleaginous suspensions may be formulated according to the known art using suitable
dispersing or wetting agents and suspending agents. The sterile injectable preparation
may also be a sterile injectable solution, suspension or emulsion in a nontoxic
parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.
Among the acceptable vehicles and solvents that may be employed are water, Ringer's
solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils
are conventionally employed as a solvent or suspending medium. For this purpose any
bland fixed oil can be employed including synthetic mono- or diglycerides. In
addition, fatty acids such as oleic acid are used in the preparation of injectables.
The injectable formulations can be sterilized, for example, by filtration
through a bacterial-retaining filter, or by incorporating sterilizing agents in the
form of sterile solid compositions which can be dissolved or dispersed in sterile water
or other sterile injectable medium prior to use.
In order to prolong the effect of the inventive NEMO-derived
polypeptides, it is often desirable to slow the absoiption of the peptides from subcutaneous
or intramuscular injection. This may be accomplished by the use of a liquid
suspension of crystalline or amorphous material with poor water solubility. The rate of
absorption of the NEMO-derived polypeptides then depends upon its rate of dissolution,
which, in turn, may depend upon crystal size and crystalline form. Alternatively,
dissolving or suspending the drug in an oil vehicle accomplishes delayed absorption of
a parenterally administered NEMO-derived polypeptides form. Injectable depot forms
are made by forming microencapsulated matrices of the NEMO-derived polypeptides
in biodegradable polymers such as polylactide-polyglycolide. Depending upon the
ratio of NEMO-derived polypeptides to polymer and the nature of the particular
polymer employed, the rate of NEMO-derived polypeptides release can be controlled.
Examples of other biodegradable polymers include poly(orthoesters) and
poly(anhydrides) Depot injectable formulations are also prepared by entrapping the
NEMO-derived polypeptides in liposomes or microemulsions which are compatible
with body tissues.
Compositions for rectal or vaginal administration are preferably
suppositories which can be prepared by mixing the NEMO-derived polypeptides of
this invention with suitable non-irritating excipients or carriers such as cocoa butter,
polyethylene glycol or a suppository wax which are solid at ambient temperature but
liquid at body temperature and therefore melt in the rectum or vaginal cavity and
release the peptide.
Solid dosage forms for oral administration include capsules, tablets,
pills, prills, powders, and granules. In such solid dosage forms, the peptide is mixed
with at least one inert, pharmaceutically acceptable excipient or carrier. In addition,
the solid dosage form may contain one or more fillers, extenders, binders, humectants,
disintegrating agents, retarding agents, absorption accelerators, wetting agents, absorbents,
or lubricants. Examples of suitable fillers or extenders include, starches, lactose,
sucrose, glucose, mannitol, and silicic acid, sodium citrate and dicalcium phosphate.
Examples of suitable binders include, microcrystalline cellulose, carboxymethylcellulose,
alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia. Glycerol is
an example of a suitable humectant. Examples of suitable disintegrating agents
include, agar-agar, calcium carbonate, potato or tapioca starch, maize starch, alginic
acid, certain silicates, and sodium carbonate. Paraffin is an example of a suitable solution-
retarding agent. As absorption accelerators, any quaternary ammonium
compound may be used. Examples of suitable wetting agents include, cetyl alcohol
and glycerol monostearate. Examples of suitable absorbents include, kaolin and
bentonite clay. Examples of suitable lubricants include, talc, calcium stearate,
magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate. In the case of
capsules, tablets and pills, the dosage form may also comprise buffering agents.
The tablets may, if desired, be coated using known methods and
excipients that may include enteric coating using for example hydroxypropylmethylcellulose
phthalate. The tablets may be formulated in a manner known to those skilled
in the art so as to give a sustained release of the NEMO-derived polypeptides of the
present invention. Such tablets may, if desired, be provided with enteric coatings by
known methods, for example by the use of cellulose acetate phthalate. They may
optionally contain opacifying agents and can also be of a composition that they release
the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract,
optionally, in a delayed manner. Examples of embedding compositions that can be
used include polymeric substances and waxes.
Similarly, capsules, for example hard or soft gelatin capsules,
containing the active peptide with or without added excipients, may be prepared by
known methods and, if desired, provided with enteric coatings in a known manner.
The contents of the capsule may be formulated using known methods so as to give
sustained release of the active NEMO-derived polypeptides. In such solid dosage
forms the active NEMO-derived polypeptides may be admixed with at least one inert
diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is
normal practice, additional substances other than inert diluents, e.g., tableting lubricants
and other tableting aids such a magnesium stearate and microcrystalline cellulose.
In the case of capsules, tablets and pills, the dosage forms may also comprise
buffering agents. They may optionally contain opacifying agents and can also be of a
composition that they release the active ingredient(s) only, or preferentially, in a
certain part of the intestinal tract, optionally, in a delayed manner. Examples of
embedding compositions that can be used include polymeric substances and waxes.
Solid compositions of a similar type may also be employed as fillers
in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar
as well as high molecular weight polyethylene glycols and the like.
If desired, the NEMO-derived polypeptides of the present invention
can be incorporated into slow release or targeted delivery systems such as polymer
matrices, liposomes and microspheres. They may be sterilized, for example, by filtration
through a bacteria-retaining filter, or by incorporating sterilizing agents in the
form of sterile solid compositions that can dissolve in sterile water, or some other
sterile injectable medium immediately before use.
The NEMO-derived polypeptides may be formulated into granules
with or without additional excipients. The granules may be ingested directly by the
patient or they may be added to a suitable liquid carrier (for example, water) before
ingestion. The granules may contain disintegrates, e.g. an effervescent couple formed
from an acid and a carbonate or bicarbonate salt to facilitate dispersion in the liquid
medium.
Dosage forms for topical or transdermal administration of the
peptide of this invention include ointments, pastes, creams, lotions, gels, powders,
solutions, sprays, inhalants or patches. Transdermal patches have the added advantage
of providing controlled delivery of a peptide to the body. The rate can be controlled by
either providing a rate controlling membrane or by dispersing the peptide in a polymer
matrix or gel. The active component is admixed under sterile conditions with a
pharmaceutically acceptable carrier and any needed preservatives or buffers as may be
required. Dissolving or dispensing the peptide in the proper medium can make such
dosage forms. Absorption enhancers can also be used to increase the flux of the
peptide across the skin. Ophthalmic formulation, eardrops, eye ointments, powders
and solutions are also contemplated as being within the scope of this invention.
Dosage forms for topical administration may comprise a matrix in
which the pharmacologically NEMO-derived polypeptides of the present invention are
dispersed so that the peptides are held in contact with the skin in order to administer
the peptides transdermally. A suitable transdermal composition may be prepared by
mixing the pharmaceutically active NEMO-derived polypeptides with a topical
vehicle, such as animal and vegetable fats, oils, petrolatum, waxes, paraffins, starch,
tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic
acid, talc and zinc oxide, or mixtures thereof, together with a potential transdermal
accelerant such as dimethyl sulphoxide or propylene glycol. Alternatively the active
NEMO-derived polypeptides may be dispersed in a pharmaceutically acceptable paste,
cream, gel or ointment base. The amount of active NEMO-derived polypeptides
contained in a topical formulation should be such that a therapeutically effective
amount of the peptides are delivered during the period of time for which the topical
formulation is intended to be on the skin.
Powders and sprays can contain, in addition to the NEMO-derived
polypeptides of this invention, excipients such as lactose, talc, silicic acid, aluminum
hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons.
The therapeutically active NEMO-derived polypeptides may be formulated
into a composition, which is dispersed as an aerosol into the patient's oral or nasal
cavity. Such aerosols may be administered from a pump pack or from a pressurized
pack containing a volatile propellant.
The therapeutically active NEMO-derived polypeptides used in the
method of the present invention may also be administered by continuous infusion
either from an external source, for example by intravenous infusion or from a source
of the NEMO-derived polypeptides placed within the body. Internal sources include
implanted reservoirs containing the NEMO-derived polypeptides to be infused which
is continuously released for example by osmosis and implants which may be (a) liquid
such as an oily suspension of the peptides to be infused for example in the form of a
very sparingly water-soluble derivative such as a dodecanoate salt or a lipophilic ester
or (b) solid in the form of an implanted support, for example of a synthetic resin or
waxy material, for the NEMO-derived polypeptides to be infused. The support may be
a single body containing the entire quantity of the peptides or a series of several
bodies each containing part of the quantity of the peptides to be delivered. The amount
of active peptides present in an internal source should be such that a therapeutically
effective amount of the peptides are delivered over a long period of time.
The invention also relates to the use of an effective amount of a
composition comprising a polypeptide fusion construct according to the invention and
one or more pharmaceutically acceptable carriers or excipients, for the preparation of
a medicament for modulating or treating a disorder regulated by the NF-xJB signaling
pathway in a subject in need thereof.
According to an advantageous embodiment of said use, said disorder
regulated by the NF-icB signaling pathway is selected from the group consisting of
inflammatory responses, oncogenesis, and viral infection.
The invention also relates to the use of an effective amount of a
composition comprising a polypeptide fusion construct according to the invention and
one or more pharmaceutically acceptable carriers or excipients, for the preparation of
a medicament for regulating cell proliferation or apoptosis in a subject in need thereof.
The invention also relates to the use of an effective amount of a
composition comprising a polypeptide fusion construct according to the invention one
or more pharmaceutically acceptable carriers or excipients, for the preparation of a
medicament for regulating B or T lymphocytes in antigenic stimulation in a subject in
need thereof.
According to an advantageous embodiment of said uses, said subject
in need thereof is a human.
According to another advantageous embodiment of said uses, said
effective amount ranges from 0.1 mg/Kg/day to 30 mg/Kg/day.
According to another advantageous embodiment of said uses, said
composition is administered in a form selected from the group consisting of oral,
rectal, nasal, parenteral, intracisternal, intravaginal, intraperitoneal, sublingual,
topical, and bucal administration.
According to another advantageous embodiment of said uses, said
composition is administered preferably intravenously.
The present invention further provides a method of identifying
polypeptides that modulate oligomerization of NEMO by:
a) identifying a candidate polypeptide sequence;
b) creating a polypeptide fusion construct by linking said candidate
polypeptide sequence to a polypeptide having a high transduction potential via a
spacer sequence;
c) contacting a cell culture with the polypeptide fusion construct; and
d) monitoring the activity of theNF-icB signaling pathway;
e) comparing the activity of the NF-KB signaling pathway in the
presence of said polypeptide fusion construct to the activity of the NF-icB signaling
pathway in the absence of said polypeptide fusion construct to determine the relative
inhibition by said polypeptide fusion construct; and
f) correlating relative inhibition by said polypeptide fusion construct to
NEMO oligomerization.
In this method, the candidate polypeptide sequence preferably has a
coiled-coil or helical structure. More preferably, the candidate polypeptide sequence
has 20-60 amino acids. It is also preferred that the candidate polypeptide sequence be
derived from NEMO.
As stated in the embodiments above, the spacer sequence may have
a length ranging from 1-35 amino acids, but shorter lengths may also be employed
[supra). Examples of the spacer sequence includes: SEQ ID NO: 9 and SEQ ID NO:
10. Additionally, an example of the polypeptide having a high transduction potential
is a polypeptide having the amino acid sequence of SEQ ID NO: 1.
In a preferred embodiment, the cell culture contains pre-B 70Z/3
lymphocytes that have been transfected with NF-xJB dependent p-glactosidase reporter
gene.
In order to ensure that the polypeptide fusion construct is actually
incorporated into the cells contained in the cell culture it is desired that the polypeptide
fusion construct have an N-terminal cysteine residue. In this manner, the
polypeptide fusion construction may be labeled by chemically reacting the cysteine
residue with a fluorophore (e.g., BODIPY) thus enabling monitoring of cellular uptake
by a technique such as FACS.
Accordingly, the method of identifying polypeptides that modulate
oligomerization of NEMO may also include the following steps:
b-1) labeling said polypeptide fusion construct; and
c-1) monitoring cellular uptake of the labeled polypeptide fusion
construct.
Further, one of skill in the art may also be able to correlate NF-icB
pathway inhibition with modulation of NEMO oligomerization by a pull down
experiment with tagged peptides to show that NEMO associates in vivo with the
peptides. The oligomeric state of this peptide associated NEMO protein could be
characterized (cross-linking, gel filtration). In vitro, Inhibition of the anisotropy
increase resulting from the association of fluorescent antennapedia labelled CC2 or
LZ peptides with CC2 or LZ peptides, mimicking NEMO oligomerization, could be
used to test compounds inhibiting NF-icB pathway in vivo.
Having generally described this invention, a further understanding
can be obtained by reference to certain specific examples, which are provided herein
for purposes of illustration only, and are not intended to be limiting unless otherwise
specified.
EXAMPLES
Materials and methods
Cell culture, stable transfections and cell lines
The grow conditions of the murine pre-B 70Z/3 were as described in
Courtois et al., 1997 Mol. cell. Biol. 70Z3-C3 stable cell lines were prepared by
electroporation as described in Courtois et al. with the plasmid cx!21acZ-kB (a kind
gift from G.R. Crabtree), bearing three tandem copies of NF-icB sites in the IL-2
promoter (Fiering et al., 1990), The human retinoblastoma cell lines Y79 were
purchased from the American Type Culture Collection (Manassas, VA) and grown in
RPM1 1640 medium supplemented with 100 U/ml penicillin, 100 |ig/ml streptomycin,
and 10 % fetal calf serum (PCS).
FACS analysis
0.5 x 106 70Z/3-C3 cells in 0.5 ml were incubated at 37°C for
different times and with various concentrations of peptides as indicated in the Figure
legends. The cell suspension was centrifugated at 1,000 x g at room temperature and
the cell pellet was then washed three times with PBS buffer (1 ml), and finally was
resuspended with 500 \i\ of PBS buffer containing 0.1 % sodium azide. Fluorescence
analysis was performed with a FACSCalibur (BD Biosciences) and a minimum of
15,000 events per sample was selected. All experiments were performed in duplicates.
Peptide synthesis and purification
Peptides were synthesized as described in Mousson et al. 2002
(Biochemistry, 41 p!3611- p!3616), by using continuous-flow Fmoc/tBu chemistry
(Chan, WC, and White, P.D. (2000); Fmoc Solid Phase Peptide Synthesis. A pratical
approach) on an Applied Biosystems (Foster City, CA) Pioneer peptide synthesiser.
All chemical reagents were purchased from Applied Biosystems. All peptides were
blocked at the N-terminus with an acetyl group and at the C-terminus with an amide.
A single extra-cysteine residue was incorporated at the N-terminus of the peptides for
subsequent specific labeling (see Table 1).
(Table Removed) (1) In all peptides the N-terminus contains a cysteine residue for convenience of specific peptide coupling with the maleimide group as
described in the "Material and Methods." The sequences of antennapedia, TAT and poly-arginine (R7 or R9) fused to the NEMO
sequence (plain text) are highlighted in bold characters. Residues that may be involved in coiled-coil sequence are underlined and those
that were replaced in the CC2 and LZ mutants and in NLM-DR are underlined in bold characters. B=Bodipy (The C-terminal Bodipy
modification has been omitted from the sequences appearing in the Sequence Listing) Within the scope of the present invention it is
contemplated that Bodipy and/or the N-terminal cysteine may be removed and used as described herein.
Crude peptides were directly purified by reverse-phase mediumpressure
liquid chromatography (MPLC) on a Nucleoprep 20 uM CIS 100 A
preparative column, using a linear gradient of acetonitrile (1%/min) in 0.08 % aqueous
trifluoroacetic acid (TFA) (pH 2) for 60 min at a flow rate of 18 ml/min. The purity of
the peptides was verified on a nucleosil 5 uM CIS 300 A analytical column, using a
linear gradient of acetonitrile (0.5 %/min) in 0.08 % aqueous TFA (pH2) for 20 min at
a 1 ml/min flow rate. Conjugation of the fluorophore bodipy®FL N-(2 aminoethyl)
maleimide (Molecular Probes) to the sulfhydryl group was under equimolar
conditions at pH 6 in 50 mM ammonium acetate buffer for 30 min in the dark. The
mixture was then loaded on a Nucleoprep 20 uM C18 100 A preparative column to
purify the BODIPYconjugated peptide. In cell death experiments, all peptides devoid
of BODIPY-labeling were subjected to treatment with iodoacetamide to prevent any
oxidation of cysteine residue. All purified peptides were then quantified by amino acid
analysis and finally characterized by using positive ion electrospray ionization mass
spectrometry (ES+). Once integrity of the peptides and coupling efficiency were
verified by mass spectrometry, the extinction coefficients of the peptides were
measured at 505 nm or at 280 nm when peptides contained aromatic residues (see
Table 1). Stability of the labeling was monitored periodically by measuring the
absorbance of peptides at 280 nm and at 505 nm and by calculating the absorbance
ratio. All peptides were dissolved in water to stocks of 2 mM.
NF-rcB inhibition assays
In a first procedure 2.2 x 105 70Z/3-C3 cells in 220 of RPMI1640
supplemented with 10% fetal calf serum (PCS) and 50 uM (3-mercaptoethanol were
placed in a 96-well plate and incubated with various concentrations of peptide (0 to 20
jj.M) at 37°C in 5% COj incubator. After two hours an equal portion (100 jil) of each
cell sample transferred in two wells containing each 10s cells. One aliquot of cells was
then treated for 5 hours with lipopolysaccharides from Salmonella abortus (Sigma) at
0.5 ug/ml final concentration, and the other one left-treated. After 5 hours, cells were
centrifuged at 400 x g for 5 min at room temperature and the cell pellets were washed
three times with cold PBS (250 ul) by centrifugation. Cells were then lysed in the
lysis buffer (25 mM tris-phosphate buffer at pH 7.8 containing 8 mM magnesium
chloride, 1 mM dithioerythreitol, 1 % Triton X-100, 15 % glycerol and a protease
inhibitor mixture (Roche)), and samples were centrifuged at 4°C for 20 min to clarify
the lysate. The supernatant was then kept on ice, and 30 ul was then assayed to
measure the p-galactosidase activity with a plate luminometer (Berthold) using the
galacton-star as chemiluminescent substrate (BD Biosciences Clontech, Bronstein et
al., 1989). Background of reaction was measured by mixing for 1 hour 30 ul of lysis
buffer with the reaction buffer (196 ul) and the galacton-star substrate (4 ul) provided
by BD Biosciences. In a second procedure and a more stringent assay, 70Z/3-C3 cells
(2.2 x 105 in 220 (al medium) were centrifuged at 400 x g at room temperature after
peptide internalization for 2 hours, and cell pellets were washed three times with 200
ul of PBS by centrifugation. Cells were then diluted three times with complete
medium, and allowed to grow for at least 24 hours. The following steps are identical
to the first procedure.
Cell death assays
The detection of cell death was performed using the MTS assay
provided by Promega (CellTiter 96® AQue0us one solution cell proliferation assay).
Briefly, 0.3 x 106 Y79 cells in 450 ul were treated with 50 ul of the wild type Ant-
CC2 and Ant-LZ peptides (0.1 to 20 uM) or their mutants Ant-CC2 (Mu), Ant-LZ
(Mu) or the Ant or left untreated in serum-free RPMI medium at 37°C. After an incubation
of 1 or 14 hours, an aliquot of the cell suspension (200 ul, 0.12 x 106 cells),
was then transferee in 96-well plates and mixed with the MTS solution (40 ul)
containing the MTS compound and the phenazine ethosulfate. Two hours after, the
quantity of formazan produced by viable cells was measured using an automated
microplate reader (Bio-TeK Instruments, INC) at 490 run absorbance. Cell survival
was observed under microscope and was estimated as a percentage of the value of
untreated controls. The background of the reaction was determined by mixing the
MTS solution with cell-free RPMI medium. To increase the sensitivity of the cell
death assay, the present inventors used peptides devoid of BODIPY-labeling because
the absorption spectra of the fluorophor overlaps with that of the formazan product.
All experiments were repeated twice and each experiment condition was repeated in
duplicate wells in each experiment.
Analytical Gel filtration experiments
The oligomeric states of peptides were determined by filtration as
described hi Traincard et al, 2003. In brief, 500 ul samples were loaded on a
Superdex 75 HR 10/30 column equilibrated in 50 mM Tris-HCl pH 8.0 containing 200
mM NaCl and 0.1 mM DDM, at a constant flow rate of 0.4 ml/min. The presence of
the DDM detergent was added in the equilibrium buffer to minimize the adsorption in
the column and to increase the peptide recovery. The column was calibrated in the
same equilibrium buffer with blue dextran 2000 (void volume), dithioerythritol (total
volume), bovine serum albumin (67 kDa, Rs == 35.2 A), ovalbumine (43 kDa, Rs =
27.5 A), chymotrypsinogen A (25 kDa, Rs = 21.1 A), ribonuclease A (13.7 kDa, Rs =
16.4 A), cytochrome C (12.4 kDa, Rs = 17.7 A) and aprotinin (6.5 KDa, Rs = 13.5 A).
Fluorescence anisotropy measurements
Anisotropy measurements were performed with a PTI Quantamaster
fluorometer equipped with polarizers for the excitation and emission beams. This
instrument uses a PMT in the L-configuration. AH experiments were carried out in a 1
cm path-length cuvette at 22°C with excitation and emission wavelengths at 495 nm
and 520 nm, respectively. The bandpass of excitation and emission monochromators
was set at 2 and 4 nm, respectively. Steady-state fluorescence anisotropy was
expressed as millianisotropy (mA) and was calculated according to the equations: (1)
A= (Iw-GIvH)/(Ivvf2GIvH); (2) G=!HV/!HH-; where A is anisotropy, G is a correction
factor for wavelength-dependent distortion and I is the fluorescence intensity
component (subscript referring to the vertical and horizontal positioning at the excitation
and emission polarizers, respectively). Experiments were at least performed twice
and each data is the result of 20 records along a 2 min period. All measurements were
carried out in 50 mM Tris-HCl buffer at pH 8 containing 150 mM KC1. The present
inventors verified that at the BODIPY-Ant-CC2 and BODIPY-Ant LZ concentration
used (luM and 0.1 uM respectively), the filter effect was negligible. The BODIPYAnt-
CC2 peptide was preincubated overnight at 22°C alone or with increasing
concentrations (1-125 uM) of CC2 prior to anisotropy measurement. The BODIPYAnt-
LZ peptide (100 nM) was preincubated overnight at 22°C alone or with 10 uM
and 100 M concentrations of CC2 (see legend Fig. 7) prior to anisotropy measurement.
The dissociation constant parameter was estimated by globally fitting the
anisotropy data to binding isotherm equation as described in Agou et al. (2004, J Biol
Chem.) using Kaleidagraph nonlinear regression software (Synergy Software, reading
PA). The binding stoichiometry, n, was estimated from the intersection of lines
(dashed lines in Figure 7) drawn through the descending and plateau region of the
anisotropy data.
Results
Rational design of NEMO derived peptides that block NF-KB activation
The present inventors previously demonstrated that the minimally
trimerization domain of NEMO comprised of the sequence 251 to 337 (Fig. 1A). This
region likely contains two coiled-coil sequences of about 35 residues denoted CC2
(residue 253-285) and LZ (301-337) at the N- and C-terminus respectively. Although
the structure of the minimal oligomerization domain has not yet been determined,
several biochemical studies combined with the fluorescence polarization method
prompted us to propose that the CC2/LZ trimer probably forms a six-stranded helical
bundle composed of closely packed CC2 and LZ coiled-coils in an antiparallel orientation
(Traincard, 2003, submitted). Furthermore PSI-BLAST searches reveal that this
domain of NEMO contains a conserved motif of 20 residues called "NEMO like
Motif (NLM) which is shared with four other proteins including ABIN-1 (Heyninck,
1999, J. Cell. Biol.), ABIN-2/NAF (Van Huffel, 2001, J. Biol. Chem), ABIN-3/LIND
(Staege, 2001, Immune genetics) and NRP/optineurin (Schwamborn, 2000, J. Biol.
Chem) (Fig. IB). Interestingly, most of these proteins including the conserved motif
of ABIN-1 (Heyninck, 2003, FEBS Letters), the C-terminal domain of NEMO (Le
Page, 2001, Virology) and ABIN-2 (Liu WK, 2003, Biochemical Journal) or ABIN-
3/LIND (Heyninck, 2003, FEBS letters) proteins have been shown to inhibit NF-icB
activation in a dominant-negative manner when overexpressed in cells. Two
additional references that disclose ABIN peptides interacting with NF-icB also warrant
mention: WO 99/57133 and WO 03/00280.
Since disrupting NEMO oligomerization represents a potential
therapeutic strategy for inhibiting NF-KB activation, the present inventors designed
NEMO-derived partner peptides that mimic either the CC2 or the LZ sequence (Table
1). It is interesting to note that, unlike the CC2 peptide, the LZ peptide also includes
the NLM motif at the N-terminal extremity. To mediate all peptide uptake into cells,
the present inventors conjugated a functional analogue at the peptide N-terminus
comprised of the 16-amino acid sequence derived from the third helix of the
Antennapedia/penetratin protein (Ant). This amphipatic helix acts as an internalization
vector (Prochiantz, 2000, Curr. Opin. Cell Biol.). Most of antennapedia fusion
peptides were labeled with the BODIPY fluorophore to analyze the transduction
potential of each peptide into the cells. Specific labeling was performed by adding a
single cystein residue at the extremity of the N-terminus and sequence integrity was
verified by mass spectrometry (see "Materials and methods" and Table 1).
Cellular uptake of NEMO derived-peptides mediated by the
antennapedia fusion peptide
The uptake of BODIPY labeled NEMO peptides into living cells
were monitored by fluorescence activated cell sorting (FACS) which is a conventional
tool used to quantify cellular internalization. Figure 2A shows FACS analyses of cells
treated with Ant-CC2 (WT), Ant-CC2 (Mu), Ant-LZ (WT) or Ant-LZ (Mu) BODIPYpeptides
for 2 h at 37°C, and were compared with those of the autofluorescence of
untreated cells and control cells treated with an equal concentration of free BODIPY
or with BODIPY-conjugated BSA. Consistent with the role of antennapedia peptide to
transduce peptides and proteins into mammalian cells, 100% of 70Z3-C3-cell line was
similarly transduced by the four different NEMO peptides, suggesting that all of the
cells in the treated population have a near identical intracellular concentration of
NEMO-derived BODIPY-peptides. Comparative analysis indicate that untreated cells
and treated cells with BODIPY-BSA or free BODIPY exhibit a similar cell fluorescence,
verifying that our extensive washing protocol before FACS analysis was
optimal to minimize any contribution of surface-bound peptide in measuring NEMO
peptide internalization (see "Materials and methods"). Thus, these data suggest that
the observed cellular fluorescence signaling mostly reflects the intracellular concentration
of transduced NEMO peptide and not a non-specific adsorption onto the
membrane surface.
Then present inventors next investigated the kinetic and concentration
dependency of cellular uptake for the Ant-CC2 BODIPY peptide keeping in mind
that the transduction of other NEMO peptides should occur in a similar fashion (Fig.
2B and 2C). FACS analysis 5 h after addition of 70Z3-C3 cell treated with 0.2,2 or 20
M.M BODIPY-Ant-CC2 peptide at 37°C demonstrate the linear dependancy of the
intracellular concentration as a function of the incubated concentration of the
antennapedia fusion peptide as widely reported in literature (Lindsay, 2002, Current
Opinion in Pharmacology). Notably, the cells treated with the Ant-CC2 at 20 uM and
at 37°C already reach maximum intracellular concentration in 30 min and remain
unchanged for up to 5 h. Since the time to induce a strong NF-icB activation in
response to LPS requires 3-5 hours of cell treatment, these results indicate that the
intracellular concentration of each peptide remains constant during the LPS stimulation.
Specific inhibition of LPS-induced NF-KB activation by cell
permeable CC2 and LZ
To analyze the inhibition potential of LPS-induced NF-icB activation
by cell permeable BODIPY-Ant-CC2 and BODIPY-Ant-LZ peptides, the present
inventors stably transfected the murine pre-B 70Z3 cell line with p!2XlacZ-kB, which
bears the p-galactosidase reporter gene under the control of the NF-icB transcription
factor. When the resulting cell line 70Z3-C3 was treated for 5 hours 'with LPS (3
p.g/ml) a 100 fold-activation of the LacZ gene was observed, indicating that our
cellular assay monitors NF-KB activation in response to LPS with extreme sensitivity
(Figure 3 A, control "no peptide). Interestingly the incubation of cells with 20 jaM of
both NEMO-derived polypeptides decreased significantly the NF-KB activation. This
lowering was stronger in the presence of BODIPY-Ant-LZ as compared to BODIPYAnt-
CC2. The inhibition effect was essentially due to the NEMO sequence because
the presence of the isolated and/or purified antennapedia peptide containing or not
containing a N-terminal BODIPY label (BODIPY-Ant or Ant) induces the same level
of NF-KB activation as the control (Fig. 3A). Note that the basal NF-KB activity
measured in the absence of LPS was very similar in all samples indicating that both
CC2 and LZ peptides abolish the responsiveness to LPS without affecting the intrinsic
basal NF-KB activity. This was essential to minimize the in vivo cytotoxity, resulting
mainly from apoptosis induced by inhibition of NF-KB (Chen, 2003, Nature Med.).
To determine whether the BODIPY-Ant-LZ or the BODIPY-Ant-
CC2 peptide is the most efficient inhibitor, the present inventors next measured the
concentration dependent inhibition of each peptide. As shown in Figure 3B, both
NEMO peptides exhibit NF-KB dose dependant inhibition of NF-icB in response to
LPS. BODIPY-Ant-LZ inhibited NF-icB to a greater extent than BODIPY-Ant-CC2
did with IC5o values of 3 ^M and 22 u.M respectively (Fig. 3C). This striking
difference could be explained by the NLM motif included in the LZ sequence.
Consistent with the intracellular nature of the NEMO target, both LZ and CC2
peptides not fused to the antennapedia protein transduction domain (PTD) exhibited
the same level of activation as the control (Fig. 3 D), confirming that NEMO derived
peptides must cross the cell membrane for inhibition of NF-KB. Taken together these
results indicate that peptides that mimic the two coiled-coil sequences of the NEMO
oligomerization domain are potent peptide inhibitors of NF-icB activation in response
to LPS.
Mutations in the hydrophobia core of the LZ and CC2 coiled-coils
disrupt their specific inhibition of the NF-KB signaling pathway
Theoretically, if BODIPY-Ant-LZ or BODIPY-Ant-CC2 peptide
inhibit NF-KB activation through specific binding to the NEMO oligomerization
domain, mutations that disrupt the coiled-coil association should therefore exhibit
impaired abilities to inhibit NF-KB inhibition, cc-helical coiled-coil interactions have
been extensively studied and most of the rules governing their specific assembly have
been well documented (Vinson, 2002, Mol. Cell. Biol.). The coiled-coil interface that
is represented by the first (a) and fourth position (d) of the heptad repeat is generally
occupied by hydrophobic amino acids. Proline or glycine is largely excluded to
preserve the helical architecture. Core polar residues are destabilizing relative to
leucine substitutions, especially when changes occur at d positions. Considering these
rules, the present inventors synthesized a variant of BODIPY-Ant-LZ containing two
mutations L - S at the d positions (BODIPY-Ant-LZ (Mu) and a variant of BODIPYAnt-
CC2 containing two mutations L —> G and one mutation I -> G at the a positions
(BODIPY-Ant-CC2 (Mu) (Fig. 4A and 4B, and Table 1). To test the effects of these
mutations on the potential inhibition of NF-KB activation, the present inventors
developed a more stringent cellular assay that consists of the internalization of the
peptides for 2 hours followed by an extensive washing of 70Z3-C3 cells to remove
any remaining peptide in the extracellular media. Cells were allowed to grow for at
least 24 hours before LPS-induced NF-icB activation. In this way, peptide interference
with the receptor binding LPS was excluded. As with the cellular assay described
above, the BODIPY-Ant-CC2 (WT) and the BODIDY-Ant-LZ (WT) also inhibited
NF-icB activation with a 1.7 and 5.8 -fold reduction respectively (Fig. 4A and 4B)
when used at a 10 jaM concentration. This indicates that the peptides do not
competively act on the receptor binding of LPS. As expected, the presence of the CC2
variant (BODIPY-Ant-CC2 (Mu)) did not affect the NF-KB activation since pgalactosidase
activity was equivalent to that of the control (Fig. 4A, no peptide). In
response to LPS, NF-icB is more strongly activated in the presence of the BODIPYAnt
LZ mutant than in the presence of wild type. However, unlike the BODIPY-Ant-
CC2 (Mu), a slight inhibition of the LZ mutant was observed when compared to the
control (15 %). When taken together, these data demonstrate that CC2 and LZ mutants
are unable to inhibit the LPS-induced NF-icB activation as effectively the wild type
did.
Inhibition of NF-KB activation is mediated by a specific coiled coil
interaction of the LZ peptide.
Computational analyzes using the program MULTICOIL (Wolf,
1997, Protein Science) predicted that greater than 5 % of all putative ORFs found in
sequenced genomes are predicted to contain coiled-coil motifs (Newman, 2000, Proc.
Natl. Acad. Sci. USA) and that approximately 2-4 % of amino acids in proteins are
estimated to adopt coiled-coil folds (Berger, 1995, Proc.Natl.Acad.Sci. USA). This
abundance raises the question if the NEMO derived- LZ peptide maintains its coiledcoil
interaction partnering specificity in vivo. To address this question, the present
inventors synthesized another coiled-coil peptide that mimics the sequence of the
GCN4 leucine zipper and tested its ability to inhibit NF-icB activation. BODIPY-Ant-
GCN4 contained the antennapedia sequence at its N-terminus and a short SKGMQ
linker (SEQ ID NO: 9) identical to the CC2 sequence for convenience of peptide
delivery (Table 1). It was also labeled at its N-terminus with BODIPY to monitor its
cellular uptake by FACS (data not shown). The GCN4 peptide displays a low
sequence similarity with the LZ sequence of NEMO (22 %) but identical residues are
mostly represented by leucines at d positions (Figure 5). These residues contribute to
most of the energy to coiled-coil oligomerization stability (Vinson, 2002, Mol. Cell.
Biol.)- Note that a positions which are important for coiled-coil specificity (Vinson,
2002, Mol. Cell. Biol) are composed of a set of different amino acids. While GCN4 is
composed of hydrophobic residues and the typical asparagine residue, the LZ of
NEMO contains two charged amino acids R and K (Figure 4B and Fig. 5A). Thus,
these residues, which are located at the coiled coil interface likely, contributes to the
selectivity of coiled coil interaction.
Figure 5B shows the effect of the BODIPY-Ant-GCN4 at a 10 uM
concentration on the inhibition of NF-KB activation in response to LPS. To compare
the effects of coiled coil sequences, the present inventors used the stringent cellular
assay described above. BODIPY-Ant-GCN4, unlike BODIPY-Ant-LZ, has no ability
to inhibit NF-icB activation since the level of NF-KB activation was near that of the
control without peptide. Taken together, these results strongly support the hypothesis
that the LZ peptide of NEMO inhibits NF-KB activation through selective coiled-coil
interactions.
The antennapedia sequence induces monomerization of NF-KB
peptidic inhibtors
The antennapedia sequence is a protein transduction domain (PTD)
which adopts an alpha-helical amphiphatic structure (Prochiantz, 2000, Curr. Opin.
Cell Biol.). When fused to the N terminus of a coiled-coil sequence like CC2 or LZ,
the antennapedia could alter the coiled-coil association by covering the hydrophobic
interface of the coiled-coil through intramolecular interactions. To examine the effect
of N-fusion of the antennapedia peptide on the oligomerization properties of the CC2
and the LZ peptides, the present inventors analyzed peptides containing or not
containing the antennapedia sequence at their N-terminus by gel filtration. As shown
in Figure 6, all peptides containing an N-terminal fusion of antennapedia co-elute with
an elution volume corresponding to their monomeric forms as compared to globular
protein markers. Note that the present inventors had to add a detergent in the buffer
below its cmc to improve peptide recoveries. When injected at the same 10 uM
concentration, CC2 wild type and LZ wild type without the antennapedia N-fusion
oligomerize. CC2 (WT) forms a trimer whereas LZ (WT) forms a dimer as recently
reported (Traincard et al). As expected, when a CC2 mutant was chemically obtained
with three of its aliphatic residues at positions replaced with glycine residues, it lost its
ability to oligomerize (bottom panel, dashed line). The effect of the two L-»S mutations
at d positions was less strong with LZ (mutant). However, the present inventors
still detected dimerization of the LZ mutant at a 10 uM concentration (dashed line)
although its association was markedly reduced by mutations as compared to the wild
type LZ (solid line). Taken together, these data indicate that the N-fusion of the
antennapedia sequence to both CC2 and LZ peptides alter homotypic coiled-coil interactions,
facilitating the monomerization of the NEMO derived-peptides. Furthermore
these results also show that residue changes at a and d position alter oligomerization
of LZ and CC2 peptides. Thus, it is likely that the synthetic peptides form a-helical
coiled-coil structures.
Homo- and heterotypic interactions of CC2 and LZ peptides with
and without the N-fusion of antennapedia sequence.
Because the N-fusion of antennapedia modifies the oligomerization
properties of the CC2 and LZ peptides, the present inventors next studied by fluorescence
polarization whether the Ant-CC2 and the Ant-LZ monomers labeled with
BODIPY could bind to the NEMO-derived polypeptides devoid of the antennapedia
sequence. These peptides CC2 and LZ may be also considered as the in vivo binding
target for both cell permeable BODIPY-Ant-CC2 and BODIPY-Ant-LZ NF-tcB
inhibitors. Figure 7 shows a typical binding isotherm for the interaction of various
concentrations of the CC2 peptide with a fixed concentration of the BODIPY-Ant-
CC2. The shape of the binding curve is not sigmoidal, indicating that CC2 binds to the
BODIPY-Ant-CC2 peptide without cooperativity. The stoichiometry calculated from
the intercept between the tangent of the initial part of the anisotropy and the asymptote
is equal to 0.8. Taking into account this stoichiometry, the dissociation constant KD is
15.2 uM. Similar results were obtained when a fixed concentration of the BODIPYAnt-
LZ was titrated with various concentrations of the CC2 peptide as the present
inventors previously reported (Inset Figure 7, Traincard et al.). Collectively, these data
demonstrate that both Ant-CC2 and Ant-LZ monomers binds in vitro to the CC2
peptide composing the minimal oligomerization domain of NEMO.
Cell death in human Retinoblastoma cell is induced by NF-KB
inhibitors Ant-CC2 and Ant-LZ, but not by their mutants Ant-CC2 (Mu) and Ant-LZ
(Mu)
It has become clear that constitutively activated NF-KB transcription
factors have been associated with several aspects of tumorigenesis (Karin review),
including most of six essential alterations in cell physiology that dictate the conversion
of normal human cells into cancer cells (Hanahan, 2000, Cell for review). This
led to a significant enthusiasm for the use of NF-KB inhibitors as a new anti-cancer
therapy. Promising results have been reported recently using proteasome inhibitors or
the SN50 peptide that blocks the nuclear translocation (Orlowski, 2002, Trends in
Molecular Medicine; Mitsiades, 2002, Blood). However, the specificity of these
agents on NF-KB inhibition have been questioned. Poulaki et al. (2002, Am J Pathol..)
showed recently that the treatment of the human retinoblastoma (Rb) cell lines Y79
with SN50 peptide induced apoptosis of cancer cells. Sequence alignment of murine
and human NEMO proteins indicate that the minimal oligomerization domain of
NEMO is strictly conserved, suggesting that similar effects of NF-KB inhibition could
be observed in rodent as well as in human cells.
Given that specific NF-KB inhibition may trigger apoptosis of cancer
cells, the present inventors examined the effects of both cell-permeable Ant-LZ and
Ant-CC2 peptides on human retinoblastoma cell viability. In these experiments, the
present inventors used NEMO-derived polypeptides without a N-terminal BODIPY
labeling to prevent any interference with the MTS assay (see "Materials and
Methods"). As shown in Figure 7, the present inventors found a dose dependence of
the Y79 cell viability when cells were treated for 3 hours with Ant-CC2 (Fig. 8A) or
Ant-LZ (Fig 8B). The effect of the Ant-LZ peptide on cell death was stronger than
that of the Ant-CC2. This induction of cell death was significant since Rb cell survival
was 20% and 65 % with the Ant-LZ and the Ant-CC2 peptides respectively when
cancer cells were treated for 3 hours at a 20 uM concentration. Remarkably, the same
cell treatment with the Ant-CC2 (Mu) (Fig.SA) or with the Ant-CC2 (Mu) (Fig.SB)
did not induce concentration cell death as did WT peptides. These effects on cell death
were essentially due to the NEMO sequence because a longer treatment of Y79 cell
lines with the antennapedia peptide did not affect cell survival (Fig. 8C). In contrast,
80 % and 55% of Y79 cell died in the presence of Ant-LZ and Ant-CC2 respectively
at 5 uM concentration (Fig. 8C). Taken together, these results indicate that specific
NF-KB inhibition by Ant-CC2 and Ant-LZ peptides induce cell death in Rb cell lines,
validating the use of specific NF-KB inhibitors as anticancer chemotherapy.
In the absence of the pro-inflammatory signals, all peptides assayed
have no detectable cytoxicity for lymphocytes B tested at a concentration up to 30 uM
according to the MTS assay, the direct observation under microscope, and the forward
scatter-FSC and side scatter-SSC parameters deduced from FACS analyses. However,
the present inventors could detect a slight cell death by FACS in a concentrationdependent
manner when LPS stimulated the pre-B lymphocytes in the presence of
NEMO-derived polypeptides. The cellular proportion of cell death was 9 % in the
absence of stimuli at a 20 uM concentration of Ant-CC2 whereas it increased at 13 %
after LPS stimulation (data not shown). This was in agreement with the role of the
NF-icB pathway in protecting cells from apoptosis. The cell death was more
pronounced and fast on the Rb cell lines Y79 in which constitutive NF-KB activity has
been reported (Poulaki, 2002, Am J Pathol..). The present inventors did not
demonstrate here by the Annexin V labeling and the TUNEL method that the NEMO
peptides-induced cell death is indeed apoptosis. Nevertheless considering the role of
the NF-icB pathway in the regulation of apoptosis, it is likely that the cell death
induced by NEMO-derived polypeptides is apoptotic in nature.
The results set forth above, regardless of the nature of the future NFKB
inhibitors (organic or peptidomimetic compounds), targeting NEMO's oligomerization
will remain a more attractive and promising strategy as compared to those of
IKK kinase activity and of NEMO-kinase association because this molecular event
strictly depend on the pro-inflammatory signal. Therefore, such drugs would interfere
less with the basal NF-KB activity in normal cells that is required for cell viability.
Peptides derived from the N-terminal region of wild-type NEMO
The present inventors explored the N-terminal region of the wildtype
NEMO, in particular the NLM conserved motif (residues 293-322 of SEQ ID
NO: 12) appearing in Figure 1 A. To this end, the following sequences were produced
(see Table 1 for the corresponding sequence):
(Table Removed)
NLM-DR is a 21 amino acid "motif (and the corresponding wild
type NLM covering the same amino acid range) derived from the larger 30 amino acid
conserved NLM motif set forth in Figure 1 A. The NLM-DR has been mutated from
the wild type NLM sequence in that the aspartic acid at residue 11 in the wild type
sequence has been replaced by an arginine (see Table 1 and SEQ ID NO: 30).
The present inventors compared NLM-DR to the corresponding
NLM peptide by measuring the respective cooperativity indices, which evidence an
advantage of NLM-DR mutated peptide over the wild-type.
The calculation of the "cooperativity indice" by means of Hill coefficient
is based on the following formula
Boundmax x Ln /(KDn + Ln)
Wherein
Boundmax is the maximum concentration of the bound ligand,
L is the concentration of the free ligand,
n is the cooperativity indice and
KD, is the affinity constant of the ligand by the protein.
The cooperative indices calculated by means of the Hill coefficient
calculation are:
For the mutant NLM peptide (NLM-DR) - a dissociation constant Kd of
170 pM and a cooperativity indice of 1.4 (1326 uM); and
For the corresponding wild-type NLM peptide (NLM) - a dissociation
constant Kd of 240 uM and a cooperativity indice of 2.1 (99642 uM).
If these values were to be compared to those obtained from the
curves without accounting for cooperativity, the affinity of NLM-DR peptide would
be 75 times higher than the affinity of wild-type NLM peptide.
The present inventors also evaluated the biologically relevant results
for the aforementioned forms of NLM-DR as they apply to the inhibition of NF-icB
activation pathway, IC50 and toxicity results (FACS). The results are presented in
Table 2:
(Table Removed)Cytotoxiciry analysed by the indicated technique is scaled from no toxicity (no) to
high toxicity (++-H-);the MTS cell proliferation assay was from Promega; n.d. = not
determined.
Numerous modifications and variations on the present invention are
possible in light of the above teachings. It is, therefore, to be understood that within
the scope of the accompanying claims, the invention may be practiced otherwise than
as specifically described herein.
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We Claim:
1. A polypeptide fusion construct that disrupts NEMO oligomerization by binding to the NEMO oligomerization domain, said construct comprising :
a. an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group comprising of SEQ ID NO: 3, SEQ ID NO:7, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39;
b. polypeptide having a high transduction potential; and
c. optionally a spacer linking said polypeptide having high transduction potential and said amino acid sequence.
2. The polypeptide as claimed in Claim 1, wherein said spacer sequence has a length ranging from 1-35 amino acids.
3. The polypeptide as claimed in Claim 1, wherein said amino acid spacer sequence is selected from the group comprising of SEQ ID NO: 9 and SEQ ID NO: 10.
4. The polypeptide as claimed in Claim 1, wherein said polypeptide having high transduction potential has an amino acid sequence of SEQ ID NO: 1.
5. The polypeptide as claimed in Claim 1, wherein the polypeptide fusion construct has the amino acid sequence selected in the group consisting of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 13 and SEQ ID NO: 15.

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Patent Number 247561
Indian Patent Application Number 2181/DELNP/2006
PG Journal Number 16/2011
Publication Date 22-Apr-2011
Grant Date 20-Apr-2011
Date of Filing 20-Apr-2006
Name of Patentee INSTITUT PASTEUR
Applicant Address 28, RUE DU, DOCTEUR ROUX, F-75015, PARIS FRANCE.
Inventors:
# Inventor's Name Inventor's Address
1 AGOU FABRICE 21 AVENUE DU BEL AIR, F-75012 PARIS FRANCE.
2 COURTOIS GILLES 157 RUE DE MÉNILMONTANT, F-75020 PARIS FRANCE.
3 ISRAEL ALAIN 20 RUE DAGUERRE, F-75014 PARIS FRANCE.
4 VERON MICHEL 16 RUE DE FOURCY, F-75004 PARIS FRANCE.
5 TRAINCARD FRANCOIS 21 RUE JULES GUESDES, F-92130 ISSY-LES-MOULINEAUX FRANCE.
6 YAMAOKA SHOJI 34 RUE DE VOUILLÉ, F-75015 PARIS FRANCE.
7 BALEUX FRANCOISE 4 RUE LHOMOND, 75005 PARIS FRANCE.
8 COÏC YVES-MARIE 10, ALLÉE DU PRÉ-MAISTRE-GILLES, 92190 MEUDON, FRANCE.
PCT International Classification Number A61K 38/17
PCT International Application Number PCT/IB2004/003352
PCT International Filing date 2004-09-24
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/505,161 2003-09-24 U.S.A.
2 60/530,418 2003-12-18 U.S.A.