Title of Invention	AN ASSAY FOR IDENTIFICATION OF A PROTEIN KINASE INHIBITOR WITH A FLUORESCENT PROBE
Abstract	This invention relates to fluorescent probes for identification of compounds binding to protein kinases, for measurement of the affinity of inhibitors of protein kinases, and determination of the active concentration of protein kinases binding to the probe. Bisubstrate-analog character of the probe enables the simultaneous evaluation of inhibitors targeted to both ATP binding site and substrate protein/peptide binding domain of the kinase. High affinity of the probe (Kd = 1.0 nM towards cAMP-dependent protein kinase) affords the application of the enzymes at low concentration which leads to the substantial decrease of the consumption of the kinase. Due to the ability of the conjugates of oligo(D-arginine) with a ATP binding site targeted inhibitors of this invention to bind with high affinity to a wide spectrum of (basophilic) kinases, a single Fluorescent probe is applicable for assessment of inhibitory potency of compounds towards a great number of protein kinases.

Title of Invention

AN ASSAY FOR IDENTIFICATION OF A PROTEIN KINASE INHIBITOR WITH A FLUORESCENT PROBE

Abstract

This invention relates to fluorescent probes for identification of compounds binding to protein kinases, for measurement of the affinity of inhibitors of protein kinases, and determination of the active concentration of protein kinases binding to the probe. Bisubstrate-analog character of the probe enables the simultaneous evaluation of inhibitors targeted to both ATP binding site and substrate protein/peptide binding domain of the kinase. High affinity of the probe (Kd = 1.0 nM towards cAMP-dependent protein kinase) affords the application of the enzymes at low concentration which leads to the substantial decrease of the consumption of the kinase. Due to the ability of the conjugates of oligo(D-arginine) with a ATP binding site targeted inhibitors of this invention to bind with high affinity to a wide spectrum of (basophilic) kinases, a single Fluorescent probe is applicable for assessment of inhibitory potency of compounds towards a great number of protein kinases.

Full Text	FORM 2 THE PATENTS ACT, 1970 {39 of 1970) & THE PATENTS RULES, 2003 COMPLETE SPECIFICATION [See section 10, Rule 13] BISUBSTRATE FLUORESCENT PROBE BINDING TO PROTEIN KINASES; UNIVERSITY OF TARTU, WHOSE ADDRESS IS ULIKOOLI 18, EE50090 TARTU, ESTONIA. THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED. 2 RELATED APPLICATION This application claims priority to Estonian provisional application number P200600030, filed on August 15, 2006, the contents of which are incorporated herein. FIELD OF THE INVENTION The invention concerns a novel fluorescent probe binding to the active site of a protein kinase and its application for screening of compounds and evaluation of inhibitors targeted both to the ATP-binding pocket or/and protein/peptide substrate-binding domain of the kinase, and methods of manufacturing of such probes. The invention also relates to the screening assay for identifying compounds that bind to and modulate the activity of a wide variety of members of protein kinase super-family. The invention relates to the assay for identification and evaluation of protein kinase inhibitors and determination of the concentration of protein kinases. BACKGROUND TO THE INVENTION Protein Kinases and Protein Kinase Inhibitors Protein kinases (PKs) play a key role in the regulation of protein functions in living cells. It has been estimated that the activity of one third of proteins is regulated through phosphorylation of one or more of serine, theorine and thyrosine residues of the protein. More than 400 human diseases (incl. cancer) have been linked to aberrant protein kinase signaling. This has made PK the second largest drug target (and fastest growing category of drugs in development) after G protein-coupled receptors. [Cohen, Nat. Rev. Drug Discov. 1 (2002) 309; Fischer, Curr. Med. Chem. 11 (2004) 1563] Protein kinases follow ternary complex kinetic mechanism in which direct transfer of the phosphoryl group from ATP to the protein substrate occurs in the active site. [Adams, Chem. Rev. 101 (2001) 2271] rt 3 Three different kind of active site-targeted protein kinase inhibitors are previously known. Firstly, despite serious selectivity problems (all 500 protein kinases and more than 1500 other proteins are able to bind purine nucleotides), and high concentration of competing ATP in the cellular milieu, the main efforts of drug companies have been directed to the development of ATP competitive inhibitors. Imatinib, a specific small molecule inhibitor of the Abl kinase, has become the first successful breakthrough in kinase-targeted cancer therapy. The second type of active site targeted inhibitors of protein kinases comprise compounds that selectively interfere with protein-protein interactions and block the binding of the substrate protein to the active site of the protein kinase. [Recent reviews: Bogoyevitch et al., Biochim. Biophys. Acta. 1754 (2005) 79; Lawrence, New Design Strategies for Ligands That Target Protein Kinase-Mediated Protein-Protein Interactions.; Pinna, A. L, Cohen, P. T. W. Eds.; 2005; p. 11J Thirdly, combination of the aforementioned approaches and development of bisubstrate-analog (biligand) inhibitors that simultaneously associate with both ATP and protein binding domains of the dual substrate enzyme has given selective and potent inhibitors of PK. Several strategies of the design of bisubstrate-analog inhibitors have been described. [For a review on the subject, see: Parang et al., Pharmacol. Therap. 93 (2002) 145]. Construction of bisubstrate-analogue (Afunctional) inhibitors could lead to enhanced specificity and potency in protein kinase inhibition. The inhibitor mimics two natural substrates/ligands and associates with two regions of a given kinase simultaneously. Most bisubstrate analogues have been designed to mimic ATP and the acceptor component. The inhibitors of this kind are covalent conjugates between one moiety inhibiting ATP binding and one moiety inhibiting binding of a protein/peptide substrate to the protein kinase. [Ricouart et al., J. Med. Chem. 34 (1991) 73; Medzihradszky et al., J. Am. Chem. Soc. 116 (1994) 9413; Patent application describing bisubstrate analog inhibitors of PK: WO0070029, WO0170770, WO03010281, WO2004110337, EE200300187] One of the present inventors has previously developed bisubstrate-analog inhibitors for protein serine/threonine kinases PKA and PKC with activities in sub-micromolar region. [Loog et al., Bioorg. Med. Chem. Lett., 9 (1999) 1447, Uri et al., 4 Bioorg. Med. Chem. Lett., 12 (2002) 2117, WO0070029, EE200300187]. These inhibitors comprise moieties of analogs of both substrates of protein kinases: ATP binding site targeted adenosine-5'-carboxylic acid (Adc) and the protein substrate domain directed oligo-(L-arginine). The design of the latter fragment was based on the knowledge that phosphorylation sites of the substrates of basophilic protein kinases (cAPK, PKC, Akt/PKB, PKG, etc.) are flanked by regions rich in arginine or/and lysine residues. [Pinna et al., Biochim. Biophys. Acta. 1314 (1996) 191] Two active fragments of the inhibitors were connected via a tether which length was optimized in structure-activity studies. After the priority date (August 15, 2006) the synthesis and characterization of bisubstrate-analog inhibitors for protein serine/threonine kinases incorporating D-arginine residues that is the subject of the present patent application was described in two published articles by inventors of the present patent application. [Enkvist et al., J.Med.Chem. 49 (2006) 7150; Viht et al., Anal.Biochem., 362 (2007) 268] Existing assay methods Development of new assay methods for evaluation of protein kinase inhibitors has run in parallel with the increase of importance of effective inhibitors for drug industry. During recent years, flexible fluorometric kinase assay methods have substituted problematic (e.g., personal risks, environmental hazards, short half-lives of 32P- and 33P-Iabeled compounds, long exposure times) radiometric methods. Fluorescent methods can be spatially and temporally more focused than radioactive methods, and as such, they are more suitable for application in high-throughput screening (HTS) assays. [Olive, Expert Rev Proteomics. 1 (2004) 327] Great majority of evaluations of kinase inhibitors is performed in the form of kinetic studies and the new, potential inhibitors are screened and characterized on the basis of their retarding effect on the rate of substrate (peptide or protein) phosphorylation reaction catalyzed by the kinase. In the case of radioactive methods the product of the phosphorylation reaction that is covalently labeled with radioactive phosphor isotope is separated from radioactive ATP and quantified. 5 The application of labor-intensive separation steps and the use of large amounts of radioactivity make these methods problematic for high throughput assays. Kinetic methods where the amount of the product of the phosphorylation reaction is established by its competition with a fluorescent binder for an antibody (a phosphopeptide formed in a kinase reaction displaces a fluorescently-labeled phosphopeptide from a phospho-specific antibody) or other phosphopeptide binding macromolecule (Immobilized Metal Assay for Phosphochemicals, 1MAP) is another class of assays in active use. [e.g., W09818956]. In this case the change in the fluorescence anisotropy resulting from the displacement of the fluorescent binder from its complex with the antibody is usually measured. Although these methods suit better for HTS assays, they still require an effective substrate, fluorescent binder and high-affinity antibody or other phosphor-binding macromolecule for the method. Large number of measurements has to be performed to characterize the inhibitory compounds with comparable K values. Alternatively, kinase inhibitors may be detected by direct or indirect measurement of the binding of the inhibitor to the kinase. Some small-molecule inhibitors can be conjugated to a fluorescent dye without loosing their binding affinity to kinase. The kinase-bound labeled inhibitor can then be displaced by competitive kinase inhibitors and the change in fluorescent characteristics measured. This interaction therefore forms the basis for a competitive binding assay of kinase inhibitors where no knowledge of the substrate or an antibody to the phosphorylated kinase substrate is required. Several papers and patent applications describe the use of fluorescent probes for determination of the binding characteristics of protein kinase inhibitors, [e.g., Chen et al., 263 (1993) 15812, WO2005/033330] Limitations of Current Binding Assays Fluorescent probes with micromolar affinity towards cAPK were disclosed by Chen et al. [Chen et al., 268 (1993) 15812] These ATP-competitive probes have complicated emission characteristics that originate from the fluorescence of both the fluorescent dye and bisindolylmaleimide ligand. Low (micromolar) affinity and complex fluorescence spectrum make it difficult to use these fluorescent 6 conjugates as probes for binding assays. [WO9906590] Other ATP competitive fluorescent probes have been described in literature, [e.g., WO2005/033330] A fluorescent probe targeted to the protein/peptide substrate binding domain of the cAMP-dependent protein kinase (cAPK), fluorescein-labeled 20 amino acid residues-containing sequence of protein kinase heat stable inhibitor protein PKI (PKI 5-24), was shown to bind to cAPK with micromolar affinity (1^=1.6 (iM) in a fluorescence polarization assay. [Shneider et al., Org. Lett. 7 (2005) 1695] Low binding affinity of the probe prevents its application for the determination of the binding constants of the protein/peptide substrate-binding domain targeted inhibitors. The main limitations of the binding assays that have been disclosed so far are the following: A. Fluorescent probes available have low affinity (usually micromolar or submicromolar) towards protein kinases that leads to substantially higher consumption of kinases when using these methods if compared to kinetic assays, and to the impossibility of the application of the probes for determination of exact binding characteristics of high-affinity inhibitors; B. Fluorescent probes available are active to a single kinase or a small family of kinases that makes it impossible to use a single probe for generic characterization of inhibitors with multiple kinases; C. Alt fluorescent probes available thus far enable the testing of compounds binding to either ATP or protein/peptide substrate binding site and as such differently from kinetic methods do not permit simultaneous screening of ATP and protein-peptide substrate binding site-targeted inhibitors. To our knowledge, prior art published by other groups most related to the current invention is described in the patent application WO2005/033330 "Fluorescent probes for use in protein kinase inhibitor binding assay". This invention discribes a fluorescent probe with affinity in high nanomolar range (Kd = 161 nM) which leads to the need for high concentration of the kinase in the assay format (200 nM STK12 kinase was used in the disclosed Example). Requested kinase 7 concentration, arising from the Kd value of the fluorescent probe, is a hundred-fold higher than the kinase concentration usually applied for kinetic measurements (ca 1 nM concentration of the enzyme is often used in kinetic assays). This leads to substantial increase of the cost of the assay. Due to sub-micromolar dissociation constant of the probe it cannot be used for determination of binding constants of inhibitors with nanomolar affinity [Fluorescence Polarization Technical Resource Guide THIRD EDITION 2004, Invitrogen Corporation. Chapter 7] The probe disclosed in WO2005/033330 is targeted to the ATP binding site of the kinase and as such the application of the probe for screening of PK inhibitors neglects the inhibitors targeted to the protein/peptide binding domain of the kinase. Furthermore, the fluorescent probe disclosed in WO2005/033330 is applicable for testing of inhibitors of a specific kinase (STK12 and close analogs). Benefits of the Presented Invention The invention described in current patent application overcomes limitations of all known fluorescent probes and is applicable in competitive displacement assays for identification and characterization of inhibitors of many protein kinases and determination of the concentration of the protein kinase. If compared to the probes from previous inventions, the Fluorescent probe of the present invention has very high affinity (Kd s 1 nM) and can be used for testing of inhibitors of a variety of protein kinases targeted to both ATP and/or protein substrate binding sites. SUMMARY OF THE INVENTION This invention relates to fluorescent probes for identification of compounds binding to protein kinases, for measurement of the affinity of inhibitors of protein kinases, and determination of the active concentration of protein kinases binding to the probe. Bisubstrate-analog character of the probe enables the simultaneous evaluation of inhibitors targeted to both ATP binding site and/or substrate protein/peptide binding domain of the kinase. High affinity of the probe (Kd = 1.0 nM towards cAPK) affords the application of the enzymes at very low concentration ( 8 determination of binding constants for inhibitors with nanomolar and micromolar affinity. Due to the ability of the conjugates of oligo(D-arginine) with a ATP binding site targeted inhibitors of this invention to bind with high affinity to a wide spectrum of (basophilic) kinases, a single Fluorescent probe is applicable to a number of protein kinases. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Titration of the fluorescent probe ARC-TAMRA (10 nM) with cAPK Ca (fluorescence spectrometer LS 55 (Perkin Elmer) with 0.5 ml quartz cell). Kd=1.1 nM was calculated with the application of nonlinear regression analysis to the relationship: A=A( + (Ab-Af)x «^Kd+KT)-V(LT + Kd ,RTH-4LTRT] 2LT (A) A is measured anisotropy; Af is the anisotropy of free ARC-TAMRA; Ab is the anisotropy of bound ARC-TAMRA; LT is the concentration of ARC-TAMRA added, 10 nM; RT is the concentration of the total kinase (cAPK Cot). Figure 2. Titration of of ARC-TAMRA (2 nM and 10 nM) with cAPK Ca (fluorescence spectrometer LS 55 (Perkin Elmer) with 0.5 ml quartz cell). Kd values of 1.1 nM and 0.94 nM were calculated for 2 nM and 10 nM of ARC-TAMRA, respectively from the relationship (A) (Figure 1). Figure 3- Determination of the concentration of the active kinase (fluorescence spectrometer LS 55 (Perkin Elmer) with 0.5 ml quartz cell). The fraction of the active (binding) form of the kinase in solution, k=0.317, was calculated by the application of nonlinear regression analysis to the relationship: A K fK Lt + Kd + k-Rt-J(Lt + Kd + k-Rt)2-4-Lf k-Rt A = Af + (Ab-Af) — (B) 2-Lt A is the measured anisotropy; Af is the anisotropy of free ARC-TAMRA; Ab is the anisotropy of bound ARC-TAMRA; LT is the concentration of ARC-TAMRA added, 10 nM; RT is the total (nominal) concentration of kinase (cAPK Ca); K^ is the 9 dissociation constant of the reaction between ARC-TAMRA and cAPK Ca (Kd=1.0 nM). Figure 4. Dependence of anisotropy (normalized values) on the concentration of inhibitors H89; PKI [inhibitory peptide PKI(5-24)], ARC [AdcAhx{L-Arg)6-NH2], and D-ARC [AdcAhx(D-Arg)6-NH2]. Assay conditions: 10 nM of ARC-TAMRA and 10 nM of kinase (cAPK Ca), fluorescence spectrometer LS 55 (Perkin Elmer) with 0.5 ml quartz cell. The Kd value of 1.0 nM was used for ARC-TAMRA. The obtained inhibitory characteristics for inhibitors were as follows: Inhibitor ICso K, L-ARC 1,3 (iM HOnM D-ARC 35 nM 2,5 nM H89 180nM 15 nM PKI (5-24) 140 nM 12 \xM Figure 5. Titration of ARC-TAMRA (2 nM and 20 nM) with cAPK Ca (fluorescence plate reader PHERAstar (BMG LABTECH), Corning 384 well Low Volume NBS microptates (30 pJ reaction volume)). Kd value of 0.4 nM was calculated from the relationship (A) (Figure 1). Figure 6. Dependence of anisotropy (normalized values) on the concentration of inhibitors H89; ARC [AdcAhx(L-Arg)fl-NH2], D-ARC [AdcAhx(D-Arg)6-NH2], and the regulatory subunit of cAMP-dependent protein kinase Rlla. Assay conditions: 2 nM of ARC-TAMRA and 3 nM of kinase (cAPK Ca), fluorescence plate reader PHERAstar (BMG LABTECH). The Kd value of 0.4 nM was used for ARC-TAMRA. Kj values of 21 nM (AdcAhx(L-Arg)6-NH2), 13 nM (H89), 1.1 nM (AdcAhx(D-Arg)6-NH2), and 0.5 nM (Rlla) were obtained. Figure 7. Titration of ARC-TAMRA (2 nM and 20 nM) with Rho-associated kinase ROCK jl (fluorescence plate reader PHERAstar, BMG LABTECH). Kd value of 4 nM was calculated for ARC-TAMRA - ROCK II complex according to the relationship (A) (Figure 1). Figure 8. Dependence of anisotropy on the concentration of inhibitors H89, Y-27632, and ARC [AdcAhx(L-Arg)6-NH2]. Assay conditions; 2 nM of ARC-TAMRA and 15 nM ROCK II, fluorescence plate reader PHERAstar (BMG LABTECH). The 10 Kd value of 4.0 nM was used for ARC-TAMRA. Ki values of 45 nM (H89), 40 nM (Y-27632) and 5 nM (AdcAhx(L-Arg)6-NH2) were obtained. DETAILED DESCRIPTION OF INVENTION This invention relates to fluorescent probes for the identification of compounds binding to protein kinases, for measurement of the affinity of inhibitors towards protein kinases and determination of the active concentration of protein kinases binding to the probe. Bisubstrate-analog character of the probes enables the evaluation of inhibitors targeted to both ATP binding site and protein/peptide substrate binding domain of the kinase. The fluorescent probe of this invention has very high affinity towards the kinase (Kd=1.0 nM) which enables the application of low concentrations of kinases in assays and determination of binding constants for inhibitors with high (low nanomolar) affinity. Displacement of the fluorescent probe by an inhibitor can be used for the determination of the values of dissociation constants K Fluorescent probe The first embodiment of the invention is a fluorescent probe with the general formula (I); (X-Y-Z)-L-F (I), wherein X-Y-Z is a bisubstrate-analog inhibitor of a protein kinase, in which X is targeted to the ATP-binding pocket of a kinase, Z binds to the protein/peptide-binding domain of the kinase, Y is a tether that connects X and Z and permits simultaneous binding of X and Z to the active site of the kinase; F is a fluorescent dye which optical characteristics are changed in the course of the binding of (X-Y-Z)-L-F to the kinase; L is a linker between the bisubstrate-analog inhibitor XYZ and fluorescent label F. The presently preferred fluorescent probe for the application in the binding assay has the structure: 11 AdcAhx(D-Arg)6-D-Lys(5-TAMRA)-C(0)NH2, ARC-TAMRA The probe, ARC-TAMRA, has high affinity (Kd=1.0 nM; Figures 1 and 2, Example 4) towards the catalytic subunit of cAMP-dependent protein kinase (cAPK Ca) and it was displaced from its complex with the kinase by ATP-competitive inhibitor H89, protein substrate competitive inhibitor peptide PKl(5-24), and bisubstrate inhibitor AdcAhx(D-Arg)6-NH2 (Example 5, Figure 3). Thus, the fluorescent probe of the present invention ARC-TAMRA is the first high-affinity probe disclosed that can be used to measure simultaneously the affinity of inhibitors binding to ATP and protein substrate binding sites of the kinase. The bisubstrate (biligand) character of the probe AdcAhx(D-Arg)6-D-Lys(5-TAMRA)-C(0}NH2 is proved with its displacement from the complex with the kinase by both ATP-binding site targeted inhibitor H89 and protein/peptide substrate binding site targeted inhibitor PKI(5-24), and also by very high affinity of the fluorescent probe. The inhibitory part of the probe AdcAhx(D-Arg)6-NH2 has high inhibitory potency to many protein kinases (Table 3). As such the fluorescent probe has the potential for application in assays concerning many basophilic protein kinases. Selective inhibition of basophilic kinaseses by AdcAhx(D-Arg)6-NH2 (Table 3) is another demonstration of the bisubstrate nature of the inhibitors of the present invention. 5-TAMRA, 5-carboxytetramethylrhodamine, is a good fluorescent label to be used in fluorescence polarization assays, as it contains the dye as a single-position 12 isomer, the probe is exited at long wavelength, it has high brightness and great photostability. Other fluorescent labels are suitable for the application as the moiety F of the fluorescent probe (X-Y-Z)-L-F. These fluorescent dyes are well known to the people skilled in the art. Principles for the choice of fluorescent labels are disclosed in [Polarization Technical Resource Guide • THIRD EDITION 2004 Invitrogen Corporation , USA www.invitroqen.com/panveral. Many such labels are described in the Handbook: [The Handbook — A Guide to Fluorescent Probes and Labeling Technologies Web Edition of The Handbook, Tenth Edition 2006 Invitrogen Corporation] Usually fluorescence labels with higher brightness (brightness of a fluorochrome is proportional to the product of its extinction coefficient and its quantum efficiency), longer excitation and emission wavelengths, and greater photostability are preferred, but the choice of the label depends on the analytical instrument used for the assay and special analytical situation. Other fluorescent dyes that may be useful in the present invention include fluorescein and fluorescein derivatives, rhodamine and rhodamine derivatives, and many other fluorescent labels that are produced and sold by different companies. Fluorescent probes of the structure (X-Y-Z)-L-F incorporating as F mineral fluorescent labels, e.g., quantum dots, may be useful for special applications. The aim of the linker L in the structure of the fluorescent probe (X-Y-Z)-L-F is the positioning of the fluorescent label F in the position where the group F causes minimal hindrance to the binding of the bisubstrate inhibitor X-Y-Z to the kinase. In the preferred fluorescent probe AdcAhx(D-Arg)6-D-Lys(5-TAMRA)-C(0)NH2 D-lysine stands for the linker L but in the case of other probes the linker may be a Ci-C2o alkylene group, a Ci-C20 alkenylene group, a C-i-C20 alkynylene group, wherein one or more of the CH2 groups present in the C-i-C20 alkylene group, C1-C20 alkenylene group, C1-C20 alkynylene group is optionally replaced with -0-, -C(O)-, -C(0)N-, -S-, -S(O)-, -SO2-, -N(R)-; R is H or C^ alkyl. In the case of some probes L may be omitted. 13 In the examples of the present invention the fluorescent dye F is connected via linker L to the moiety Z of bisubstrate inhibitor X-Y-Z, but L may also be attached to any of the X, Y and Z moieties at various locations. As it will be shown in later parts of the invention other types of bisubstrate inhibitors may form the active ingredient X-Y-Z of the fluorescent probe. An example of another fluorescent probe structure is the compound where Hidaka's ATP competitive PK inhibitor H9 is conjugated with hexa(D-arginine) peptide via flexible hexanoic acid tether. The compound 26 H9-(CH2)5C(0)(D-Arg)6-NH2 with very high inhibitory potency (IC5o=5.3 nM; Ki=1-2 nM; Example 2, Table 2) was connected through D-lysine linker to the fluorescent label 5-TAMRA to obtain high-affinity fluorescent probe of the present invention H9-Hex{D-Arg)6-D-Lys(5-TAMRA)-C(0)NH2: As it will be described further in Example 2 H9-Hex(D-Arg)6NH2 (Compound 26) has very high affinity towards the kinase cAPK. Inhibitory selectivities of bisubstrate inhibitors AdcAhx(D-Arg)6-NH2 and H9-Hex(D-Arg)6NH2 towards basophilic kinases are somewhat different (Example 3) and the derived fluorescent probes AdcAhx(D-Arg)6-D-Lys(5-TAMRA)-C(0)NH2 and H9-Hex(D-Arg)6-D-l_ys(5-TAMRA)-C(0)NH2 (synthesis of both probes id described in Example 1) may find application in different assay formats. Although the preferred method of the invention is to use the fluorescent probe with fluorescence polarization measurement, an environmentally sensitive fluorescent probe is another choice of the present invention: change in fluorescent intensity as the result of the binding of the probe to the kinase would be measured rather than the change in the fluorescence polarization. A preferred fluorescent probe for these measurements is AdcAhx(D-Arg)6-D-Lys(NBD)-NH2. 14 The probe contains NBD (7-Nitrobenz-2-Oxa-1,3-Diazole) dye which fluorescence intensity is strongly dependent on environment. The probe has very high affinity towards cAPK (Kd ca 1 nM). The synthesis of the fluorescent probe AdcAhx(D-Arg)6-D-Lys{NBD)-NH2 is described in Example 1. People skilled in the art know that there are other possibilities (in addition to fluorescence polarization and fluorescence intensity measurements) to measure the change in fluorescence of a probe as a resuft of its association with a kinase. The methods like fluorescence correlation spectroscopy, fluorescence resonance energy transfer (FRET), fluorescence lifetime, photo-bleaching and photo-activation, flurescence intensity distribution analysis and their combinations can be used for the monitoring the equilibrium of the reaction between a fluorescent probe and a protein. These methods are well characterized in literature [e.g., White et al. Adv. Drug Deliv. Rev. 57 (2005) 17] and may find application in kinase associatiation studies connected to the fluorescent probes disclosed in the present inventon. The probes of the present invention can be used with classical fluoresence techniques and confocal single-molecul techniques in single- and multi-parameter imaging formats [Tinnefeld et al., Angew. Chem. Int. Ed. 44 (2005) 2642]. Authors of the present invention have shown that adenosine - oligoarginine conjugates are cell membrane permeable [Uri et al., Bioorg. Med. Chem. Lett. 12 (2002) 2117], therefore the fluorescent probes of the present invention are applicable in experiments with live cells, tissues and organisms. Preferred embodyments of the present invention, fluorescent probes AdcAhx(D-Arg)6-D-Lys(5-TAMRA)-C(0)NH2, H9-Hex(D-Arg)6-D-Lys(5-TAMRA)-C(0)NH2, AdcAhx(D-Arg)6-D-l_ys(NBD)-NH2 contain D-arginines, differently from previously disclosed 15 bisubstrate inhibitors which incorporate L-isomers of amino acids. As such these probes are persistent towards enzymatic degradation. [Elmquist, Biol. Chem. 384 (2003) 387] and well suited for applications in biological systems. In biological systems fluorescent probes of the present invention could be used together with kinases fused to fluorescent proteins for dual color imaging and protein-inhibitor interaction studies based on measurement of fluorescence resonance energy transfer (FRET). The fluorescence polarization assay The preferred assay method for the application of the fluorescent probes of the present invention is fluorescence polarization or fluorescence anisotropy. Both terms are used synonymously to describe molecular, interactions in solution. [Fluorescence Polarization Technical Resource Guide • Analysis of FP Binding Data Invitrogen Corporation • USA • www.invitrogen.com/panvera] This technique measures the change in rotational speed of a ligand during its excited lifetime upon binding to its receptor. Fluorescence polarization assays are homogenious and as such they do not require separation steps like chromatography, filtration, centrifugation, precipitation or electrophoresis. Due to the ratiometric nature of tha assay fluorescence polarization is well suited to assay miniaturization, and the assays can be performed with equal success in a cuvette of a fluorescence spectrometer and in low \x\ volumes in wells of 1536-well microtiter plates with a stable plate reader with a fluorescence polarization detector. In the embodiments of the present invention fluorescence spectrometer LS 55 (Perkin Elmer) with 0.5 ml quartz cell or fluorescence plate reader PHERAstar (BMG LABTECH) with Corning 384 well Low Volume NBS microplates (30 (J reaction volume) were used for assays. In the experimental setup used here a fluorescence polarization signal is greatly increased when fluorophore is slowed down drastically in its rotational speed. This occurs upon binding of a small fluorescently labelled probe of the present invention to significantly larger molecule of the target protein kinase. The degree of polarization is determined by measuring the fluorescence intensities of parallel and perpendicular emitted light with respect to the plane of linearly polarized excitation light. The preferred embodiment of the Fluorescent probe of the present invention 16 ARC-TAMRA, AdcAhx(D-Arg)6-D-Lys(5-TAMRA)-C(0)NH2, has MW of 1869 and the MW of the kinase, cAPK Ca, is ca 40000. This leads to big differences in the anisotropy values of the solution of free ARC-TAMRA (Af = 40 mA) and the solution of the complexed with the kinase fluorescent probe ARC TAMRA (Ab = 240 mA). Due to mathematical simplicity, anisotropy values are preferred because it is easier to deconvolute anisotropy values into component values than it is with polarization values. Fluorescent probe ARC-TAMRA involved in binding events exist in a mixture of only two states: bound or free. With only two species, the anisotropy additivity equation has the shape: A = FfAf + FbAb, where: Ff + Fb= 1; A = observed anisotropy value Ff = fraction of fluorescent ligand that is free Fb = fraction of fluorescent ligand that is bound Af = anisotropy of the free fluorescent ligand Ab = anisotropy of the bound fluorescent ligand The top and bottom plateaus of the semi-log equilibrium binding isotherm (anisotropy vs. Log total receptor concentration) define the anisotropy of the free and bound states, Af and Ab. With the observed anisotropy, A, we can calculate the fraction of Bound and Free fluorescent ligand for a given anisotropy value. The change in fluorescence anisotropy was plotted against the kinase concentration [cAPK Ca]. The data were fitted to the equation: Lt + Kd + Rt - J(Lt + Kd + Rt)2-4-LfRt A = Af + (Ab-Af) ^ 2-Lt where, A is measured anisotropy; Af is the anisotropy of free ARC-TAMRA; Ab is the anisotropy of bound ARC-TAMRA; LT is the concentration of ARC-TAMRA added; RT is the total concentration of active kinase (cAPK Ca). 17 Characterization of the fluorescent probe ARC-TAMRA in binding experiments with the catalytic subunit of cAMP-dependent protein kinase is characterized in Example 4 and Figures 1 and 2. Kd of 1.0 nM was determined for the fluorescent probe ARC-TAMRA in these assays. Competition Experiments The present invention embodies an assay in which the binding characteristics of the test compounds are determined on the basis of their ability to displace the fluorescent probe from its complex with the kinase in a fluorescence polarization assay. This is described in Example 5 and shown on Figure 3. By virtue of the bisubstrate (biligand) character of the fluorescent probe of the present invention it is displaced from the complex with the protein kinase with both ATP binding site targeted inhibitors and protein substrate binding site targeted inhibitors. As it has been shown in Figure 3 three different types of inhibitors are able to displace the fluorescent probe ARC-TAMRA from its complex with the kinase cAPK Ca: a) ATP competitive inhibitor H89; b) Protein substrate competitive inhibitor PKl(5-24); c) Bisubstrate-analog inhibitors AdcAhx(L-Arg)6-NH2 and AdcAhx(D-Arg)6-NH2 The obtained result (Example 5) reveals that the fluorescent probe of the present invention can be used for evaluation of compounds binding to different binding pockets of the active site of the kinase. Thus, the displacement method of the present invention catches up with kinetic methods in detecting both types of inhibitors in a single assay. This has not been previously possible with fluorescent probes based on ATP competitive or protein substrate competitive inhibitors. The high affinity of the fluorescent probe of the present invention (K 18 used for the determination of binding constants for inhibitors with low nanomolar affinity). Also, the high affinity of the fluorescent probe ARC-TAMRA Taking into the account the selectivity profile of the bisbstrate inhibitors of the present invention AdcAhx(D-Arg)6-NH2 and H9-Hex(D-Arg)6NH2 (Table 2) the corresponding fluorescent probes are applicable to characterization of the binding of inhibitors to many kinases with basophilic substrate profile. Additionally, the potential targets of the fluorescent probes of the present invention are different mutated forms of basophilic protein kinases, truncated forms of kinases inactive states of protein kinases, and pseudokinases. Determination of the concentration of the active form of the kinase Determination of the concentration of the special form of a kinase is of paramount importance for many enzyme applications. In kinetic assays the kinase amount in the assay volume is characterized on the activity basis and the molar concentration of the kinase is often not known. Majority of the methods in use for determination of enzyme concentration (Bradford, Lowry, SDS electrophoresis) give the total concentration of the protein (not the concentration of its active form), are inaccurate and need unacceptably large amounts of the protein for analysis. The application of the fluorescent probe of the present invention ARC-TAMRA enables a simple procedure for the determination of the concentration of the active (binding to the fluorescent probe) form of the kinase in the sample. The fraction of the active (binding) form of the kinase in solution, k, was calculated with the application of nonlinear regression analysis to the relationship: 19 where A is the measured anisotropy; Af is the anisotropy of free ARC-TAMRA; Ab is the anisotropy of bound to the kinase form of ARC-TAMRA; LT is the concentration of ARC-TAMRA added, 10 nM; RT is the total concentration of active kinase (cAPK Ca); Kd is the dissociation constant of the reaction between ARC-TAMRA and cAPK Ca(Kd=1.0 nM). The application of the fluorescent probe ARC-TAMRA for the determination of the concentration of cAPK Ca is described in Example 6. It was established that there was good proportionality between the concentration of the binding protein (as established with the fluorescent probe ARC-TAMRA) and the phosphorylation activity of the solution of catalytic subunit of cAMP-dependent protein kinase. The reduction of kinase activity enzyme samples with time was in accord with the determined amount of kinase associated with ARC-TAMRA. Optimization of the structure of bisubstrate inhibitor X-Y-Z To be widely applicable in bioanalytical methods the fluorescent probe should have possibly highest affinity towards its target protein. It has been shown that the higher the affinity of the fluorescent ligand, the wider the range of inhibitor potency that can be resolved [Huang, J. Biomol. Screen. 8 (2003) 34]. The lowest inhibitor Kj value that can be resolved in an FP-based binding assay is approximately equal to the Kd value o1 the fluorescent probe. Starting from the compound AdcAhx(L-Arg)6 with IC50 of 400 nM [Vint et al., Anal. Biochem. 340 (2005) 165; Loog et al., Bioorg. Med. Chem. Lett. 9 (1999) 1447] modifications in the structure of different moieties of the conjugates were performed. Flexible and productive solid phase synthetic methods were developed for the preparation of conjugates of oligoarginine peptides with adenine, adenosine, adenosine-5'-carboxylic acid (Adc), and 5-isoquinolinesulfonic acid for structure-activity studies (Schemes 1-4). Different combinations of fragments were incorporated into conjugates and the novel compounds were tested as inhibitors of cAPK (cAMP-dependent prtotein kinase). 20 New synthetic methods: synthesis of fluorescent probes and bisubstrate inhibitors for SAR studies The synthesis of the first class of compounds containing Adc (2-8, Scheme 1) was performed with the application of Rink amide resin instead of Wang resin. The structures of tether and peptide components were varied. It was decided to preserve the optimal towards cAPK length of the linker corresponding to that of 6-aminohexanoic acid (equivalent to seven chemical bonds) between the nucleoside and peptide moieties. The optimal length of the tether may be different for other basophilic kinases. Additionally, the tether may contain different heteroatoms and the simple hydrocarbon chain may be substituted with more complicate straight or branched linking units. The diversity of the structure of the peptide part of the conjugates was achieved by the variation of length and chirality of oligoarginine chain, as represented by peptide moieties of (L-Arg)4-NH2 (2), (L-Arg)6-NH2 (3), (D-Arg)4-NH2 (4) and (D-Arg)6-NH2 (5). The second group of compounds contained adenosine and 5'-amino-5'-deoxyadenosine derivatives (Scheme 2). The reaction of 4-nitrophenyl chloroformate with 2',3'-isopropylidene adenosine resulted in the activated carbonate, which gave adenosine-5'-urethanes in the reaction with amines. The length of the linker between nucleoside and peptide moieties was varied. Peptides containing a free amine-group near the C-terminus (e.g., the side chain of lysine or a diamine linker) were prepared on solid phase, cleaved and purified by HPLC. A special class of biligand inhibitors deprived of ribose moiety, conjugates of adenine and oligoarginine, was synthesized by connecting adenine at C8 or N9 position to a peptide via a linker chain. Precursors of these molecules containing a linker with a free carboxylate group were synthesized in solution. Coupling of the precursors with the resin-bound peptides resulted in conjugates with peptides after cleavage and deprotection with TFA (Scheme 3). Conjugates of isoquinolinesulfonamide and oligoarginine were synthesized to compare their activity and selectivity to those of analogous adenosine-containing 21 compounds. If compared to the previously synthesised isoquinolinesulfonyl peptides [Ricouart et al., J. Med. Chem. 34 (1991) 73], several changes were introduced into the structures and the synthesis was carried out on solid-phase with the application of Fmoc-peptide and peptoid chemistry procedures. The use of Rink amide resin excluded the presence of negatively charged C-terminal carboxylate group and resulted in products in the form of their C-terminal amides. The conventional Fmoc-peptide chemistry allowed the circumvention of the harsh HF treatment in the final step of Boc-chemistry procedures. The (p-Ala)-Ser linker of previous conjugates was replaced with 6-aminohexanoic acid, which eliminated potentially phosphorylatable serine residue, removed unnecessary chiral centre and simplified the overall synthetic procedure. Resin-bound oligoarginine peptides were acylated at N-terminus with 6-bromohexanoic acid and the following reaction with 5-isoquinolinesulfonyl ethylenediamine and cleavage with TFA led to the 5-isoquinolinesulfonyl ethylenediamine containing conjugate (Scheme 4). A special embodiment of the present patent application is the group of bisubstrate-analog inhibitors incorporating D-arginine-rich peptides. The high affinity of that kind of conjugates towards cAPK (Table 2) and many other basophilic protein kinases (Table 3) was not known previously. This result is surprising by virtue of the fact that cAPK has a strong requirement for L-configuration both at the phosphorylatable serine residue as well as at the N-terminal arginine residues of the substrate. [Eller, Biochem. Int. 25 (1991) 453] High binding energy may result from beneficial positioning of structurally flexible interacting partners, bisubstrate inhibitor and the kinase, in their complex. Introduction of arginine residues into ARC in the form of their D-configuration converts the peptide motif into retro-inverso counterpart of the C-terminally appended oligo(L-arginine) peptide. Retro-inverso peptide can be regarded as a derivative of a normal peptide in which the relative amino acid side chain topology is maintained, while the backbone termini and direction of the peptide bonds are reversed.This may lead to substantial synergism of the binding energy of the bisubstrate inhibitor if compared to the sum of the binding energies of its constituents, single-site directed inhibitors. Further specification of the interaction mechanism will be possible after solving the crystal structures of the complexes of these inhibitors with protein kinases. 22 Using the methods described above, one skilled in the art could readily prepare various bisubstrate analog inhibitors for structure-activity studies. Other building blocks (e.g., purines, pyrimidines, purine and pyrimidine nucleosides, bisindolylmaleimides, isoquinolines, staurosporines, balanol, etc.) with affinity towards the kinase under investigation can be introduced into the X moiety of the conjugates X-Y-Z and the new conjugates tested for activity. Likewise, the structure of the peptide part Z and the structure of the tether part Y of the bisubstrate inhibitors X-Y-Z could be further optimized to achieve conjugates with the highest affinity towards a special kinase. The optimal structure of the organic tether (Y) may vary between different protein kinases and is also dependent on the selection of X and Z. Y should be selected among groups providing a chain comprising from 1-50 atoms, for instance 1-30 atoms, such as1-18 atoms. In addition to the chain as such there may be groups projecting from the chain. The chain may comprise one or more groups selected from amides (-CONR1-, where R' is selected from the side groups present in amino acids), amines (-NR"-, where R", for instance, is selected from lower alkyl (C1.5 alkyl)), azo (-N=N-), ether(-O-), thioether (-S-), bivalent hydrocarbon groups etc. The chain may be built up of amino acid residues of the a-,-or p-, y-or s-type or of the L- or the D-type or mixtures of these. The chain may carry one or more positive and/or negative groups, for instance primary, secondary, tertiary and quaternary ammonium groups, respective carboxy and sulfonate groups. In case L is a peptide chain it may contain from 1-15 amino acid residues or even more. One type of linkers comprises a hydrocarbon chain which (a) is straight branched or cyclic and possibly is interrupted at one or more positions by an oxygen or a nitrogen and/or (b) has one or more carbons that are substituted with an NR1R2 or an-OR3 group in which R1.3 is selected amongst CMO alkyl. One kind of linker complying with this is a po!y(ethylene glycol) linker with formula -(CH2CH20)n-, where n may be an integer 1-10. Y may be attached to the X and Z moieties at various locations. In the case of cAMP-dependent protein kinase the optimization of the length of Y has been performed previously with inhibitors of lower affinity incorporating L-arginine residues. [Loog et al., Bioorg. Med. Chem. Lett., 9 (1999) 1447] Structure- 23 activity studies showed that for the conjugate with X = adenosine-5'-carboxyiic acid and 2 = hexa(L-arginine) the optimal tether Y is 6-aminohexanoic acid [NH2(CH2)nCOOH, n=5). The same length of the tether turned out to be optimal for the high-affinity bisubstrate inhibitors of the present invention (Example 2, Table 2). The conjugates with the highest affinity can be linked to a fluorescent dye and new fluorescent probes with requested properties will be obtained. Different synthetic methods that were introduced for the preparation of bisubstrate-analog inhibitors are described in the Schemes 1 - 4. 24 Scheme 1. Synthesis of the bisubstrate-anaiog inhibitors, conjugates of Adc and a peptide Tether Peptide 2 NH-(CH2)5CO (L-Arg)4-NH2 3 NH-(CH2)5CO (L-Arg)6-NH2 4 NH-(CH2)5CO (D-Arg)4-NH2 5 NH-(CH2)5CO (D-Arg)6-NH2 6 N(CH3)-(CH2)sCO (L-Arg)4-NH2 7 NH-CH2-Ph-4-CO (L-Arg)4-NH2 8 NH-Phe-*e/aAIa-CO (L-Arg)4-NH2 Scheme 2. Synthesis of the bisubstrate-anaiog inhibitors, conjugates of adenosine and a peptide Scheme 3. Synthesis of the bisubstrate-anaiog inhibitors, conjugates of adenine and a peptide Scheme 4. Synthesis of the bisubstrate-anaiog inhibitors, conjugates of isoquinolinesulfonamide derivative H9 and a peptide 25 Structure-activity studies with cAMP-deoendent protein kinase (cAPK) The inhibitory potency of the synthesized conjugates was tested towards cAMP-dependent protein kinase (cAPK), a well characterized representative of basophilic protein kinases. In the case of Adc-peptide conjugates the amidation of peptide C-terminus increased the potency of bisubstrate inhibitors 2-3 fold. This may be caused by the compensation of the positive charge of an arginine residue that participates in a favourable contact with the kinase by the negative charge of the carboxylate group. Substantial modifications in the structure of the tether between adenosine and peptide moieties of conjugates were well tolerated by cAPK (Table 2, compounds 2, 6, 8), while some small structural modifications led to significant decrease of activity. Variation of the structure of the conjugates also included the connection of adenosine to the C-terminus of the oligoarginine chain and the incorporation of residues of D-arginine. Inhibitors with peptide moieties connected to the nucleoside part by C- and N-termini had similar affinities in the case of compounds containing peptides with L-amino acid residues, whereas the compound comprising the peptide with D-arginine residues connected via the C-terminus had 500-fold lower activity than the counterpart with N-terminal peptide (5). The bisubstrate character of the inhibitor 5 is supported by the high inhibitory potency of the compound if compared to the submillimolar potencies of the single site-targeted constituents of the conjugate, adenosine and oligo-(D-arginine). The inhibitory potency of the fluorescent probe ARC-TAMRA, AdcAhx(D-Arg)e-D-Lys(5-TAMRA)-NH2, was also established with the inhibition assay. The obtained IC50 value of inhibition 6.2 nM is even lower than that of the corresponding bisubstrate inhibitor AdcAhx(D-Arg)6NH2 (1C50 = 8.3 nM). The result shows that the addition of the linker (D-lysine) and the fluorescent dye (5-TAMRA) to the special position of the btligand inhibitor does not interfere substantially with its binding to the kinase. 26 Selectivity study Selectivity testing was performed (Example 3) for two compounds, 5 and 26, that had revealed the highest inhibitory potency towards cAPK Ca. The panel of kinases included all 52 kinases available for testing. To the best of our knowledge, the selectivity profiling has not been carried out with a wide panel of PK before for bisubstrate-type inhibitors. To make the inhibitory potencies comparable, assays were run at the ATP concentrations which were close to the Km value of the kinase. The results are presented {Example 3) at 1 uM concentration of the inhibitor as the percent of residual activity of the kinase relative to that in control incubations where the inhibitor was omitted. The activity profiles of two compounds were mostly similar. Both conjugates inhibited strongly ROCK-II, MSK1, PKB(3, PKBAph, and PRK2 (residual activity £ 3%). Compound 26 inhibited almost completely MAPKAP-K1a/rsk1, p70S6K, and MAPKAP-K1b, while residual activity in the presence of 5 was 7-28% for these kinases. The biggest difference between the two compounds was observed for the inhibition of MAPKAP-K1a/rsk1 where the corresponding values were 0 and 28% for 26 and 5, respectively. CAMK-1, PKA, PIM2, and PKD1 retained less than 10% of activity in the presence of 26. The significant difference between the two compounds was also found for PKD1 (the residual activities were 54% for 5 and 9% for 26). Checkpoint kinases (CHK1 and CHK2) were both inhibited more strongly by 5 than by 26. Several kinases were weakly inhibited by biligand inhibitors, while some of them were not inhibited at all, e.g., acidophilic kinases represented by casein kinases CK1 and CK2 retained full activity in the presence of 5 as well as 26. As illustrated by data in Table 2, oligo-(D-arginine) containing bifunctional inhibitors were not specific to any particular protein kinase but slowed down the reactions catalyzed by basophilic kinases, especially of the AGC (cAMP dependent, cGMP dependent, and protein kinase C family) and CAMK (calmodulin-dependent protein kinase) groups. Of the 14 PK inhibited by more than 85% by compound 26 at 1 micromolar concentration, 11 kinases belong to the AGC group and 3 kinases to the CAMK group. All these 14 kinases have been reported to be of basophilic type which 27 points to the biligand character of the inhibitors where the inhibitory potency of the conjugates is influenced by binding to both substrate-binding sites of the kinase. The only AGC group kinase that falls out of this selection is PDK, an AGC kinase which expresses badly defined consensus sequences and lacks strong Arg preference at any position. On the basis of the analysis of the results of the selectivity study of two inhibitors with 52 kinases, previously known substrate consensus sequences and other structural characteristics we have selected 59 protein kinases from the AGC family that are potential binders to the fluorescent probes disclosed in the present invention (Table 1). Table 1. 59 kinases from AGC group (classification by [Manning et al., Science 298 (2002) 1912]) which on the basis of the analysis of their substrate consensus sequences and other characteristics are potential binders to the fluorescent probes disclosed in the present invention Number Kinase Ar.r.gRftinn Grouo Familv Suhfamilv 1 AKT1 SK018 AGC AKT 2 AKT2 SK019 AGC AKT 3 AKT3 SK020 AGC AKT 4 DMPK1 SK111 AGC DMPK GEK 5 DMPK2 SK112 AGC DMPK GEK 6 MRCKb SK241 AGC DMPK GEK 7 MRCKa SK299 AGC DMPK GEK 8 MRCKps SK660 AGC DMPK GEK 8 ROCK2 SK263 AGC DMPK ROCK 10 ROCK1 SK331 AGC DMPK ROCK 11 CRIK SK695 AGC DMPK 12 MAST3 SK196 AGC MAST 13 MAST2 SK216 AGC MAST 14 MAST1 SK345 AGC MAST 15 MASTL SK455 AGC MAST 16 MAST4 SK701 AGC MAST 17 NDR1 SK249 AGC NDR 28 18 LATS1 SK441 AGC NDR 19 LATS2 SK442 AGC NDR 20 NDR2 SK500 AGC NDR 21 PKACa SK300 AGC PKA 22 PKACb SK301 AGC PKA 23 PKACg SK302 AGC PKA 24 PRKX SK313 AGC PKA 25 PRKXps SK319 AGC PKA 26 PRKY SK320 AGC PKA 27 PKCa SK303 AGC PKC Alpha 28 PKCb SK304 AGC PKC Alpha 29 PKCg SK307 AGC PKC Alpha 30 PKCd SK305 AGC PKC Delta 31 PKCt SK310 AGC PKC Delta 32 PKCh SK270 AGC PKC Eta 33 PKCe SK306 AGC PKC Eta 34 PKCi SK308 AGC PKC lota 35 PKCz SK311 AGC PKC iota 36 PKCips SK711 AGC PKC iota 37 PKG1 SK073 AGC PKG 38 PKG2 SK075 AGC PKG 39 PKN1 SK317 AGC PKN 40 PKN2 SK318 AGC PKN 41 PKN3 SK511 AGC PKN 42 MSK1 SK242 AGC RSK MSK 43 MSK2 SK243 AGC RSK MSK 44 p70S6K SK265 AGC RSK p70 45 p70S6Kb SK266 AGC RSK p70 46 p70S6Kps2 SK640 AGC RSK p70 47 p70S6Kps1 SK740 AGC RSK p70 48 RSK1 SK336 AGC RSK RSK 49 RSK2 SK337 AGC RSK RSK 50 RSK3 SK338 AGC RSK RSK 51 RSK4 SK518 AGC RSK 52 SgK494 SK491 AGC RSK 53 RSKL2 SK473 AGC RSKL 54 RSKL1 SK517 AGC RSKL 55 SGK SK346 AGC SGK 55 SGK2 SK523 AGC SGK 56 SGK3 SK525 AGC SGK 57 YANK3 SK469 AGC YANK 58 YANK2 SK481 AGC YANK 59 YANK1 SK624 AGC YANK Profiling with the 52-kinase selectivity panel (Example 3) reveals strong tendency of the arginine-rich conjugates to inhibit specifically basophilic protein kinases that points to the active participation of both functionary moieties in the formation of the binary complex with the kinase. In addition to the majority of AGC kinases, many kinases of CAMK and STE groups and some kinases not falling into major groups (classification by [Manning et al., Science 298 (2002) 1912]) have also been shown to be basophilic protein kinases [Pinna et al., Biochim. Biophys. Acta. 1314 (1996) 191; Zhu et al., J. Biol. Chem. 280 (2005) 36372]. All these kinases could be targets of fluorescent probes of the present invention and the probes can be used in analytical methods concerning these kinases. Additionally, the targets of the fluorescent probes of the present invention are different mutated forms of basophilic protein kinases, truncated forms of kinases inactive states of protein kinases, and pseudokinases. EXPERIMENTAL PART Abbreviations Adc - 5'-adenosine carboxylic acid or 1-(6-amino-9H-purin-9-yl)-1-deoxy-n-D- ribofuranuronic acid (CAS 3415-09-6); Ahx - 6-aminohexanoic acid; Arg - arginine; ARC - adenosine-arginine conjugate; 30 ATP - adenosine-5'-triphosphate; Boc - tert-butoxycarbonyl; cAMP - cyclic adenosine 3',5'-monophosphate; cAPK - cAMP-dependent protein kinase; DIC- 1,3-diisopropylcarbodiimide; DIPEA - N.N-diisopropylethylamine; DMF - dimethylformamide; DMSO - dimethyl sulfoxide; DTT-dithiothreitol Fmoc - 9-fluorenylmethoxycarbonyl; HOBt - 1-hydroxybenzotriazole; HPLC - high performance liquid chromatography; Ip: 2\3'-0-isopropylidene; MALDITOF MS, matrix assisted laser desorption ionisation time-of-flight mass spectrometry; NMR - nuclear magnetic resonance; Pbf - 2,2,4l6,7-pentamethyldihydrobenzofuran-5-sulfonyl; PK-protein kinase; PKA - cAMP-dependent protein kinase; RP - reverse phase; TFA - triffuoroacetic acid; TLC - thin layer chromatography. EXAMPLES EXAMPLE 1. SYNTHETIC PROCEDURES Solid phase synthesis procedures Peptide fragments were prepared by using traditional Fmoc solid-phase peptide synthesis methods on Rink amide MBHA resin (Schemes 1 - 4). Protected amino acids (3 equivalents) were dissolved in DMF and activated with BOP/HOBt (2.94 eq each) in DMF//V-methylmorpholine. Coupling solutions were added to the resin and shaked for 40-60 min. The completeness of each step was monitored with Kaiser-test, which was followed by deprotection of Fmoc-group by 20% piperidine 31 solution in DMF (20 min). Fmoc-protected linker, 6-Fmoc-aminohexanoic acid, was attached to the peptide part following the same protocol. The N-terminal Fmoc group was removed with 20% piperidine solution in DMF (20 min). Adenosine-5'-carboxylic acid was attached to the resin-bound peptide in the form of its isopropylidene protected derivative (3 equivalents) in DMF and activated with BOP/HOBt (2.94 eq each) in DMF/diisopropylethylamine. The protection groups were removed and the conjugate cleaved from the resin with 2 h treatment with 90% trifluoroacetic acid (5% triisopropylsilane, 5% water). The conjugate was purified with C18 reversed phase HPLC and lyophilized. Synthesis of the fluorescent probe AdcAhx(D-Arg)R-D-Lvs(5-TAMRA)-NH? (ARC-TAMRA) AdcAhx(D-Arg)6-D-Lys-C(0)NH2 in the form of its TFA salt (2.5 mg.1 jimo!) was dissolved in DMSO. 5-TAMRA, SE (5-Carboxytetramethylrhodamine, succinimidyl ester) (Anaspec; 0.53 mg, 1 (imol) in DMF (50 ^l) and DIEA (10 nl) were added. After 3 h reaction the solvents were removed in the freeze dryer and the product, AdcAhx(D-Arg)6-D-Lys(5-TAMRA)-C(0)NH2, purified by HPLC with C18 reversed phase column. MW (MALDI TOF, M+H+) = 1870; ^(absorption) = 559 nm; Xmaxtemission) = 582 nm. Adc-Ahx-(D-Arg6)-[D-Lys(5-TAMRA)]-NH2 has excitation and emission maxima at 559 nm and 582 nm (50 mM HEPES, 200 mM NaCI, 0,5 g/l BSA, 5 mM DTT, 0.05% TWEEN 20, MilliQ water; pH 7.4), respectively. The spectra were recorded on Perkin Elmer fluorescence spectrometer LS 55. Synthesis of the fluorescent probe AdcAhx(D-Arg)g-D-Lys(NBDVNH? -NHKD-Arg)6-(D-Irys)-C(=0)-NH2 o AdcAhx(D-Arg)6-D-Lys-C(0)NH2 in the form of its TFA salt (2.5 rng,1 mmol) was dissolved in DMSO (100 y\). 4-Fluoro-7-nitrobenzofurazan (NBD-F) (Anaspec; 0.2 32 mg, 1.0 nmo!) in DMF (100 \x\) was added. After 3h reaction at room temperature the solution was put into the freeze-dryer and the solvents were removed. HPLC purification on a C18 reversed phase column with water-ACN gradient gave the product in the form of dark powder. MW (MALDI TOF, M+H+) = 1751. X(max) at 480 nm in water, X(max) at 466 nm in methanol. Synthesis of the fluorescent probe H9-Hex-(D-Argk-D-Lvs(5-TAMRA)-C(Q)NH? Bromohexanoic acid (10 eq) was activated with DIC (5 eq) at 0 °C in DMF and added to the resin [Arg(Pbf)]6Lys(Boc)-[MBHA amide resin]. After 45 min agitation the resin was washed. The resulting alkyl bromide was reacted with 5-isoquinolinesulfonyl ethylenediamine (H9) in DMSO for 12 h at 50°C. Secondary amino group of the resin-bound conjugate was protected with Fmoc-CI (1h, room temp; 3 eq., 10eq. DIEA, DMF). The product was cleaved from the resin and isopropylidene and Boc groups were removed by 2 h treatment with 90% TFA (5% water, 5% diisopropylsilane) to produce IQS-NH-(CH2)2-N(Fmoc)-(CH2)5C(=0)-(D-Arg)6-D-Lys-NH2. After cleavage from the resin (90% TFA, 5% water, 5% TIS) and purification by reversed phase HPLC (water-ACN gradient, 0.1% TFA) the product was reacted with 5-TAMRA, SE (1 eq.) for 3h in the mixture of DMF and DMSO (1:1) in the presence of DIEA (5 eq.). After cleavage from the resin (90% TFA, 5% water, 5% TIS) and purification by reversed phase HPLC (water-ACN gradient, 0.1% TFA), the modified peptide H9-Hex-(D-Arg)6-(D-Lys)-NH2was reacted with 5-TAMRA, SE in DMSO (100 nl) in the presence of DIEA (10 \i\) for 3 h at roomt0. H9-Hex(D-Arg)6-D-Lys(5-TAMRA)-C(0)NH2 was purified with HPLC on a C18 reversed phase column. Dark red powder was obtained after freeze drying; ^max(absorption) = 559 nm; a.max(emission) = 582 nm. 33 EXAMPLE 2. Optimization of the structure of the bisubstrate-analoq inhibitor: determination of inhibition characteristics ICgn towards cAPK Ca 0 The IC5o values of the inhibitors were measured as previously described. [Viht et al., Anal. Biochem. 340 (2005) 165] The inhibitors in various concentrations were incubated at 30°C in Hepes buffer (50 mM, pH=7.5) containing cAPK Ca (Biaffin AG; about 1 nM), TAMRA-kemptide (10, 30 or 100 uM), ATP (100 uM or 1 mM), magnesium acetate (10 mM) and bovine serum albumin (nhibitr0.2 mg/mL). ATP was added last to initiate the phosphorylation reaction. At fixed time points, the reaction was stopped by 20-fold dilution with 75 mM phosphoric acid and obtained solutions were analyzed by normal phase TLC (without fluorescence indicator, eluted with 1-butanol/ pyridine/ acetic acid/ water, 15/10 /12 /12 by volume). The visualization and quantification of the fluorescent spots were carried out by fluorescence imaging. Data were processed with Graphpad Prism software (version 4, GraphPad). Table 2. Values of IC50 for the conjugates of oligoarginines and adenosine-5'-carboxylic acid (structures 2-8 from Scheme 1), conjugates of oligoarginines and isoquinoline sulfonamides (25 and 26), fluorescent probe ARC-TAMRA, and some single site-targeted inhibitors of the phosphorylation reaction catalyzed by cAPK Ca Compound Structure IC50 (MM) A AdcAhx(L-Arg)6OH Viht et al., Anal. Biochem. 340 (2005) 165 0.40 ±0.12 2 2.0 ±0.3 34 35 IC50-s are measured in the presence of 100 uM ATP and 30 uM TAMRA-kemptide. EXAMPLE 3. Ado 350 ± 40 H89 0.10 + 0.02 (0.85 ±0.09) H9 3.7 ±0.3 ARC-TAMRA AdcAhx(D-Arg)6-D-Lys Selectivity of bisubstrate-analog inhibitors of PK To establish the selectivity of two most potent inhibitors of cAPK (5 and 26, Table 2) towards other kinases their inhibitory effect was determined in a panel of 52 kinases. The assay of protein kinases was performed at 30°C as described previously [Davies et al., Biochem. J. 351 (2000) 95; Murray et al., Biochem. J. 384 (2004) 477]. The results of the testing are expressed as the % of residual activity of the kinase in the presence of the inhibitor at 1 micromolar concentration. Table .3. Residual activities of protein kinases in the presence of biligand inhibitors AdcAhx(D-Arg)e-NH2t 5, and H9-Hex(D-Arg)6-NH2, 26 protein kinase kinase group ATP (pM) residual activity3 (%) compound 5 compound 26 ROCK-II AGC 20 0(±0) 0(±0) MAPKAP-K1a/rsk-1 AGC 50 28 (± 7) 0(±1) SGK AGC 20 16 (±0) 1(±0) MSK1 AGC 20 2(±0) 1(±1) 36 PKBAph AGC 5 2(±0) 2(±0) P70 S6K AGC 20 7 (±2) 2(±0) MAPKAP-K1b/rsk-2 AGC 50 12 (±0) 2(±0) PRK2 AGC 5 2(±1) 2(±0) PKBb AGC 50 3(±0) 3(±0) CAMK-1 CAMK 50 15 (±2) 6(±0) PKA AGC 20 11 (±7) 6 (±2) PIM2 CAMK 5 38 (± 3) 9(±0) PKD1 CAMK 50 54 (± 8) 9(±1) PKCa AGC 20 31 (± 7) 15 (±5) CHK2 CAMK 20 14 (±2) 22 (± 5) MST2 STE 20 77 (± 3) 24 (± 0) CHK1 CAMK 20 16 (±4) 31 (± 1) MNK2 CAMK 50 99 (± 9) 31 (± 3) AMPK CAMK 50 48 (± 6) 39 (± 4) ERK8 CMGC 5 50 (± 9) 52 (± 0) Aurora B other 20 103 (±5) 53 (±1) MARIO CAMK 5 40 (± 3) 53 (+ 4) PDK1 AGC 20 62 (± 4) 56 (±1) NEK7 other 20 58 (±1) 56 (± 9) PBK TK 50 88 (± 5) 67 (± 4) MNK1 CAMK 50 102 (±1) 68 (± 9) DYRKIa CMGC 50 72 (± 6) 69 (±1) MAPKAP-K3 CAMK 20 67 (± 0) 69 (± 8) CSK TK 20 48 (± 9) 70 (±1) CDK2/cyclin A CMGC 20 88 (± 7) 73 (± 3) JNK/SAPK1C CMGC 20 70 (± 7) 75 (+ 4) PLK1 other 5 81 (± 10) 76 (± 2) 37 SAPK2a/p38 CMGC 50 79 (± 2) 76 (± 7) SAPK2b/p38B2 CMGC 20 85 (± 0) 80 (± 2) MAPKAP-K2 CAMK 20 85 (± 6) 80 (± 2) MKK1 STE 5 77 (± 9) 84 (± 6) IKKb other 5 94 (±11) 87 (±1) CK2 other 5 104 (±9) 87 (± 7) Src TK 50 84 (±9) 88 (± 4) Lck TK 50 70 (± 3) 88 (± 3) smMLCK CAMK 50 72 (± 0) 90 (± 5) eEF2K atypical 5 92 (± 9) 91 (± 8) JNK3 CMGC 50 91 (± 6) 92 (± 9) SAPK4/p38d CMGC 5 107 (±1) 93 (±1) GSK3b CMGC 5 56 (± 4) 94 (± 9) MAPK2/ERK2 CMGC 50 100 (±3) 95 (± 7) CK1 CK1 20 106 (±8) 97 (± 4) PRAK CAMK 20 89 (±4) 97 (± 8) NEK2a other 50 82 (± 2) 98 (± 6) SAPK3/p38g CMGC 5 111 (±4) 99 (±1) SRPK1 CMGC 50 100 (±3) 99 (± 2) NEK6 other 50 94 (± 8) 100 (±8) EXAMPLE 4. Characterization of the complex between the fluorescent probe ARC-TAMRA and the kinase cAPK Ca. ARC-TAMRA, Adc-Ahx-(D-Arg6)-[D-Lys(5-TAMRA)]-NH2, has excitation and emission maxima at 559 nm and 582 nm (50 mM HEPES, 200 mM NaCI, 0,5 g/l BSA, 5 mM DTT, 0.05% TWEEN 20, MilliQ water; pH 7.4), respectively. The spectra were recorded on Perkin Elmer fluorescence spectrometer LS 55. 38 The binding of the fluorescent probe ARC-TAMRA to the kinase cAPK Cot (Biaffin) was studied at 10 nM or 2nM ARC-TAMRA concentration (excitation at 557 nm, slitwidth 10 nm; emission at 585 nm, slitwidth 10 nm, detection of fluorescence polarization). 10 nM solution of ARC-TAMRA in 500 \|il of buffer (50 mM HEPES, 200 mM NaCI, 0.5 g/l BSA, 5 mM DTT, 0.05% TWEEN 20, MilliQ water; pH 7.4) was titrated with the solution of kinase cAPK Ca (Biaffin). 1-3 ul quantities of various dilutions of kinase solution were added to 500 pi of 2.0 nM or 10.0 nM solution of ARC-TAMRA, and fluorescence anisotropy was measured after each addition of the kinase solution after 10 min delay time. The change in fluorescence anisotropy was plotted against the kinase concentration [cAPK Ca]. The data were fitted to the equation: Lt + Kd + Rt-A/(Lt + Kd + Rt)2-4-Lt-Rt A = Af + (Ab-Af) — 2-Lt where, A is measured anisotropy; Af is the anisotropy of free ARC-TAMRA; Ab is the anisotropy of bound ARC-TAMRA; LT is the concentration of ARC-TAMRA added, 10 nM; RT is the total concentration of active kinase (cAPK Ca). K Kd value of 1.1 nM was calculated with 2 nM of ARC-TAMRA (FIG 2). EXAMPLE 5. Competition experiments for determination of the binding constants of inhibitors A complex was created between 10 nM of ARC-TAMRA and 10 nM of cAPK Ca in 0.500 ml of buffer (50 mM HEPES, 200 mM NaCI, 0.5 g/l BSA, 5 mM DTT, 0.05% TWEEN 20, MilliQ water; pH 7.4). Different compounds were screened for binding to cAPK Ca against fixed concentrations of ARC-TAMRA and cAPK Ca (FIG 4). 1-3 pi quantities of various dilutions of the solution of the inhibitor were added to 500 pi solution of ARC-TAMRA complex with the kinase and fluorescence anisotropy was measured after each addition of the kinase solution after 10 min delay time. 39 The following values of IC5o and Kj were obtained for competitive binding of inhibitors to the ARC-TAMRA - kinase complex: Inhibitor ic50 Ki L-ARC 1,3 nM 110 nM D-ARC 35 nM 2,5 nM H89 180 nM 15 nM PKI (5-24) 140 uM 12 nM Abbrevations for the compounds: N-(2-[p-bromocinnarnylamino-]ethyl)-5-isoquinoline-sulfonamide (H89); AdcAhx(L-Arg)6-NH2 (L-ARC); AdcAhx(D-Arg)6-NH2 (D-ARC); PKI peptide (5-24). Binding affinity constants (Kj values) of inhibitors were calculated [Nikolovska-Coleska et al., Anal. Biochem. 332 (2004) 261] according to the relationship where [l]50 is the concentration of the free inhibitor at 50% inhibition, [L]50 is the concentration of the free labeled ligand at 50% inhibition, [P]0 is the concentration of the free protein at 0% inhibition, and K Very similar Ki values were obtained for the same inhibitors in the conditions were the complex was created at lower concentrations of the interacting partners, between 2 nM ARC-TAMRA and 5 nM cAPK Ca. EXAMPLE 6. Determination of the concentration of the active form of the kinase ARC-TAMRA, Adc-Ahx-(D-Arg6)-[D-Lys(5-TAMRA)]-NH2, was used as the fluorescence probe: excitation at 558 nm (bandwidth 10 nm) and emission at 585 nm (bandwidth 10 nm): 50 mM HEPES, 200 mM NaCI, 0.5 g/l BSA, 5 mM DTT, 0.05% TWEEN 20, MilliQ water; pH 7.4. The spectra were recorded on Perkin Elmer fluorescence spectrometer LS 55. 10 nM solution of ARC-TAMRA in 500 nl of buffer (50 mM HEPES, 200 mM NaCI, 0,5 g/l BSA, 5 mM DTT, 0.05% TWEEN 20, MilliQ water; pH 7.4) was titrated with 40 the solution of kinase cAPK Ca(Biaffin). 1-3 ul quantities of various dilutions of kinase solution were added to 500 ul of 2.0 nM or 10.0 nM solution of ARC-TAMRA, and fluorescence anisotropy was measured after each addition of the kinase solution after 10 min delay time. The change in fluorescence anisotropy was plotted (FIG 3) against the kinase concentration [cAPK Ca]. The fraction of the active (binding) form of the kinase in solution, k, was calculated by the application of nonlinear regression analysis to the relationship [Data were processed with Graphpad Prism software (version 4, GraphPad)]: Lt + Kd + k-Rt-V(Lt + Kd + k-Rt)2-4-Lt-k-Rt A = Af + (Ab-Af) - 2-Lt where A is the measured anisotropy; Af is the anisotropy of free ARC-TAMRA; Ab is the anisotropy of bound ARC-TAMRA; LT is the concentration of ARC-TAMRA added, 10 nM; RT is the total concentration of active kinase (cAPK Ca); Kd is the dissociation constant of the reaction between ARC-TAMRA and cAPK Ca (Kd=1.0 nM). The obtained k value (Figure 3) of 0.317 shows that 317 % of the nominal kinase is in the active (binding to the probe) form in this sample of kinase. EXAMPLE 7. Hiqh-throuahput binding assay for inhibitors of the kinase cAPK Ca The high sensitivity of fluorescence platereader PHERAstar (BMG LABTECH) enabled the realization of displacement experiments at 1 nM concentration of the fluorescent probe ARC-TAMRA, Adc-Ahx-(D-Arg6)-[D-Lys(5-TAMRA)]-NH2. Corning 384 well Low Volume NBS microplates (20 p} or 30 JLLI reaction volumes) were used for assays. Due to the polar and charged character of the fluorescent probe the selection of the well material of microplate is of great importance and specially designed coatings for fluorescence measurements with polar peptides and proteins are better suited than ordinary glass or polystyrene based well materials. The application of latter materials may lead to adsorbation of the probes 41 of the present inventions to the surface of the well of the microliter plate and misrepresentation of results of the measurements. Characterization of the fluorescent probe Titrations of 2 nM and 20 nM ARC-TAMRA solutions were performed with cAPK CD. 2-fold serial dilutions of the kinase were used leading to concentrations of the kinase between 100 nM and 0.05 nM and 20 jiL reaction volumes on a 384-well microliter plate. Before the measurement of fluorescence anisotropies the plate was rotated (400 rpm) for 10 minutes at 30°C. Optical conditions for the measurements were set as the following: 540 nm excitation and 590 nm emission (20 nm bandpass filters). To find the value of Kd, titration curves were analyzed by a scientific curve-fitting program, e.g., GraphPad Prism 4.03, with nonlinear regression, using the function below: , K {k K Lt + Kd + k.Rt-J(Lt + Kd + k.Rt)2-4-Lt-k-Rt A = Af + (Ab - Af) ^ , 2-Lt where A is measured anisotropy, Af - anisotropy value for free ARC-Photo, Ab - anisotropy value for bound ARC-Photo, Lt - total concentration of ARC-Photo, Rt - nominal total concentration of enzyme, k - concentration of active enzyme divided by nominal concentration of enzyme, Kd - dissociation constant for ARC-Photo complex with enzyme. Kd value of 0.4 was determined for the complex (FIG 5). Determination of the inhibition constants (Ki) To determine the inhibition constant (KJ, 3-fold serial dilutions of a given inhibitor are performed analogously as for titration experiments. Before the measurement of fluorescence anisotropies the plate is rotated (400 rpm) for 10 minutes at 30°C. Kj values of 13 nM for ATP-competitive inhibitor H89, 0.5 nM for substrate protein-competitive inhibitor Rlla (regulatory subunit of cAPK), 21 nM for bisubstrate inhibitor AdcAhx(L-Arg)6-NH2 and 1.1 nM for bisubstrate inhibitor AdcAhx(D-Arg)6-NH2were determined (FIG 6). The obtained Kj values for competitive binding are in good accord with the inhibitory constants for the same compounds of the phosphorylation reaction catalyzed by cAPK Cot (Example 2) and inhibitory constants determined with the 42 same fluorescent probe with a fluorescence spectrometer In a 0.5 ml cell (Example 5). EXAMPLE 8. Characterization of the complex between the fluorescent probe ARC-TAMRA and the Rho-associated kinase ROCK II and determination of the inhibition constants for inhibitors Experiments were performed in the wells of 384-well microtiter plates and with the measurements of fluorescence anisotropy with the platereader PHERAStar according to the protocols described in the Example 7. The titration of the fluorescent probe ARC-TAMRA with the kinase ROCK II enabled the determination of dissociation constant Kd of the complex between ARC-TAMRA and the kinase as of 4 nM (FIG 7). The displacement of the fluorescent probe ARC-TAMRA from the complex with the kinase (formed between 2nM of ARC-Photo and 15 nM of ROCK II) by different inhibitors of ROCK enabled the characterization of the inhibitors with Ki values of 45 nM, 40 nM, and 5 nM for H89, Y-27632, and L-ARC, respectively (FIG 8). Similar results were obtained with the application of the other Rho-associated kinase ROCK I, having high structural similarity to ROCK II. 43 WE CLAIM 1. A fluorescent probe with the general formula (I): (X-Y-Z)-L-F (1), wherein - X-Y-Z is a bisubstrate-analog inhibitor of a protein kinase, in which X is a compound that binds to the ATP-binding pocket of the kinase and; - Z is a compound that binds to the protein/peptide-binding domain of the kinase; - Y is a an organic tether that connects X and Z and permits simultaneous binding of X and Z to the active site of the kinase; - F is a fluorescent dye which optical characteristics are changed in the course of the binding of (X-Y-Z)-L-F to the kinase; - L is a linker (between the bisubstrate-analog inhibitor XYZ and fluorescent label F) formed of a hydrocarbon chain, of other type of organic molecule or of part of organic molecule. L may be omitted in the probe and F directly bound to inhibitor XYZ. 2. The fluorescent probe of claim 1, wherein the structure of XYZ comprises ATP or ATP mimics and a peptide or a peptide mimics. 3. The fluorescent probe of claim 1, wherein X is adenosine or adenosine-based compound. 4. The fluorescent probe of claim 1, wherein X is purine or purine-based compound. 5. The fluorescent probe of claim 1, wherein X is isoquinoline or isoquinoline-based compound. 6. The fluorescent probe of claim 1, wherein Z is a peptide or a peptide mimics. 44 12. The fluorescent probe of claim 1, wherein the structure of the probe is 13. The fluorescent probe of claim 1, wherein the structure of the probe is 7. The fluorescent probe of claim 1, wherein Z is a arginine-rich peptide or a arginine-rich peptide mimics containing guanidino groups, e.g., peptoid. 8. The fluorescent probe of claim 1, wherein Z is a peptide containing D-arginine residues. 9. The fluorescent probe of claim 1, wherein Z is a peptide containing 6 D-arginine residues. 10. The fluorescent probe of claim 1, wherein Y is a hydrocarbon chain of 1-20 carbon atoms, or a alkylene chain where any of C atoms is replaced with one or more of amide groups or ester groups or O, N, P and S atoms. Y may be a straight or branched chain molecule consisting of peptides or peptoids or their mimics and of their combinations. 11. The fluorescent probe of claim 1, wherein the structure of the probe is 14. An assay for the identification of a kinase inhibitor by displacement of the fluorescent probe of any of claims 1-13 from its complex with the kinase by an inhibitor compound comprising the steps of: 1) establishing the Kd for the complex of the fluorescent probe with the kinase; 2) contacting the fluorescent probe with the kinase and measuring the fluorescence signal of the formed complex; 3) incubation of the complex formed in the previous step with the potential inhibitory compound or a mixture of compounds and measuring of the fluorescence signal of the probe; 4) comparing the fluorescence signals from step 2) and step 3): difference in these fluorescence signals indicates the presence of a active compound in the probe of the step 3). 15. A method for the determination of binding characteristics of protein kinase inhibitors by displacement of the fluorescent probe of any of claims 1-13 from its complex with the kinase by an inhibitor compound comprising the steps of: 1) establishing the Kd for the complex of the fluorescent probe with the kinase; 2) contacting the fluorescent probe with the kinase and measuring the fluorescence signal of the formed complex; 3) incubating of the complex formed in the previous step with a series of different concentrations of the inhibitory compound and measuring of the fluorescence signal at each concentration of the inhibitor; 46 4) calculating of the Kd of the inhibitory compound for its binding to the active site of the kinase proceeding from the Kd for the complex of the fluorescent probe with the kinase and the results of the measurements of the step 3). 16. Method according to claim 15 wherein the probe is the fluorescent probe of any of claims 1-13 where the signal is measured as a shift in fluorescence polarization of the probe. 17. Method of claim 15 wherein the screening is performed on a multi-well plate. 18. Method for the determination of binding characteristics for compounds targeting the ATP-binding pocket of the kinase by displacement of the fluorescent probe of any of claims 1-13 from its complex with the kinase by an inhibitor compound comprising the steps of: 1) establishing the Kd for the complex of the fluorescent probe with the kinase; 2) contacting the fluorescent probe with the kinase and measuring the fluorescence signal of the formed complex; 3) incubating of the complex formed in the previous step with a series of different concentrations of the inhibitory compound and measuring of the fluorescence signal at each concentration of the inhibitor; 4) calculating of the Kd of the inhibitory compound for its binding to the ATP binding site of the kinase proceeding from the Kd for the complex of the fluorescent probe with the kinase and the results of the measurements of the step 3. 19. Method for the determination of binding characteristics for compounds targeting the protein/peptide substrate binding domain of the kinase by displacement of the fluorescent probe of any of claims 1-13 from its complex with the kinase by an inhibitor compound comprising the steps of: 1) establishing the Kd for the complex of the fluorescent probe with the kinase; 47 2) contacting the fluorescent probe with the kinase and measuring the fluorescence signal of the formed complex; 3) incubating of the complex formed in the previous step with a series of different concentrations of the inhibitory compound and measuring of the fluorescence signal at each concentration of the inhibitor; 4) calculating of the Kd of the inhibitory compound for its binding to the substrate protein/peptie binding domain of the kinase proceeding from the Kd for the complex of the fluorescent probe with the kinase and the results of the measurements of the step 3. 20. Method according to claim 15 wherein the protein kinase is a basophilic protein kinase. 21. Method according to claim 15 wherein the kinase is cAMP-dependent protein kinase. 22. Method according to claim 15 wherein the kinase is Rho-associated kinase (ROCK). 23. A method for quantifying the kinase in a sample characterized by bringing the fluorescent probe according to any of claims 1-13 into contact with the sample and measuring of the fluorescence anisotropy from the probe: 1) establishing the Kd for the complex of the fluorescent probe according to the claim 1 with the kinase; 2) contacting the fluorescent probe with a series of samples of the kinase at different dilutions and measuring the fluorescence signal of the formed complex of the diluted samples; 3) calculating the fraction of the active (binding) form of the kinase in solution, k, according to the relationship 48 where A is the measured anisotropy; Af is the anisotropy of the free fluorescent probe; Abis the anisotropy of the bound fluorescent probe; LT is the concentration of the fluorescent probe added; RT is the total concentration of the kinase; Kd is the dissociation constant of the reaction between the. fluorescent probe and the kinase. 24. A kit for the methods of identification of inhibitors of protein kinases or determination of binding characteristics of inhibitors as defined in any one of claims 14, 15 or 23, comprising a fluorescent probe of any one of claims 1-13. 25. A bisubstrate-analog inhibitor of basophilic protein kinase with the general formula (II): X-Y-Z (II), in which X is a compound that binds to the ATP-binding pocket of the kinase and; - Z is a peptide or peptide analog incorporating 1-10 D-arginine residues; - Y is an organic tether that connects X and Z and permits simultaneous binding of X and Z to the active site of the kinase. Dated this 16th day of March, 2009.

Full Text

FORM 2
THE PATENTS ACT, 1970 {39 of 1970)
& THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
[See section 10, Rule 13]
BISUBSTRATE FLUORESCENT PROBE BINDING TO PROTEIN KINASES;

UNIVERSITY OF TARTU, WHOSE ADDRESS
IS ULIKOOLI 18, EE50090 TARTU, ESTONIA.

THE FOLLOWING SPECIFICATION
PARTICULARLY DESCRIBES THE INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED.

2
RELATED APPLICATION
This application claims priority to Estonian provisional application number P200600030, filed on August 15, 2006, the contents of which are incorporated herein.
FIELD OF THE INVENTION
The invention concerns a novel fluorescent probe binding to the active site of a protein kinase and its application for screening of compounds and evaluation of inhibitors targeted both to the ATP-binding pocket or/and protein/peptide substrate-binding domain of the kinase, and methods of manufacturing of such probes.
The invention also relates to the screening assay for identifying compounds that bind to and modulate the activity of a wide variety of members of protein kinase super-family. The invention relates to the assay for identification and evaluation of protein kinase inhibitors and determination of the concentration of protein kinases.
BACKGROUND TO THE INVENTION
Protein Kinases and Protein Kinase Inhibitors
Protein kinases (PKs) play a key role in the regulation of protein functions in living cells. It has been estimated that the activity of one third of proteins is regulated through phosphorylation of one or more of serine, theorine and thyrosine residues of the protein. More than 400 human diseases (incl. cancer) have been linked to aberrant protein kinase signaling. This has made PK the second largest drug target (and fastest growing category of drugs in development) after G protein-coupled receptors. [Cohen, Nat. Rev. Drug Discov. 1 (2002) 309; Fischer, Curr. Med. Chem. 11 (2004) 1563]
Protein kinases follow ternary complex kinetic mechanism in which direct transfer of the phosphoryl group from ATP to the protein substrate occurs in the active site. [Adams, Chem. Rev. 101 (2001) 2271]
r*t

3
Three different kind of active site-targeted protein kinase inhibitors are previously known. Firstly, despite serious selectivity problems (all 500 protein kinases and more than 1500 other proteins are able to bind purine nucleotides), and high concentration of competing ATP in the cellular milieu, the main efforts of drug companies have been directed to the development of ATP competitive inhibitors. Imatinib, a specific small molecule inhibitor of the Abl kinase, has become the first successful breakthrough in kinase-targeted cancer therapy.
The second type of active site targeted inhibitors of protein kinases comprise compounds that selectively interfere with protein-protein interactions and block the binding of the substrate protein to the active site of the protein kinase. [Recent reviews: Bogoyevitch et al., Biochim. Biophys. Acta. 1754 (2005) 79; Lawrence, New Design Strategies for Ligands That Target Protein Kinase-Mediated Protein-Protein Interactions.; Pinna, A. L, Cohen, P. T. W. Eds.; 2005; p. 11J
Thirdly, combination of the aforementioned approaches and development of bisubstrate-analog (biligand) inhibitors that simultaneously associate with both ATP and protein binding domains of the dual substrate enzyme has given selective and potent inhibitors of PK. Several strategies of the design of bisubstrate-analog inhibitors have been described. [For a review on the subject, see: Parang et al., Pharmacol. Therap. 93 (2002) 145]. Construction of bisubstrate-analogue (Afunctional) inhibitors could lead to enhanced specificity and potency in protein kinase inhibition. The inhibitor mimics two natural substrates/ligands and associates with two regions of a given kinase simultaneously. Most bisubstrate analogues have been designed to mimic ATP and the acceptor component. The inhibitors of this kind are covalent conjugates between one moiety inhibiting ATP binding and one moiety inhibiting binding of a protein/peptide substrate to the protein kinase. [Ricouart et al., J. Med. Chem. 34 (1991) 73; Medzihradszky et al., J. Am. Chem. Soc. 116 (1994) 9413; Patent application describing bisubstrate analog inhibitors of PK: WO0070029, WO0170770, WO03010281, WO2004110337, EE200300187]
One of the present inventors has previously developed bisubstrate-analog inhibitors for protein serine/threonine kinases PKA and PKC with activities in sub-micromolar region. [Loog et al., Bioorg. Med. Chem. Lett., 9 (1999) 1447, Uri et al.,

4 Bioorg. Med. Chem. Lett., 12 (2002) 2117, WO0070029, EE200300187]. These inhibitors comprise moieties of analogs of both substrates of protein kinases: ATP binding site targeted adenosine-5'-carboxylic acid (Adc) and the protein substrate domain directed oligo-(L-arginine). The design of the latter fragment was based on the knowledge that phosphorylation sites of the substrates of basophilic protein kinases (cAPK, PKC, Akt/PKB, PKG, etc.) are flanked by regions rich in arginine or/and lysine residues. [Pinna et al., Biochim. Biophys. Acta. 1314 (1996) 191] Two active fragments of the inhibitors were connected via a tether which length was optimized in structure-activity studies.
After the priority date (August 15, 2006) the synthesis and characterization of bisubstrate-analog inhibitors for protein serine/threonine kinases incorporating D-arginine residues that is the subject of the present patent application was described in two published articles by inventors of the present patent application. [Enkvist et al., J.Med.Chem. 49 (2006) 7150; Viht et al., Anal.Biochem., 362 (2007) 268]
Existing assay methods
Development of new assay methods for evaluation of protein kinase inhibitors has run in parallel with the increase of importance of effective inhibitors for drug industry. During recent years, flexible fluorometric kinase assay methods have substituted problematic (e.g., personal risks, environmental hazards, short half-lives of 32P- and 33P-Iabeled compounds, long exposure times) radiometric methods. Fluorescent methods can be spatially and temporally more focused than radioactive methods, and as such, they are more suitable for application in high-throughput screening (HTS) assays. [Olive, Expert Rev Proteomics. 1 (2004) 327]
Great majority of evaluations of kinase inhibitors is performed in the form of kinetic studies and the new, potential inhibitors are screened and characterized on the basis of their retarding effect on the rate of substrate (peptide or protein) phosphorylation reaction catalyzed by the kinase. In the case of radioactive methods the product of the phosphorylation reaction that is covalently labeled with radioactive phosphor isotope is separated from radioactive ATP and quantified.

5 The application of labor-intensive separation steps and the use of large amounts of radioactivity make these methods problematic for high throughput assays.
Kinetic methods where the amount of the product of the phosphorylation reaction is established by its competition with a fluorescent binder for an antibody (a phosphopeptide formed in a kinase reaction displaces a fluorescently-labeled phosphopeptide from a phospho-specific antibody) or other phosphopeptide binding macromolecule (Immobilized Metal Assay for Phosphochemicals, 1MAP) is another class of assays in active use. [e.g., W09818956]. In this case the change in the fluorescence anisotropy resulting from the displacement of the fluorescent binder from its complex with the antibody is usually measured. Although these methods suit better for HTS assays, they still require an effective substrate, fluorescent binder and high-affinity antibody or other phosphor-binding macromolecule for the method. Large number of measurements has to be performed to characterize the inhibitory compounds with comparable K* values.
Alternatively, kinase inhibitors may be detected by direct or indirect measurement of the binding of the inhibitor to the kinase. Some small-molecule inhibitors can be conjugated to a fluorescent dye without loosing their binding affinity to kinase. The kinase-bound labeled inhibitor can then be displaced by competitive kinase inhibitors and the change in fluorescent characteristics measured. This interaction therefore forms the basis for a competitive binding assay of kinase inhibitors where no knowledge of the substrate or an antibody to the phosphorylated kinase substrate is required. Several papers and patent applications describe the use of fluorescent probes for determination of the binding characteristics of protein kinase inhibitors, [e.g., Chen et al., 263 (1993) 15812, WO2005/033330]
Limitations of Current Binding Assays
Fluorescent probes with micromolar affinity towards cAPK were disclosed by Chen et al. [Chen et al., 268 (1993) 15812] These ATP-competitive probes have complicated emission characteristics that originate from the fluorescence of both the fluorescent dye and bisindolylmaleimide ligand. Low (micromolar) affinity and complex fluorescence spectrum make it difficult to use these fluorescent

6
conjugates as probes for binding assays. [WO9906590] Other ATP competitive fluorescent probes have been described in literature, [e.g., WO2005/033330]
A fluorescent probe targeted to the protein/peptide substrate binding domain of the cAMP-dependent protein kinase (cAPK), fluorescein-labeled 20 amino acid residues-containing sequence of protein kinase heat stable inhibitor protein PKI (PKI 5-24), was shown to bind to cAPK with micromolar affinity (1^=1.6 (iM) in a fluorescence polarization assay. [Shneider et al., Org. Lett. 7 (2005) 1695] Low binding affinity of the probe prevents its application for the determination of the binding constants of the protein/peptide substrate-binding domain targeted inhibitors.
The main limitations of the binding assays that have been disclosed so far are the following:
A. Fluorescent probes available have low affinity (usually micromolar or
submicromolar) towards protein kinases that leads to substantially higher
consumption of kinases when using these methods if compared to kinetic
assays, and to the impossibility of the application of the probes for
determination of exact binding characteristics of high-affinity inhibitors;
B. Fluorescent probes available are active to a single kinase or a small family
of kinases that makes it impossible to use a single probe for generic
characterization of inhibitors with multiple kinases;
C. Alt fluorescent probes available thus far enable the testing of compounds
binding to either ATP or protein/peptide substrate binding site and as such
differently from kinetic methods do not permit simultaneous screening of
ATP and protein-peptide substrate binding site-targeted inhibitors.
To our knowledge, prior art published by other groups most related to the current invention is described in the patent application WO2005/033330 "Fluorescent probes for use in protein kinase inhibitor binding assay". This invention discribes a fluorescent probe with affinity in high nanomolar range (Kd = 161 nM) which leads to the need for high concentration of the kinase in the assay format (200 nM STK12 kinase was used in the disclosed Example). Requested kinase

7 concentration, arising from the Kd value of the fluorescent probe, is a hundred-fold higher than the kinase concentration usually applied for kinetic measurements (ca 1 nM concentration of the enzyme is often used in kinetic assays). This leads to substantial increase of the cost of the assay. Due to sub-micromolar dissociation constant of the probe it cannot be used for determination of binding constants of inhibitors with nanomolar affinity [Fluorescence Polarization Technical Resource Guide THIRD EDITION 2004, Invitrogen Corporation. Chapter 7]
The probe disclosed in WO2005/033330 is targeted to the ATP binding site of the kinase and as such the application of the probe for screening of PK inhibitors neglects the inhibitors targeted to the protein/peptide binding domain of the kinase. Furthermore, the fluorescent probe disclosed in WO2005/033330 is applicable for testing of inhibitors of a specific kinase (STK12 and close analogs).
Benefits of the Presented Invention
The invention described in current patent application overcomes limitations of all known fluorescent probes and is applicable in competitive displacement assays for identification and characterization of inhibitors of many protein kinases and determination of the concentration of the protein kinase. If compared to the probes from previous inventions, the Fluorescent probe of the present invention has very high affinity (Kd s 1 nM) and can be used for testing of inhibitors of a variety of protein kinases targeted to both ATP and/or protein substrate binding sites.
SUMMARY OF THE INVENTION
This invention relates to fluorescent probes for identification of compounds binding to protein kinases, for measurement of the affinity of inhibitors of protein kinases, and determination of the active concentration of protein kinases binding to the probe. Bisubstrate-analog character of the probe enables the simultaneous evaluation of inhibitors targeted to both ATP binding site and/or substrate protein/peptide binding domain of the kinase.
High affinity of the probe (Kd = 1.0 nM towards cAPK) affords the application of the enzymes at very low concentration (
8 determination of binding constants for inhibitors with nanomolar and micromolar affinity. Due to the ability of the conjugates of oligo(D-arginine) with a ATP binding site targeted inhibitors of this invention to bind with high affinity to a wide spectrum of (basophilic) kinases, a single Fluorescent probe is applicable to a number of protein kinases.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Titration of the fluorescent probe ARC-TAMRA (10 nM) with cAPK Ca (fluorescence spectrometer LS 55 (Perkin Elmer) with 0.5 ml quartz cell). Kd=1.1 nM was calculated with the application of nonlinear regression analysis to the relationship:
A=A( + (Ab-Af)x «^Kd+KT)-V(LT + Kd ,RTH-4LTRT]
2LT (A)
A is measured anisotropy; Af is the anisotropy of free ARC-TAMRA; Ab is the anisotropy of bound ARC-TAMRA; LT is the concentration of ARC-TAMRA added, 10 nM; RT is the concentration of the total kinase (cAPK Cot).
Figure 2. Titration of of ARC-TAMRA (2 nM and 10 nM) with cAPK Ca (fluorescence spectrometer LS 55 (Perkin Elmer) with 0.5 ml quartz cell). Kd values of 1.1 nM and 0.94 nM were calculated for 2 nM and 10 nM of ARC-TAMRA, respectively from the relationship (A) (Figure 1).
Figure 3- Determination of the concentration of the active kinase (fluorescence spectrometer LS 55 (Perkin Elmer) with 0.5 ml quartz cell). The fraction of the active (binding) form of the kinase in solution, k=0.317, was calculated by the application of nonlinear regression analysis to the relationship:
A K fK Lt + Kd + k-Rt-J(Lt + Kd + k-Rt)2-4-Lf k-Rt
A = Af + (Ab-Af) — (B)
2-Lt
A is the measured anisotropy; Af is the anisotropy of free ARC-TAMRA; Ab is the anisotropy of bound ARC-TAMRA; LT is the concentration of ARC-TAMRA added, 10 nM; RT is the total (nominal) concentration of kinase (cAPK Ca); K^ is the

9 dissociation constant of the reaction between ARC-TAMRA and cAPK Ca (Kd=1.0 nM).
Figure 4. Dependence of anisotropy (normalized values) on the concentration of inhibitors H89; PKI [inhibitory peptide PKI(5-24)], ARC [AdcAhx{L-Arg)6-NH2], and D-ARC [AdcAhx(D-Arg)6-NH2]. Assay conditions: 10 nM of ARC-TAMRA and 10 nM of kinase (cAPK Ca), fluorescence spectrometer LS 55 (Perkin Elmer) with 0.5 ml quartz cell. The Kd value of 1.0 nM was used for ARC-TAMRA. The obtained inhibitory characteristics for inhibitors were as follows:

Inhibitor ICso K,
L-ARC 1,3 (iM HOnM
D-ARC 35 nM 2,5 nM
H89 180nM 15 nM
PKI (5-24) 140 nM 12 \xM
Figure 5. Titration of ARC-TAMRA (2 nM and 20 nM) with cAPK Ca (fluorescence plate reader PHERAstar (BMG LABTECH), Corning 384 well Low Volume NBS microptates (30 pJ reaction volume)). Kd value of 0.4 nM was calculated from the relationship (A) (Figure 1).
Figure 6. Dependence of anisotropy (normalized values) on the concentration of inhibitors H89; ARC [AdcAhx(L-Arg)fl-NH2], D-ARC [AdcAhx(D-Arg)6-NH2], and the regulatory subunit of cAMP-dependent protein kinase Rlla. Assay conditions: 2 nM of ARC-TAMRA and 3 nM of kinase (cAPK Ca), fluorescence plate reader PHERAstar (BMG LABTECH). The Kd value of 0.4 nM was used for ARC-TAMRA. Kj values of 21 nM (AdcAhx(L-Arg)6-NH2), 13 nM (H89), 1.1 nM (AdcAhx(D-Arg)6-NH2), and 0.5 nM (Rlla) were obtained.
Figure 7. Titration of ARC-TAMRA (2 nM and 20 nM) with Rho-associated kinase ROCK jl (fluorescence plate reader PHERAstar, BMG LABTECH). Kd value of 4 nM was calculated for ARC-TAMRA - ROCK II complex according to the relationship (A) (Figure 1).
Figure 8. Dependence of anisotropy on the concentration of inhibitors H89, Y-27632, and ARC [AdcAhx(L-Arg)6-NH2]. Assay conditions; 2 nM of ARC-TAMRA and 15 nM ROCK II, fluorescence plate reader PHERAstar (BMG LABTECH). The

10 Kd value of 4.0 nM was used for ARC-TAMRA. Ki values of 45 nM (H89), 40 nM (Y-27632) and 5 nM (AdcAhx(L-Arg)6-NH2) were obtained.
DETAILED DESCRIPTION OF INVENTION
This invention relates to fluorescent probes for the identification of compounds binding to protein kinases, for measurement of the affinity of inhibitors towards protein kinases and determination of the active concentration of protein kinases binding to the probe. Bisubstrate-analog character of the probes enables the evaluation of inhibitors targeted to both ATP binding site and protein/peptide substrate binding domain of the kinase. The fluorescent probe of this invention has very high affinity towards the kinase (Kd=1.0 nM) which enables the application of low concentrations of kinases in assays and determination of binding constants for inhibitors with high (low nanomolar) affinity. Displacement of the fluorescent probe by an inhibitor can be used for the determination of the values of dissociation constants K Fluorescent probe
The first embodiment of the invention is a fluorescent probe with the general formula (I);
(X-Y-Z)-L-F (I),
wherein X-Y-Z is a bisubstrate-analog inhibitor of a protein kinase, in which X is targeted to the ATP-binding pocket of a kinase, Z binds to the protein/peptide-binding domain of the kinase, Y is a tether that connects X and Z and permits simultaneous binding of X and Z to the active site of the kinase; F is a fluorescent dye which optical characteristics are changed in the course of the binding of (X-Y-Z)-L-F to the kinase; L is a linker between the bisubstrate-analog inhibitor XYZ and fluorescent label F.
The presently preferred fluorescent probe for the application in the binding assay has the structure:

11
AdcAhx(D-Arg)6-D-Lys(5-TAMRA)-C(0)NH2, ARC-TAMRA
The probe, ARC-TAMRA, has high affinity (Kd=1.0 nM; Figures 1 and 2, Example 4) towards the catalytic subunit of cAMP-dependent protein kinase (cAPK Ca) and it was displaced from its complex with the kinase by ATP-competitive inhibitor H89, protein substrate competitive inhibitor peptide PKl(5-24), and bisubstrate inhibitor AdcAhx(D-Arg)6-NH2 (Example 5, Figure 3). Thus, the fluorescent probe of the present invention ARC-TAMRA is the first high-affinity probe disclosed that can be used to measure simultaneously the affinity of inhibitors binding to ATP and protein substrate binding sites of the kinase. The bisubstrate (biligand) character of the probe AdcAhx(D-Arg)6-D-Lys(5-TAMRA)-C(0}NH2 is proved with its displacement from the complex with the kinase by both ATP-binding site targeted inhibitor H89 and protein/peptide substrate binding site targeted inhibitor PKI(5-24), and also by very high affinity of the fluorescent probe.
The inhibitory part of the probe AdcAhx(D-Arg)6-NH2 has high inhibitory potency to many protein kinases (Table 3). As such the fluorescent probe has the potential for application in assays concerning many basophilic protein kinases. Selective inhibition of basophilic kinaseses by AdcAhx(D-Arg)6-NH2 (Table 3) is another demonstration of the bisubstrate nature of the inhibitors of the present invention.
5-TAMRA, 5-carboxytetramethylrhodamine, is a good fluorescent label to be used in fluorescence polarization assays, as it contains the dye as a single-position

12 isomer, the probe is exited at long wavelength, it has high brightness and great photostability.
Other fluorescent labels are suitable for the application as the moiety F of the fluorescent probe (X-Y-Z)-L-F. These fluorescent dyes are well known to the people skilled in the art. Principles for the choice of fluorescent labels are disclosed in [Polarization Technical Resource Guide • THIRD EDITION 2004 Invitrogen Corporation , USA www.invitroqen.com/panveral. Many such labels are described in the Handbook: [The Handbook — A Guide to Fluorescent Probes and Labeling Technologies Web Edition of The Handbook, Tenth Edition 2006 Invitrogen Corporation]
Usually fluorescence labels with higher brightness (brightness of a fluorochrome is proportional to the product of its extinction coefficient and its quantum efficiency), longer excitation and emission wavelengths, and greater photostability are preferred, but the choice of the label depends on the analytical instrument used for the assay and special analytical situation. Other fluorescent dyes that may be useful in the present invention include fluorescein and fluorescein derivatives, rhodamine and rhodamine derivatives, and many other fluorescent labels that are produced and sold by different companies. Fluorescent probes of the structure (X-Y-Z)-L-F incorporating as F mineral fluorescent labels, e.g., quantum dots, may be useful for special applications.
The aim of the linker L in the structure of the fluorescent probe (X-Y-Z)-L-F is the positioning of the fluorescent label F in the position where the group F causes minimal hindrance to the binding of the bisubstrate inhibitor X-Y-Z to the kinase. In the preferred fluorescent probe AdcAhx(D-Arg)6-D-Lys(5-TAMRA)-C(0)NH2 D-lysine stands for the linker L but in the case of other probes the linker may be a Ci-C2o alkylene group, a Ci-C20 alkenylene group, a C-i-C20 alkynylene group, wherein one or more of the CH2 groups present in the C-i-C20 alkylene group, C1-C20 alkenylene group, C1-C20 alkynylene group is optionally replaced with -0-, -C(O)-, -C(0)N-, -S-, -S(O)-, -SO2-, -N(R)-; R is H or C^ alkyl. In the case of some probes L may be omitted.

13 In the examples of the present invention the fluorescent dye F is connected via linker L to the moiety Z of bisubstrate inhibitor X-Y-Z, but L may also be attached to any of the X, Y and Z moieties at various locations.
As it will be shown in later parts of the invention other types of bisubstrate inhibitors may form the active ingredient X-Y-Z of the fluorescent probe. An example of another fluorescent probe structure is the compound where Hidaka's ATP competitive PK inhibitor H9 is conjugated with hexa(D-arginine) peptide via flexible hexanoic acid tether. The compound 26 H9-(CH2)5C(0)(D-Arg)6-NH2 with very high inhibitory potency (IC5o=5.3 nM; Ki=1-2 nM; Example 2, Table 2) was connected through D-lysine linker to the fluorescent label 5-TAMRA to obtain high-affinity fluorescent probe of the present invention
H9-Hex{D-Arg)6-D-Lys(5-TAMRA)-C(0)NH2:

As it will be described further in Example 2 H9-Hex(D-Arg)6NH2 (Compound 26) has very high affinity towards the kinase cAPK. Inhibitory selectivities of bisubstrate inhibitors AdcAhx(D-Arg)6-NH2 and H9-Hex(D-Arg)6NH2 towards basophilic kinases are somewhat different (Example 3) and the derived fluorescent probes AdcAhx(D-Arg)6-D-Lys(5-TAMRA)-C(0)NH2 and H9-Hex(D-Arg)6-D-l_ys(5-TAMRA)-C(0)NH2 (synthesis of both probes id described in Example 1) may find application in different assay formats.
Although the preferred method of the invention is to use the fluorescent probe with fluorescence polarization measurement, an environmentally sensitive fluorescent probe is another choice of the present invention: change in fluorescent intensity as the result of the binding of the probe to the kinase would be measured rather than the change in the fluorescence polarization. A preferred fluorescent probe for these measurements is AdcAhx(D-Arg)6-D-Lys(NBD)-NH2.

14
The probe contains NBD (7-Nitrobenz-2-Oxa-1,3-Diazole) dye which fluorescence intensity is strongly dependent on environment. The probe has very high affinity towards cAPK (Kd ca 1 nM). The synthesis of the fluorescent probe AdcAhx(D-Arg)6-D-Lys{NBD)-NH2 is described in Example 1.
People skilled in the art know that there are other possibilities (in addition to fluorescence polarization and fluorescence intensity measurements) to measure the change in fluorescence of a probe as a resuft of its association with a kinase. The methods like fluorescence correlation spectroscopy, fluorescence resonance energy transfer (FRET), fluorescence lifetime, photo-bleaching and photo-activation, flurescence intensity distribution analysis and their combinations can be used for the monitoring the equilibrium of the reaction between a fluorescent probe and a protein. These methods are well characterized in literature [e.g., White et al. Adv. Drug Deliv. Rev. 57 (2005) 17] and may find application in kinase associatiation studies connected to the fluorescent probes disclosed in the present inventon. The probes of the present invention can be used with classical fluoresence techniques and confocal single-molecul techniques in single- and multi-parameter imaging formats [Tinnefeld et al., Angew. Chem. Int. Ed. 44 (2005) 2642].
Authors of the present invention have shown that adenosine - oligoarginine conjugates are cell membrane permeable [Uri et al., Bioorg. Med. Chem. Lett. 12 (2002) 2117], therefore the fluorescent probes of the present invention are applicable in experiments with live cells, tissues and organisms. Preferred embodyments of the present invention, fluorescent probes AdcAhx(D-Arg)6-D-Lys(5-TAMRA)-C(0)NH2, H9-Hex(D-Arg)6-D-Lys(5-TAMRA)-C(0)NH2, AdcAhx(D-Arg)6-D-l_ys(NBD)-NH2 contain D-arginines, differently from previously disclosed

15
bisubstrate inhibitors which incorporate L-isomers of amino acids. As such these probes are persistent towards enzymatic degradation. [Elmquist, Biol. Chem. 384 (2003) 387] and well suited for applications in biological systems. In biological systems fluorescent probes of the present invention could be used together with kinases fused to fluorescent proteins for dual color imaging and protein-inhibitor interaction studies based on measurement of fluorescence resonance energy transfer (FRET).
The fluorescence polarization assay
The preferred assay method for the application of the fluorescent probes of the present invention is fluorescence polarization or fluorescence anisotropy. Both terms are used synonymously to describe molecular, interactions in solution. [Fluorescence Polarization Technical Resource Guide • Analysis of FP Binding Data Invitrogen Corporation • USA • www.invitrogen.com/panvera] This technique measures the change in rotational speed of a ligand during its excited lifetime upon binding to its receptor.
Fluorescence polarization assays are homogenious and as such they do not require separation steps like chromatography, filtration, centrifugation, precipitation or electrophoresis. Due to the ratiometric nature of tha assay fluorescence polarization is well suited to assay miniaturization, and the assays can be performed with equal success in a cuvette of a fluorescence spectrometer and in low \x\ volumes in wells of 1536-well microtiter plates with a stable plate reader with a fluorescence polarization detector. In the embodiments of the present invention fluorescence spectrometer LS 55 (Perkin Elmer) with 0.5 ml quartz cell or fluorescence plate reader PHERAstar (BMG LABTECH) with Corning 384 well Low Volume NBS microplates (30 (J reaction volume) were used for assays.
In the experimental setup used here a fluorescence polarization signal is greatly increased when fluorophore is slowed down drastically in its rotational speed. This occurs upon binding of a small fluorescently labelled probe of the present invention to significantly larger molecule of the target protein kinase. The degree of polarization is determined by measuring the fluorescence intensities of parallel and perpendicular emitted light with respect to the plane of linearly polarized excitation light. The preferred embodiment of the Fluorescent probe of the present invention

16 ARC-TAMRA, AdcAhx(D-Arg)6-D-Lys(5-TAMRA)-C(0)NH2, has MW of 1869 and the MW of the kinase, cAPK Ca, is ca 40000. This leads to big differences in the anisotropy values of the solution of free ARC-TAMRA (Af = 40 mA) and the solution of the complexed with the kinase fluorescent probe ARC TAMRA (Ab = 240 mA).
Due to mathematical simplicity, anisotropy values are preferred because it is
easier to deconvolute anisotropy values into component values than it is with
polarization values.
Fluorescent probe ARC-TAMRA involved in binding events exist in a mixture of
only two states: bound or free. With only two species, the anisotropy additivity
equation has the shape:
A = FfAf + FbAb,
where: Ff + Fb= 1;
A = observed anisotropy value
Ff = fraction of fluorescent ligand that is free
Fb = fraction of fluorescent ligand that is bound
Af = anisotropy of the free fluorescent ligand
Ab = anisotropy of the bound fluorescent ligand
The top and bottom plateaus of the semi-log equilibrium binding isotherm
(anisotropy vs. Log total receptor concentration) define the anisotropy of the free
and bound states, Af and Ab. With the observed anisotropy, A, we can calculate
the fraction of Bound and Free fluorescent ligand for a given anisotropy value.
The change in fluorescence anisotropy was plotted against the kinase
concentration [cAPK Ca]. The data were fitted to the equation:
Lt + Kd + Rt - J(Lt + Kd + Rt)2-4-LfRt
A = Af + (Ab-Af) ^
2-Lt
where, A is measured anisotropy; Af is the anisotropy of free ARC-TAMRA; Ab is the anisotropy of bound ARC-TAMRA; LT is the concentration of ARC-TAMRA added; RT is the total concentration of active kinase (cAPK Ca).

17 Characterization of the fluorescent probe ARC-TAMRA in binding experiments with the catalytic subunit of cAMP-dependent protein kinase is characterized in Example 4 and Figures 1 and 2. Kd of 1.0 nM was determined for the fluorescent probe ARC-TAMRA in these assays.
Competition Experiments
The present invention embodies an assay in which the binding characteristics of the test compounds are determined on the basis of their ability to displace the fluorescent probe from its complex with the kinase in a fluorescence polarization assay. This is described in Example 5 and shown on Figure 3.
By virtue of the bisubstrate (biligand) character of the fluorescent probe of the present invention it is displaced from the complex with the protein kinase with both ATP binding site targeted inhibitors and protein substrate binding site targeted inhibitors. As it has been shown in Figure 3 three different types of inhibitors are able to displace the fluorescent probe ARC-TAMRA from its complex with the kinase cAPK Ca:
a) ATP competitive inhibitor H89;
b) Protein substrate competitive inhibitor PKl(5-24);
c) Bisubstrate-analog inhibitors AdcAhx(L-Arg)6-NH2 and AdcAhx(D-Arg)6-NH2
The obtained result (Example 5) reveals that the fluorescent probe of the present invention can be used for evaluation of compounds binding to different binding pockets of the active site of the kinase. Thus, the displacement method of the present invention catches up with kinetic methods in detecting both types of inhibitors in a single assay. This has not been previously possible with fluorescent probes based on ATP competitive or protein substrate competitive inhibitors.
The high affinity of the fluorescent probe of the present invention (K
18 used for the determination of binding constants for inhibitors with low nanomolar affinity).
Also, the high affinity of the fluorescent probe ARC-TAMRA Taking into the account the selectivity profile of the bisbstrate inhibitors of the present invention AdcAhx(D-Arg)6-NH2 and H9-Hex(D-Arg)6NH2 (Table 2) the corresponding fluorescent probes are applicable to characterization of the binding of inhibitors to many kinases with basophilic substrate profile.
Additionally, the potential targets of the fluorescent probes of the present invention are different mutated forms of basophilic protein kinases, truncated forms of kinases inactive states of protein kinases, and pseudokinases.
Determination of the concentration of the active form of the kinase
Determination of the concentration of the special form of a kinase is of paramount importance for many enzyme applications. In kinetic assays the kinase amount in the assay volume is characterized on the activity basis and the molar concentration of the kinase is often not known. Majority of the methods in use for determination of enzyme concentration (Bradford, Lowry, SDS electrophoresis) give the total concentration of the protein (not the concentration of its active form), are inaccurate and need unacceptably large amounts of the protein for analysis.
The application of the fluorescent probe of the present invention ARC-TAMRA enables a simple procedure for the determination of the concentration of the active (binding to the fluorescent probe) form of the kinase in the sample.
The fraction of the active (binding) form of the kinase in solution, k, was calculated with the application of nonlinear regression analysis to the relationship:

19

where A is the measured anisotropy; Af is the anisotropy of free ARC-TAMRA; Ab is the anisotropy of bound to the kinase form of ARC-TAMRA; LT is the concentration of ARC-TAMRA added, 10 nM; RT is the total concentration of active kinase (cAPK Ca); Kd is the dissociation constant of the reaction between ARC-TAMRA and cAPK Ca(Kd=1.0 nM). The application of the fluorescent probe ARC-TAMRA for the determination of the concentration of cAPK Ca is described in Example 6.
It was established that there was good proportionality between the concentration of the binding protein (as established with the fluorescent probe ARC-TAMRA) and the phosphorylation activity of the solution of catalytic subunit of cAMP-dependent protein kinase. The reduction of kinase activity enzyme samples with time was in accord with the determined amount of kinase associated with ARC-TAMRA.
Optimization of the structure of bisubstrate inhibitor X-Y-Z
To be widely applicable in bioanalytical methods the fluorescent probe should have possibly highest affinity towards its target protein. It has been shown that the higher the affinity of the fluorescent ligand, the wider the range of inhibitor potency that can be resolved [Huang, J. Biomol. Screen. 8 (2003) 34]. The lowest inhibitor Kj value that can be resolved in an FP-based binding assay is approximately equal to the Kd value o1 the fluorescent probe. Starting from the compound AdcAhx(L-Arg)6 with IC50 of 400 nM [Vint et al., Anal. Biochem. 340 (2005) 165; Loog et al., Bioorg. Med. Chem. Lett. 9 (1999) 1447] modifications in the structure of different moieties of the conjugates were performed. Flexible and productive solid phase synthetic methods were developed for the preparation of conjugates of oligoarginine peptides with adenine, adenosine, adenosine-5'-carboxylic acid (Adc), and 5-isoquinolinesulfonic acid for structure-activity studies (Schemes 1-4). Different combinations of fragments were incorporated into conjugates and the novel compounds were tested as inhibitors of cAPK (cAMP-dependent prtotein kinase).

20 New synthetic methods: synthesis of fluorescent probes and bisubstrate inhibitors for SAR studies
The synthesis of the first class of compounds containing Adc (2-8, Scheme 1) was performed with the application of Rink amide resin instead of Wang resin. The structures of tether and peptide components were varied. It was decided to preserve the optimal towards cAPK length of the linker corresponding to that of 6-aminohexanoic acid (equivalent to seven chemical bonds) between the nucleoside and peptide moieties. The optimal length of the tether may be different for other basophilic kinases. Additionally, the tether may contain different heteroatoms and the simple hydrocarbon chain may be substituted with more complicate straight or branched linking units.
The diversity of the structure of the peptide part of the conjugates was achieved by the variation of length and chirality of oligoarginine chain, as represented by peptide moieties of (L-Arg)4-NH2 (2), (L-Arg)6-NH2 (3), (D-Arg)4-NH2 (4) and (D-Arg)6-NH2 (5).
The second group of compounds contained adenosine and 5'-amino-5'-deoxyadenosine derivatives (Scheme 2). The reaction of 4-nitrophenyl chloroformate with 2',3'-isopropylidene adenosine resulted in the activated carbonate, which gave adenosine-5'-urethanes in the reaction with amines. The length of the linker between nucleoside and peptide moieties was varied. Peptides containing a free amine-group near the C-terminus (e.g., the side chain of lysine or a diamine linker) were prepared on solid phase, cleaved and purified by HPLC.
A special class of biligand inhibitors deprived of ribose moiety, conjugates of adenine and oligoarginine, was synthesized by connecting adenine at C8 or N9 position to a peptide via a linker chain. Precursors of these molecules containing a linker with a free carboxylate group were synthesized in solution. Coupling of the precursors with the resin-bound peptides resulted in conjugates with peptides after cleavage and deprotection with TFA (Scheme 3).
Conjugates of isoquinolinesulfonamide and oligoarginine were synthesized to compare their activity and selectivity to those of analogous adenosine-containing

21
compounds. If compared to the previously synthesised isoquinolinesulfonyl peptides [Ricouart et al., J. Med. Chem. 34 (1991) 73], several changes were introduced into the structures and the synthesis was carried out on solid-phase with the application of Fmoc-peptide and peptoid chemistry procedures. The use of Rink amide resin excluded the presence of negatively charged C-terminal carboxylate group and resulted in products in the form of their C-terminal amides. The conventional Fmoc-peptide chemistry allowed the circumvention of the harsh HF treatment in the final step of Boc-chemistry procedures. The (p-Ala)-Ser linker of previous conjugates was replaced with 6-aminohexanoic acid, which eliminated potentially phosphorylatable serine residue, removed unnecessary chiral centre and simplified the overall synthetic procedure. Resin-bound oligoarginine peptides were acylated at N-terminus with 6-bromohexanoic acid and the following reaction with 5-isoquinolinesulfonyl ethylenediamine and cleavage with TFA led to the 5-isoquinolinesulfonyl ethylenediamine containing conjugate (Scheme 4).
A special embodiment of the present patent application is the group of bisubstrate-analog inhibitors incorporating D-arginine-rich peptides. The high affinity of that kind of conjugates towards cAPK (Table 2) and many other basophilic protein kinases (Table 3) was not known previously. This result is surprising by virtue of the fact that cAPK has a strong requirement for L-configuration both at the phosphorylatable serine residue as well as at the N-terminal arginine residues of the substrate. [Eller, Biochem. Int. 25 (1991) 453] High binding energy may result from beneficial positioning of structurally flexible interacting partners, bisubstrate inhibitor and the kinase, in their complex. Introduction of arginine residues into ARC in the form of their D-configuration converts the peptide motif into retro-inverso counterpart of the C-terminally appended oligo(L-arginine) peptide. Retro-inverso peptide can be regarded as a derivative of a normal peptide in which the relative amino acid side chain topology is maintained, while the backbone termini and direction of the peptide bonds are reversed.This may lead to substantial synergism of the binding energy of the bisubstrate inhibitor if compared to the sum of the binding energies of its constituents, single-site directed inhibitors. Further specification of the interaction mechanism will be possible after solving the crystal structures of the complexes of these inhibitors with protein kinases.

22
Using the methods described above, one skilled in the art could readily prepare various bisubstrate analog inhibitors for structure-activity studies. Other building blocks (e.g., purines, pyrimidines, purine and pyrimidine nucleosides, bisindolylmaleimides, isoquinolines, staurosporines, balanol, etc.) with affinity towards the kinase under investigation can be introduced into the X moiety of the conjugates X-Y-Z and the new conjugates tested for activity. Likewise, the structure of the peptide part Z and the structure of the tether part Y of the bisubstrate inhibitors X-Y-Z could be further optimized to achieve conjugates with the highest affinity towards a special kinase.
The optimal structure of the organic tether (Y) may vary between different protein kinases and is also dependent on the selection of X and Z. Y should be selected among groups providing a chain comprising from 1-50 atoms, for instance 1-30 atoms, such as1-18 atoms. In addition to the chain as such there may be groups projecting from the chain. The chain may comprise one or more groups selected from amides (-CONR1-, where R' is selected from the side groups present in amino acids), amines (-NR"-, where R", for instance, is selected from lower alkyl (C1.5 alkyl)), azo (-N=N-), ether(-O-), thioether (-S-), bivalent hydrocarbon groups etc. The chain may be built up of amino acid residues of the a-,-or p-, y-or s-type or of the L- or the D-type or mixtures of these. The chain may carry one or more positive and/or negative groups, for instance primary, secondary, tertiary and quaternary ammonium groups, respective carboxy and sulfonate groups. In case L is a peptide chain it may contain from 1-15 amino acid residues or even more. One type of linkers comprises a hydrocarbon chain which (a) is straight branched or cyclic and possibly is interrupted at one or more positions by an oxygen or a nitrogen and/or (b) has one or more carbons that are substituted with an NR1R2 or an-OR3 group in which R1.3 is selected amongst CMO alkyl. One kind of linker complying with this is a po!y(ethylene glycol) linker with formula -(CH2CH20)n-, where n may be an integer 1-10. Y may be attached to the X and Z moieties at various locations.
In the case of cAMP-dependent protein kinase the optimization of the length of Y has been performed previously with inhibitors of lower affinity incorporating L-arginine residues. [Loog et al., Bioorg. Med. Chem. Lett., 9 (1999) 1447] Structure-

23
activity studies showed that for the conjugate with X = adenosine-5'-carboxyiic acid and 2 = hexa(L-arginine) the optimal tether Y is 6-aminohexanoic acid [NH2(CH2)nCOOH, n=5). The same length of the tether turned out to be optimal for the high-affinity bisubstrate inhibitors of the present invention (Example 2, Table 2).
The conjugates with the highest affinity can be linked to a fluorescent dye and new fluorescent probes with requested properties will be obtained.
Different synthetic methods that were introduced for the preparation of bisubstrate-analog inhibitors are described in the Schemes 1 - 4.

24
Scheme 1. Synthesis of the bisubstrate-anaiog inhibitors, conjugates of Adc and a peptide

Tether Peptide
2 NH-(CH2)5CO (L-Arg)4-NH2
3 NH-(CH2)5CO (L-Arg)6-NH2
4 NH-(CH2)5CO (D-Arg)4-NH2
5 NH-(CH2)5CO (D-Arg)6-NH2
6 N(CH3)-(CH2)sCO (L-Arg)4-NH2
7 NH-CH2-Ph-4-CO (L-Arg)4-NH2
8 NH-Phe-*e/aAIa-CO (L-Arg)4-NH2

Scheme 2. Synthesis of the bisubstrate-anaiog inhibitors, conjugates of adenosine and a peptide

Scheme 3. Synthesis of the bisubstrate-anaiog inhibitors, conjugates of adenine and a peptide

Scheme 4. Synthesis of the bisubstrate-anaiog inhibitors, conjugates of isoquinolinesulfonamide derivative H9 and a peptide

25
Structure-activity studies with cAMP-deoendent protein kinase (cAPK)
The inhibitory potency of the synthesized conjugates was tested towards cAMP-dependent protein kinase (cAPK), a well characterized representative of basophilic protein kinases. In the case of Adc-peptide conjugates the amidation of peptide C-terminus increased the potency of bisubstrate inhibitors 2-3 fold. This may be caused by the compensation of the positive charge of an arginine residue that participates in a favourable contact with the kinase by the negative charge of the carboxylate group. Substantial modifications in the structure of the tether between adenosine and peptide moieties of conjugates were well tolerated by cAPK (Table 2, compounds 2, 6, 8), while some small structural modifications led to significant decrease of activity.
Variation of the structure of the conjugates also included the connection of adenosine to the C-terminus of the oligoarginine chain and the incorporation of residues of D-arginine.
Inhibitors with peptide moieties connected to the nucleoside part by C- and N-termini had similar affinities in the case of compounds containing peptides with L-amino acid residues, whereas the compound comprising the peptide with D-arginine residues connected via the C-terminus had 500-fold lower activity than the counterpart with N-terminal peptide (5). The bisubstrate character of the inhibitor 5 is supported by the high inhibitory potency of the compound if compared to the submillimolar potencies of the single site-targeted constituents of the conjugate, adenosine and oligo-(D-arginine).
The inhibitory potency of the fluorescent probe ARC-TAMRA, AdcAhx(D-Arg)e-D-Lys(5-TAMRA)-NH2, was also established with the inhibition assay. The obtained IC50 value of inhibition 6.2 nM is even lower than that of the corresponding bisubstrate inhibitor AdcAhx(D-Arg)6NH2 (1C50 = 8.3 nM). The result shows that the addition of the linker (D-lysine) and the fluorescent dye (5-TAMRA) to the special position of the btligand inhibitor does not interfere substantially with its binding to the kinase.

26
Selectivity study
Selectivity testing was performed (Example 3) for two compounds, 5 and 26, that had revealed the highest inhibitory potency towards cAPK Ca. The panel of kinases included all 52 kinases available for testing. To the best of our knowledge, the selectivity profiling has not been carried out with a wide panel of PK before for bisubstrate-type inhibitors. To make the inhibitory potencies comparable, assays were run at the ATP concentrations which were close to the Km value of the kinase. The results are presented {Example 3) at 1 uM concentration of the inhibitor as the percent of residual activity of the kinase relative to that in control incubations where the inhibitor was omitted.
The activity profiles of two compounds were mostly similar. Both conjugates inhibited strongly ROCK-II, MSK1, PKB(3, PKBAph, and PRK2 (residual activity £ 3%). Compound 26 inhibited almost completely MAPKAP-K1a/rsk1, p70S6K, and MAPKAP-K1b, while residual activity in the presence of 5 was 7-28% for these kinases. The biggest difference between the two compounds was observed for the inhibition of MAPKAP-K1a/rsk1 where the corresponding values were 0 and 28% for 26 and 5, respectively.
CAMK-1, PKA, PIM2, and PKD1 retained less than 10% of activity in the presence of 26. The significant difference between the two compounds was also found for PKD1 (the residual activities were 54% for 5 and 9% for 26). Checkpoint kinases (CHK1 and CHK2) were both inhibited more strongly by 5 than by 26. Several kinases were weakly inhibited by biligand inhibitors, while some of them were not inhibited at all, e.g., acidophilic kinases represented by casein kinases CK1 and CK2 retained full activity in the presence of 5 as well as 26. As illustrated by data in Table 2, oligo-(D-arginine) containing bifunctional inhibitors were not specific to any particular protein kinase but slowed down the reactions catalyzed by basophilic kinases, especially of the AGC (cAMP dependent, cGMP dependent, and protein kinase C family) and CAMK (calmodulin-dependent protein kinase) groups. Of the 14 PK inhibited by more than 85% by compound 26 at 1 micromolar concentration, 11 kinases belong to the AGC group and 3 kinases to the CAMK group. All these 14 kinases have been reported to be of basophilic type which

27
points to the biligand character of the inhibitors where the inhibitory potency of the conjugates is influenced by binding to both substrate-binding sites of the kinase. The only AGC group kinase that falls out of this selection is PDK, an AGC kinase which expresses badly defined consensus sequences and lacks strong Arg preference at any position.
On the basis of the analysis of the results of the selectivity study of two inhibitors with 52 kinases, previously known substrate consensus sequences and other structural characteristics we have selected 59 protein kinases from the AGC family that are potential binders to the fluorescent probes disclosed in the present invention (Table 1).
Table 1. 59 kinases from AGC group (classification by [Manning et al., Science
298 (2002) 1912]) which on the basis of the analysis of their substrate consensus
sequences and other characteristics are potential binders to the fluorescent probes
disclosed in the present invention
Number Kinase Ar.r.gRftinn Grouo Familv Suhfamilv

1 AKT1 SK018 AGC AKT
2 AKT2 SK019 AGC AKT
3 AKT3 SK020 AGC AKT
4 DMPK1 SK111 AGC DMPK GEK
5 DMPK2 SK112 AGC DMPK GEK
6 MRCKb SK241 AGC DMPK GEK
7 MRCKa SK299 AGC DMPK GEK
8 MRCKps SK660 AGC DMPK GEK
8 ROCK2 SK263 AGC DMPK ROCK
10 ROCK1 SK331 AGC DMPK ROCK
11 CRIK SK695 AGC DMPK
12 MAST3 SK196 AGC MAST
13 MAST2 SK216 AGC MAST
14 MAST1 SK345 AGC MAST
15 MASTL SK455 AGC MAST
16 MAST4 SK701 AGC MAST
17 NDR1 SK249 AGC NDR

28

18 LATS1 SK441 AGC NDR
19 LATS2 SK442 AGC NDR
20 NDR2 SK500 AGC NDR
21 PKACa SK300 AGC PKA
22 PKACb SK301 AGC PKA
23 PKACg SK302 AGC PKA
24 PRKX SK313 AGC PKA
25 PRKXps SK319 AGC PKA
26 PRKY SK320 AGC PKA
27 PKCa SK303 AGC PKC Alpha
28 PKCb SK304 AGC PKC Alpha
29 PKCg SK307 AGC PKC Alpha
30 PKCd SK305 AGC PKC Delta
31 PKCt SK310 AGC PKC Delta
32 PKCh SK270 AGC PKC Eta
33 PKCe SK306 AGC PKC Eta
34 PKCi SK308 AGC PKC lota
35 PKCz SK311 AGC PKC iota
36 PKCips SK711 AGC PKC iota
37 PKG1 SK073 AGC PKG
38 PKG2 SK075 AGC PKG
39 PKN1 SK317 AGC PKN
40 PKN2 SK318 AGC PKN
41 PKN3 SK511 AGC PKN
42 MSK1 SK242 AGC RSK MSK
43 MSK2 SK243 AGC RSK MSK
44 p70S6K SK265 AGC RSK p70
45 p70S6Kb SK266 AGC RSK p70
46 p70S6Kps2 SK640 AGC RSK p70
47 p70S6Kps1 SK740 AGC RSK p70
48 RSK1 SK336 AGC RSK RSK
49 RSK2 SK337 AGC RSK RSK
50 RSK3 SK338 AGC RSK RSK

51 RSK4 SK518 AGC RSK
52 SgK494 SK491 AGC RSK
53 RSKL2 SK473 AGC RSKL
54 RSKL1 SK517 AGC RSKL
55 SGK SK346 AGC SGK
55 SGK2 SK523 AGC SGK
56 SGK3 SK525 AGC SGK
57 YANK3 SK469 AGC YANK
58 YANK2 SK481 AGC YANK
59 YANK1 SK624 AGC YANK
Profiling with the 52-kinase selectivity panel (Example 3) reveals strong tendency of the arginine-rich conjugates to inhibit specifically basophilic protein kinases that points to the active participation of both functionary moieties in the formation of the binary complex with the kinase. In addition to the majority of AGC kinases, many kinases of CAMK and STE groups and some kinases not falling into major groups (classification by [Manning et al., Science 298 (2002) 1912]) have also been shown to be basophilic protein kinases [Pinna et al., Biochim. Biophys. Acta. 1314 (1996) 191; Zhu et al., J. Biol. Chem. 280 (2005) 36372]. All these kinases could be targets of fluorescent probes of the present invention and the probes can be used in analytical methods concerning these kinases.
Additionally, the targets of the fluorescent probes of the present invention are different mutated forms of basophilic protein kinases, truncated forms of kinases inactive states of protein kinases, and pseudokinases.
EXPERIMENTAL PART
Abbreviations
Adc - 5'-adenosine carboxylic acid or 1-(6-amino-9H-purin-9-yl)-1-deoxy-n-D-
ribofuranuronic acid (CAS 3415-09-6);
Ahx - 6-aminohexanoic acid;
Arg - arginine;
ARC - adenosine-arginine conjugate;

30 ATP - adenosine-5'-triphosphate; Boc - tert-butoxycarbonyl; cAMP - cyclic adenosine 3',5'-monophosphate; cAPK - cAMP-dependent protein kinase; DIC- 1,3-diisopropylcarbodiimide; DIPEA - N.N-diisopropylethylamine; DMF - dimethylformamide; DMSO - dimethyl sulfoxide; DTT-dithiothreitol Fmoc - 9-fluorenylmethoxycarbonyl; HOBt - 1-hydroxybenzotriazole; HPLC - high performance liquid chromatography; Ip: 2\3'-0-isopropylidene;
MALDITOF MS, matrix assisted laser desorption ionisation time-of-flight mass spectrometry;
NMR - nuclear magnetic resonance; Pbf - 2,2,4l6,7-pentamethyldihydrobenzofuran-5-sulfonyl;
PK-protein kinase;
PKA - cAMP-dependent protein kinase;
RP - reverse phase;
TFA - triffuoroacetic acid;
TLC - thin layer chromatography.
EXAMPLES
EXAMPLE 1. SYNTHETIC PROCEDURES
Solid phase synthesis procedures
Peptide fragments were prepared by using traditional Fmoc solid-phase peptide synthesis methods on Rink amide MBHA resin (Schemes 1 - 4). Protected amino acids (3 equivalents) were dissolved in DMF and activated with BOP/HOBt (2.94 eq each) in DMF//V-methylmorpholine. Coupling solutions were added to the resin and shaked for 40-60 min. The completeness of each step was monitored with Kaiser-test, which was followed by deprotection of Fmoc-group by 20% piperidine

31
solution in DMF (20 min). Fmoc-protected linker, 6-Fmoc-aminohexanoic acid, was attached to the peptide part following the same protocol. The N-terminal Fmoc group was removed with 20% piperidine solution in DMF (20 min). Adenosine-5'-carboxylic acid was attached to the resin-bound peptide in the form of its isopropylidene protected derivative (3 equivalents) in DMF and activated with BOP/HOBt (2.94 eq each) in DMF/diisopropylethylamine. The protection groups were removed and the conjugate cleaved from the resin with 2 h treatment with 90% trifluoroacetic acid (5% triisopropylsilane, 5% water). The conjugate was purified with C18 reversed phase HPLC and lyophilized.
Synthesis of the fluorescent probe AdcAhx(D-Arg)R-D-Lvs(5-TAMRA)-NH? (ARC-TAMRA)
AdcAhx(D-Arg)6-D-Lys-C(0)NH2 in the form of its TFA salt (2.5 mg.1 jimo!) was dissolved in DMSO. 5-TAMRA, SE (5-Carboxytetramethylrhodamine, succinimidyl ester) (Anaspec; 0.53 mg, 1 (imol) in DMF (50 ^l) and DIEA (10 nl) were added. After 3 h reaction the solvents were removed in the freeze dryer and the product, AdcAhx(D-Arg)6-D-Lys(5-TAMRA)-C(0)NH2, purified by HPLC with C18 reversed phase column. MW (MALDI TOF, M+H+) = 1870; ^(absorption) = 559 nm; Xmaxtemission) = 582 nm.
Adc-Ahx-(D-Arg6)-[D-Lys(5-TAMRA)]-NH2 has excitation and emission maxima at 559 nm and 582 nm (50 mM HEPES, 200 mM NaCI, 0,5 g/l BSA, 5 mM DTT, 0.05% TWEEN 20, MilliQ water; pH 7.4), respectively. The spectra were recorded on Perkin Elmer fluorescence spectrometer LS 55. Synthesis of the fluorescent probe AdcAhx(D-Arg)g-D-Lys(NBDVNH?
-NHKD-Arg)6-(D-Irys)-C(=0)-NH2
o
AdcAhx(D-Arg)6-D-Lys-C(0)NH2 in the form of its TFA salt (2.5 rng,1 mmol) was dissolved in DMSO (100 y\). 4-Fluoro-7-nitrobenzofurazan (NBD-F) (Anaspec; 0.2

32
mg, 1.0 nmo!) in DMF (100 \x\) was added. After 3h reaction at room temperature the solution was put into the freeze-dryer and the solvents were removed. HPLC purification on a C18 reversed phase column with water-ACN gradient gave the product in the form of dark powder. MW (MALDI TOF, M+H+) = 1751. X(max) at 480 nm in water, X(max) at 466 nm in methanol.
Synthesis of the fluorescent probe H9-Hex-(D-Argk-D-Lvs(5-TAMRA)-C(Q)NH?

Bromohexanoic acid (10 eq) was activated with DIC (5 eq) at 0 °C in DMF and added to the resin [Arg(Pbf)]6Lys(Boc)-[MBHA amide resin]. After 45 min agitation the resin was washed. The resulting alkyl bromide was reacted with 5-isoquinolinesulfonyl ethylenediamine (H9) in DMSO for 12 h at 50°C. Secondary amino group of the resin-bound conjugate was protected with Fmoc-CI (1h, room temp; 3 eq., 10eq. DIEA, DMF). The product was cleaved from the resin and isopropylidene and Boc groups were removed by 2 h treatment with 90% TFA (5% water, 5% diisopropylsilane) to produce
IQS-NH-(CH2)2-N(Fmoc)-(CH2)5C(=0)-(D-Arg)6-D-Lys-NH2. After cleavage from the resin (90% TFA, 5% water, 5% TIS) and purification by reversed phase HPLC (water-ACN gradient, 0.1% TFA) the product was reacted with 5-TAMRA, SE (1 eq.) for 3h in the mixture of DMF and DMSO (1:1) in the presence of DIEA (5 eq.). After cleavage from the resin (90% TFA, 5% water, 5% TIS) and purification by reversed phase HPLC (water-ACN gradient, 0.1% TFA), the modified peptide H9-Hex-(D-Arg)6-(D-Lys)-NH2was reacted with 5-TAMRA, SE in DMSO (100 nl) in the presence of DIEA (10 \i\) for 3 h at roomt0. H9-Hex(D-Arg)6-D-Lys(5-TAMRA)-C(0)NH2 was purified with HPLC on a C18
reversed phase column. Dark red powder was obtained after freeze drying;
^max(absorption) = 559 nm; a.max(emission) = 582 nm.

33
EXAMPLE 2.

Optimization of the structure of the bisubstrate-analoq inhibitor: determination of inhibition characteristics ICgn towards cAPK Ca
0
The IC5o values of the inhibitors were measured as previously described. [Viht et al., Anal. Biochem. 340 (2005) 165] The inhibitors in various concentrations were incubated at 30°C in Hepes buffer (50 mM, pH=7.5) containing cAPK Ca (Biaffin AG; about 1 nM), TAMRA-kemptide (10, 30 or 100 uM), ATP (100 uM or 1 mM), magnesium acetate (10 mM) and bovine serum albumin (nhibitr0.2 mg/mL). ATP was added last to initiate the phosphorylation reaction. At fixed time points, the reaction was stopped by 20-fold dilution with 75 mM phosphoric acid and obtained solutions were analyzed by normal phase TLC (without fluorescence indicator, eluted with 1-butanol/ pyridine/ acetic acid/ water, 15/10 /12 /12 by volume). The visualization and quantification of the fluorescent spots were carried out by fluorescence imaging. Data were processed with Graphpad Prism software (version 4, GraphPad).
Table 2. Values of IC50 for the conjugates of oligoarginines and adenosine-5'-carboxylic acid (structures 2-8 from Scheme 1), conjugates of oligoarginines and isoquinoline sulfonamides (25 and 26), fluorescent probe ARC-TAMRA, and some single site-targeted inhibitors of the phosphorylation reaction catalyzed by cAPK Ca

Compound Structure IC50 (MM)
A AdcAhx(L-Arg)6OH Viht et al., Anal. Biochem. 340 (2005) 165 0.40 ±0.12
2 2.0 ±0.3

34

35

IC50-s are measured in the presence of 100 uM ATP and 30 uM TAMRA-kemptide. EXAMPLE 3.

Ado 350 ± 40
H89 0.10 + 0.02 (0.85 ±0.09)
H9 3.7 ±0.3
ARC-TAMRA AdcAhx(D-Arg)6-D-Lys Selectivity of bisubstrate-analog inhibitors of PK
To establish the selectivity of two most potent inhibitors of cAPK (5 and 26, Table 2) towards other kinases their inhibitory effect was determined in a panel of 52 kinases. The assay of protein kinases was performed at 30°C as described previously [Davies et al., Biochem. J. 351 (2000) 95; Murray et al., Biochem. J. 384 (2004) 477]. The results of the testing are expressed as the % of residual activity of the kinase in the presence of the inhibitor at 1 micromolar concentration.
Table .3. Residual activities of protein kinases in the presence of biligand inhibitors AdcAhx(D-Arg)e-NH2t 5, and H9-Hex(D-Arg)6-NH2, 26

protein kinase kinase group ATP (pM) residual activity3 (%)

compound 5 compound 26
ROCK-II AGC 20 0(±0) 0(±0)
MAPKAP-K1a/rsk-1 AGC 50 28 (± 7) 0(±1)
SGK AGC 20 16 (±0) 1(±0)
MSK1 AGC 20 2(±0) 1(±1)

36

PKBAph AGC 5 2(±0) 2(±0)
P70 S6K AGC 20 7 (±2) 2(±0)
MAPKAP-K1b/rsk-2 AGC 50 12 (±0) 2(±0)
PRK2 AGC 5 2(±1) 2(±0)
PKBb AGC 50 3(±0) 3(±0)
CAMK-1 CAMK 50 15 (±2) 6(±0)
PKA AGC 20 11 (±7) 6 (±2)
PIM2 CAMK 5 38 (± 3) 9(±0)
PKD1 CAMK 50 54 (± 8) 9(±1)
PKCa AGC 20 31 (± 7) 15 (±5)
CHK2 CAMK 20 14 (±2) 22 (± 5)
MST2 STE 20 77 (± 3) 24 (± 0)
CHK1 CAMK 20 16 (±4) 31 (± 1)
MNK2 CAMK 50 99 (± 9) 31 (± 3)
AMPK CAMK 50 48 (± 6) 39 (± 4)
ERK8 CMGC 5 50 (± 9) 52 (± 0)
Aurora B other 20 103 (±5) 53 (±1)
MARIO CAMK 5 40 (± 3) 53 (+ 4)
PDK1 AGC 20 62 (± 4) 56 (±1)
NEK7 other 20 58 (±1) 56 (± 9)
PBK TK 50 88 (± 5) 67 (± 4)
MNK1 CAMK 50 102 (±1) 68 (± 9)
DYRKIa CMGC 50 72 (± 6) 69 (±1)
MAPKAP-K3 CAMK 20 67 (± 0) 69 (± 8)
CSK TK 20 48 (± 9) 70 (±1)
CDK2/cyclin A CMGC 20 88 (± 7) 73 (± 3)
JNK/SAPK1C CMGC 20 70 (± 7) 75 (+ 4)
PLK1 other 5 81 (± 10) 76 (± 2)

37

SAPK2a/p38 CMGC 50 79 (± 2) 76 (± 7)
SAPK2b/p38B2 CMGC 20 85 (± 0) 80 (± 2)
MAPKAP-K2 CAMK 20 85 (± 6) 80 (± 2)
MKK1 STE 5 77 (± 9) 84 (± 6)
IKKb other 5 94 (±11) 87 (±1)
CK2 other 5 104 (±9) 87 (± 7)
Src TK 50 84 (±9) 88 (± 4)
Lck TK 50 70 (± 3) 88 (± 3)
smMLCK CAMK 50 72 (± 0) 90 (± 5)
eEF2K atypical 5 92 (± 9) 91 (± 8)
JNK3 CMGC 50 91 (± 6) 92 (± 9)
SAPK4/p38d CMGC 5 107 (±1) 93 (±1)
GSK3b CMGC 5 56 (± 4) 94 (± 9)
MAPK2/ERK2 CMGC 50 100 (±3) 95 (± 7)
CK1 CK1 20 106 (±8) 97 (± 4)
PRAK CAMK 20 89 (±4) 97 (± 8)
NEK2a other 50 82 (± 2) 98 (± 6)
SAPK3/p38g CMGC 5 111 (±4) 99 (±1)
SRPK1 CMGC 50 100 (±3) 99 (± 2)
NEK6 other 50 94 (± 8) 100 (±8)
EXAMPLE 4.
Characterization of the complex between the fluorescent probe ARC-TAMRA and the kinase cAPK Ca.
ARC-TAMRA, Adc-Ahx-(D-Arg6)-[D-Lys(5-TAMRA)]-NH2, has excitation and emission maxima at 559 nm and 582 nm (50 mM HEPES, 200 mM NaCI, 0,5 g/l BSA, 5 mM DTT, 0.05% TWEEN 20, MilliQ water; pH 7.4), respectively. The spectra were recorded on Perkin Elmer fluorescence spectrometer LS 55.

38 The binding of the fluorescent probe ARC-TAMRA to the kinase cAPK Cot (Biaffin) was studied at 10 nM or 2nM ARC-TAMRA concentration (excitation at 557 nm, slitwidth 10 nm; emission at 585 nm, slitwidth 10 nm, detection of fluorescence polarization).
10 nM solution of ARC-TAMRA in 500 |il of buffer (50 mM HEPES, 200 mM NaCI, 0.5 g/l BSA, 5 mM DTT, 0.05% TWEEN 20, MilliQ water; pH 7.4) was titrated with the solution of kinase cAPK Ca (Biaffin). 1-3 ul quantities of various dilutions of kinase solution were added to 500 pi of 2.0 nM or 10.0 nM solution of ARC-TAMRA, and fluorescence anisotropy was measured after each addition of the kinase solution after 10 min delay time. The change in fluorescence anisotropy was plotted against the kinase concentration [cAPK Ca]. The data were fitted to the equation:
Lt + Kd + Rt-A/(Lt + Kd + Rt)2-4-Lt-Rt
A = Af + (Ab-Af) —
2-Lt
where, A is measured anisotropy; Af is the anisotropy of free ARC-TAMRA; Ab is
the anisotropy of bound ARC-TAMRA; LT is the concentration of ARC-TAMRA
added, 10 nM; RT is the total concentration of active kinase (cAPK Ca).
K Kd value of 1.1 nM was calculated with 2 nM of ARC-TAMRA (FIG 2).
EXAMPLE 5.
Competition experiments for determination of the binding constants of inhibitors
A complex was created between 10 nM of ARC-TAMRA and 10 nM of cAPK Ca in 0.500 ml of buffer (50 mM HEPES, 200 mM NaCI, 0.5 g/l BSA, 5 mM DTT, 0.05% TWEEN 20, MilliQ water; pH 7.4). Different compounds were screened for binding to cAPK Ca against fixed concentrations of ARC-TAMRA and cAPK Ca (FIG 4). 1-3 pi quantities of various dilutions of the solution of the inhibitor were added to 500 pi solution of ARC-TAMRA complex with the kinase and fluorescence anisotropy was measured after each addition of the kinase solution after 10 min delay time.

39 The following values of IC5o and Kj were obtained for competitive binding of inhibitors to the ARC-TAMRA - kinase complex:

Inhibitor ic50 Ki
L-ARC 1,3 nM 110 nM
D-ARC 35 nM 2,5 nM
H89 180 nM 15 nM
PKI (5-24) 140 uM 12 nM
Abbrevations for the compounds: N-(2-[p-bromocinnarnylamino-]ethyl)-5-isoquinoline-sulfonamide (H89); AdcAhx(L-Arg)6-NH2 (L-ARC); AdcAhx(D-Arg)6-NH2 (D-ARC); PKI peptide (5-24).
Binding affinity constants (Kj values) of inhibitors were calculated [Nikolovska-Coleska et al., Anal. Biochem. 332 (2004) 261] according to the relationship

where [l]50 is the concentration of the free inhibitor at 50% inhibition, [L]50 is the concentration of the free labeled ligand at 50% inhibition, [P]0 is the concentration of the free protein at 0% inhibition, and K Very similar Ki values were obtained for the same inhibitors in the conditions were the complex was created at lower concentrations of the interacting partners, between 2 nM ARC-TAMRA and 5 nM cAPK Ca.
EXAMPLE 6.
Determination of the concentration of the active form of the kinase
ARC-TAMRA, Adc-Ahx-(D-Arg6)-[D-Lys(5-TAMRA)]-NH2, was used as the fluorescence probe: excitation at 558 nm (bandwidth 10 nm) and emission at 585 nm (bandwidth 10 nm): 50 mM HEPES, 200 mM NaCI, 0.5 g/l BSA, 5 mM DTT, 0.05% TWEEN 20, MilliQ water; pH 7.4. The spectra were recorded on Perkin Elmer fluorescence spectrometer LS 55.
10 nM solution of ARC-TAMRA in 500 nl of buffer (50 mM HEPES, 200 mM NaCI, 0,5 g/l BSA, 5 mM DTT, 0.05% TWEEN 20, MilliQ water; pH 7.4) was titrated with

40 the solution of kinase cAPK Ca(Biaffin). 1-3 ul quantities of various dilutions of kinase solution were added to 500 ul of 2.0 nM or 10.0 nM solution of ARC-TAMRA, and fluorescence anisotropy was measured after each addition of the kinase solution after 10 min delay time. The change in fluorescence anisotropy was plotted (FIG 3) against the kinase concentration [cAPK Ca]. The fraction of the active (binding) form of the kinase in solution, k, was calculated by the application of nonlinear regression analysis to the relationship [Data were processed with Graphpad Prism software (version 4, GraphPad)]:
Lt + Kd + k-Rt-V(Lt + Kd + k-Rt)2-4-Lt-k-Rt
A = Af + (Ab-Af) -
2-Lt
where A is the measured anisotropy; Af is the anisotropy of free ARC-TAMRA; Ab is the anisotropy of bound ARC-TAMRA; LT is the concentration of ARC-TAMRA added, 10 nM; RT is the total concentration of active kinase (cAPK Ca); Kd is the dissociation constant of the reaction between ARC-TAMRA and cAPK Ca (Kd=1.0 nM).
The obtained k value (Figure 3) of 0.317 shows that 317 % of the nominal kinase is in the active (binding to the probe) form in this sample of kinase.
EXAMPLE 7.
Hiqh-throuahput binding assay for inhibitors of the kinase cAPK Ca
The high sensitivity of fluorescence platereader PHERAstar (BMG LABTECH) enabled the realization of displacement experiments at 1 nM concentration of the fluorescent probe ARC-TAMRA, Adc-Ahx-(D-Arg6)-[D-Lys(5-TAMRA)]-NH2. Corning 384 well Low Volume NBS microplates (20 p} or 30 JLLI reaction volumes) were used for assays. Due to the polar and charged character of the fluorescent probe the selection of the well material of microplate is of great importance and specially designed coatings for fluorescence measurements with polar peptides and proteins are better suited than ordinary glass or polystyrene based well materials. The application of latter materials may lead to adsorbation of the probes

41 of the present inventions to the surface of the well of the microliter plate and misrepresentation of results of the measurements.
Characterization of the fluorescent probe
Titrations of 2 nM and 20 nM ARC-TAMRA solutions were performed with cAPK CD. 2-fold serial dilutions of the kinase were used leading to concentrations of the kinase between 100 nM and 0.05 nM and 20 jiL reaction volumes on a 384-well microliter plate. Before the measurement of fluorescence anisotropies the plate was rotated (400 rpm) for 10 minutes at 30°C. Optical conditions for the measurements were set as the following: 540 nm excitation and 590 nm emission (20 nm bandpass filters). To find the value of Kd, titration curves were analyzed by a scientific curve-fitting program, e.g., GraphPad Prism 4.03, with nonlinear regression, using the function below:
, K {k K Lt + Kd + k.Rt-J(Lt + Kd + k.Rt)2-4-Lt-k-Rt
A = Af + (Ab - Af) ^ ,
2-Lt
where A is measured anisotropy, Af - anisotropy value for free ARC-Photo, Ab -
anisotropy value for bound ARC-Photo, Lt - total concentration of ARC-Photo, Rt -
nominal total concentration of enzyme, k - concentration of active enzyme divided
by nominal concentration of enzyme, Kd - dissociation constant for ARC-Photo
complex with enzyme. Kd value of 0.4 was determined for the complex (FIG 5).
Determination of the inhibition constants (Ki)
To determine the inhibition constant (KJ, 3-fold serial dilutions of a given inhibitor are performed analogously as for titration experiments. Before the measurement of fluorescence anisotropies the plate is rotated (400 rpm) for 10 minutes at 30°C. Kj values of 13 nM for ATP-competitive inhibitor H89, 0.5 nM for substrate protein-competitive inhibitor Rlla (regulatory subunit of cAPK), 21 nM for bisubstrate inhibitor AdcAhx(L-Arg)6-NH2 and 1.1 nM for bisubstrate inhibitor AdcAhx(D-Arg)6-NH2were determined (FIG 6).
The obtained Kj values for competitive binding are in good accord with the inhibitory constants for the same compounds of the phosphorylation reaction catalyzed by cAPK Cot (Example 2) and inhibitory constants determined with the

42
same fluorescent probe with a fluorescence spectrometer In a 0.5 ml cell (Example 5).
EXAMPLE 8.
Characterization of the complex between the fluorescent probe ARC-TAMRA and the Rho-associated kinase ROCK II and determination of the inhibition constants for inhibitors
Experiments were performed in the wells of 384-well microtiter plates and with the measurements of fluorescence anisotropy with the platereader PHERAStar according to the protocols described in the Example 7. The titration of the fluorescent probe ARC-TAMRA with the kinase ROCK II enabled the determination of dissociation constant Kd of the complex between ARC-TAMRA and the kinase as of 4 nM (FIG 7).
The displacement of the fluorescent probe ARC-TAMRA from the complex with the kinase (formed between 2nM of ARC-Photo and 15 nM of ROCK II) by different inhibitors of ROCK enabled the characterization of the inhibitors with Ki values of 45 nM, 40 nM, and 5 nM for H89, Y-27632, and L-ARC, respectively (FIG 8). Similar results were obtained with the application of the other Rho-associated kinase ROCK I, having high structural similarity to ROCK II.

43
WE CLAIM
1. A fluorescent probe with the general formula (I):
(X-Y-Z)-L-F (1),
wherein
- X-Y-Z is a bisubstrate-analog inhibitor of a protein kinase, in which X is a compound that binds to the ATP-binding pocket of the kinase and;
- Z is a compound that binds to the protein/peptide-binding domain of the kinase;
- Y is a an organic tether that connects X and Z and permits simultaneous binding of X and Z to the active site of the kinase;
- F is a fluorescent dye which optical characteristics are changed in the course of the binding of (X-Y-Z)-L-F to the kinase;
- L is a linker (between the bisubstrate-analog inhibitor XYZ and fluorescent label F) formed of a hydrocarbon chain, of other type of organic molecule or of part of organic molecule. L may be omitted in the probe and F directly bound to inhibitor XYZ.

2. The fluorescent probe of claim 1, wherein the structure of XYZ comprises ATP or ATP mimics and a peptide or a peptide mimics.
3. The fluorescent probe of claim 1, wherein X is adenosine or adenosine-based compound.
4. The fluorescent probe of claim 1, wherein X is purine or purine-based compound.
5. The fluorescent probe of claim 1, wherein X is isoquinoline or isoquinoline-based compound.
6. The fluorescent probe of claim 1, wherein Z is a peptide or a peptide mimics.

44
12. The fluorescent probe of claim 1, wherein the structure of the probe is
13. The fluorescent probe of claim 1, wherein the structure of the probe is
7. The fluorescent probe of claim 1, wherein Z is a arginine-rich peptide or a arginine-rich peptide mimics containing guanidino groups, e.g., peptoid.
8. The fluorescent probe of claim 1, wherein Z is a peptide containing D-arginine residues.
9. The fluorescent probe of claim 1, wherein Z is a peptide containing 6 D-arginine residues.
10. The fluorescent probe of claim 1, wherein Y is a hydrocarbon chain of 1-20 carbon atoms, or a alkylene chain where any of C atoms is replaced with one or more of amide groups or ester groups or O, N, P and S atoms. Y may be a straight or branched chain molecule consisting of peptides or peptoids or their mimics and of their combinations.
11. The fluorescent probe of claim 1, wherein the structure of the probe is

14. An assay for the identification of a kinase inhibitor by displacement of the
fluorescent probe of any of claims 1-13 from its complex with the kinase by an
inhibitor compound comprising the steps of:
1) establishing the Kd for the complex of the fluorescent probe with the kinase;
2) contacting the fluorescent probe with the kinase and measuring the fluorescence signal of the formed complex;
3) incubation of the complex formed in the previous step with the potential inhibitory compound or a mixture of compounds and measuring of the fluorescence signal of the probe;
4) comparing the fluorescence signals from step 2) and step 3): difference in these fluorescence signals indicates the presence of a active compound in the probe of the step 3).
15. A method for the determination of binding characteristics of protein kinase
inhibitors by displacement of the fluorescent probe of any of claims 1-13 from its
complex with the kinase by an inhibitor compound comprising the steps of:
1) establishing the Kd for the complex of the fluorescent probe with the kinase;
2) contacting the fluorescent probe with the kinase and measuring the fluorescence signal of the formed complex;
3) incubating of the complex formed in the previous step with a series of different concentrations of the inhibitory compound and measuring of the fluorescence signal at each concentration of the inhibitor;

46 4) calculating of the Kd of the inhibitory compound for its binding to the active site of the kinase proceeding from the Kd for the complex of the fluorescent probe with the kinase and the results of the measurements of the step 3).
16. Method according to claim 15 wherein the probe is the fluorescent probe of any of claims 1-13 where the signal is measured as a shift in fluorescence polarization of the probe.
17. Method of claim 15 wherein the screening is performed on a multi-well plate.
18. Method for the determination of binding characteristics for compounds targeting the ATP-binding pocket of the kinase by displacement of the fluorescent probe of any of claims 1-13 from its complex with the kinase by an inhibitor compound comprising the steps of:

1) establishing the Kd for the complex of the fluorescent probe with the kinase;
2) contacting the fluorescent probe with the kinase and measuring the fluorescence signal of the formed complex;
3) incubating of the complex formed in the previous step with a series of different concentrations of the inhibitory compound and measuring of the fluorescence signal at each concentration of the inhibitor;
4) calculating of the Kd of the inhibitory compound for its binding to the ATP binding site of the kinase proceeding from the Kd for the complex of the fluorescent probe with the kinase and the results of the measurements of the step 3.
19. Method for the determination of binding characteristics for compounds
targeting the protein/peptide substrate binding domain of the kinase by
displacement of the fluorescent probe of any of claims 1-13 from its complex with
the kinase by an inhibitor compound comprising the steps of:
1) establishing the Kd for the complex of the fluorescent probe with the kinase;

47
2) contacting the fluorescent probe with the kinase and measuring the fluorescence signal of the formed complex;
3) incubating of the complex formed in the previous step with a series of different concentrations of the inhibitory compound and measuring of the fluorescence signal at each concentration of the inhibitor;
4) calculating of the Kd of the inhibitory compound for its binding to the substrate protein/peptie binding domain of the kinase proceeding from the Kd for the complex of the fluorescent probe with the kinase and the results of the measurements of the step 3.

20. Method according to claim 15 wherein the protein kinase is a basophilic protein kinase.
21. Method according to claim 15 wherein the kinase is cAMP-dependent protein kinase.
22. Method according to claim 15 wherein the kinase is Rho-associated kinase (ROCK).
23. A method for quantifying the kinase in a sample characterized by bringing the fluorescent probe according to any of claims 1-13 into contact with the sample and measuring of the fluorescence anisotropy from the probe:

1) establishing the Kd for the complex of the fluorescent probe according to the claim 1 with the kinase;
2) contacting the fluorescent probe with a series of samples of the kinase at different dilutions and measuring the fluorescence signal of the formed complex of the diluted samples;
3) calculating the fraction of the active (binding) form of the kinase in solution, k, according to the relationship

48
where A is the measured anisotropy; Af is the anisotropy of the free fluorescent probe; Abis the anisotropy of the bound fluorescent probe; LT is the concentration of the fluorescent probe added; RT is the total concentration of the kinase; Kd is the dissociation constant of the reaction between the. fluorescent probe and the kinase.
24. A kit for the methods of identification of inhibitors of protein kinases or determination of binding characteristics of inhibitors as defined in any one of claims 14, 15 or 23, comprising a fluorescent probe of any one of claims 1-13.
25. A bisubstrate-analog inhibitor of basophilic protein kinase with the general formula (II):
X-Y-Z (II),
in which X is a compound that binds to the ATP-binding pocket of the kinase and;
- Z is a peptide or peptide analog incorporating 1-10 D-arginine residues;
- Y is an organic tether that connects X and Z and permits simultaneous binding of X and Z to the active site of the kinase.
Dated this 16th day of March, 2009.

AN ASSAY FOR IDENTIFICATION OF A PROTEIN KINASE INHIBITOR WITH A FLUORESCENT PROBE

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