WO2008078085A1

WO2008078085A1 - Method

Info

Publication number: WO2008078085A1
Application number: PCT/GB2007/004924
Authority: WO
Inventors: Andrew Paul Garner; Geoffrey Allan Holdgate
Original assignee: Astrazeneca Ab; Astrazeneca Uk Limited
Priority date: 2006-12-22
Filing date: 2007-12-20
Publication date: 2008-07-03

Abstract

This invention relates to a method of screening for a compound which inhibits an autophosphorylating kinase. In particular, the invention relates to a method of screening simultaneously for both inhibitors of catalysis and compounds that prevent the activation of an autophosphorylating kinase. More particularly the invention relates to a method of screening simultaneously for both inhibitors of catalysis and compounds that prevent the activation of the autophosphorylating receptor tyrosine kinase c-Met.

Description

METHOD

Protein tyrosine kinases form a large family of enzymes containing a conserved catalytic core (Hubbard (2000) Annu. Rev. Biochem. 69, 373-398) that catalyses the phosphorylation of specific tyrosine amino acid residues in protein substrates (Manning et al (2002) Science 98, 1912-1934). This phosphorylation event often regulates the function of the protein and is thus key to numerous aspects of signal transduction and cellular regulation. Tyrosine kinases can be separated into two distinct classes: receptor tyrosine kinases and non-receptor tyrosine kinases. Receptor tyrosine kinases typically contain an extracellular domain for ligand interactions, a trans-membrane domain and an intracellular domain that possesses kinase activity. Receptor tyrosine kinases span the cell membrane and are thus able to convert an extracellular signal into an intracellular phosphorylation event, allowing them to transmit biochemical signals which influence cellular behaviour. c-Met is a receptor tyrosine kinase which acts as the cellular receptor for hepatocyte growth factor (HGF/ scatter factor), a dimeric glycoprotein that is synthesized as a single-chain precursor called pro-HGF and comprises a 50 kDa α-chain and a 145 kDa β-chain. When HGF non-covalently binds to the extracellular domain of c-Met, receptor oligomerisation occurs (Gherardi, E., et al. (2006), Proc. Natl. Acad. Sci. USA 103, 4046-4051). This results in phosphorylation of a number of sites within c-Met, such as tyrosine residues γ^1234/5 which lie within the c-Met activation loop (a flexible region of amino acids outside of the active site whose conformation influences kinase activity) and tyrosine residues γ^1349/56 which form part of a structurally unique protein docking site. This phosphorylation on key residues results in an increase in catalytic rate of approximately three orders of magnitude. Many other protein kinases have also been demonstrated to be activated by phosphorylation of the activation loop. Studies on several kinases, for example PKA, InRK, cdk2, ERK2, PKC, c-Src and v- Fps, have shown the level of activation to be in the region of 2 - 5 orders of magnitude (Steinberg, R.' et al (1993), MoLCeIl. Biol. 13, 2332 - 2341; Orr, J. & Newton, A. (1994), J. Biol. Chem. 296, 27715 - 27718; Adams, J., et al (1995), Biochemistry 34, 2447 - 2454; Boerner, R., et al (1996), Biochemistry 35, 9519 - 9525; Saylor, P., et al (1998) Biochemistry 37, 17875 - 17881; Hagopian, J., et al (2001), J.Biol. Chem. 276, 275 - 280; Prowse, C. & s Lew, J. (2001), J. Biol. Chem. 276, 99 - 103; Ablooglu, A. & Kohanski, R. (2001),

Biochemistry 40, 504 - 513). In many instances mono-phosphorylation of the activation loop is sufficient to promote kinase activity although examples of incorporation on 2 or 3 sites are also found (Ferrell, J. & Bhatt, R. (1997), J. Biol. Chem. 272, 19008 - 19016; Burack, W. & Sturgill, T. (1997) Biochemistry 36, 5929 - 5933; Wei, L. et al (1995), J. Biol. Chem. 270,o 8122 - 8130). Phosphorylation and consequent activation can occur both auto-catalytically, as in c-Met, or via heterologous protein kinases (Morgan, D. (1995) Nature 374, 131 - 134; Chou, M. et al (1998), Curr. Biol. 8, 1069 - 1077).

A significant number of clinical studies have shown that both c-Met and HGF are frequently aberrantly expressed in aggressive carcinomas, in other types of human solids tumours, and in their metastases (reviewed in Truslino et al.; Birchmeier, C, et al. (2003), Nature Rev. MoI. Cell. Biol. 4, 915-925; Maulik, G., et al. (2002), Cytokine & Growth Factor Rev. 13, 41-59; and Danilkovitch-Miagkova, A. & Zbar, B. (2002) J. Clin. Invest. 109, 863- 867). Furthermore, the presence of c-Met or HGF in clinical samples often correlates with poor patient prognosis (reviewed in Truslino et al.) suggesting that c-Met activation promoteso tumour growth and metastatic spread. c-Met therefore represents an attractive target in the pursuit of therapies for the treatment of cancer.

Kinase inhibition by small molecules has historically been achieved by inhibiting the catalysis of activated enzymes in kinase pathways by preventing the binding of ATP. To achieve this, inhibitors have typically bound in a site that is used by the purine moiety of ATP5 thus preventing ATP from binding. These types of compounds are thus inhibitors of kinase catalysis, or more simply inhibitors of catalysis. Recently, interest in achieving inhibition in kinase pathways has also encompassed compounds binding to non-activated kinases, and in sites other than the purine site (reviewed in Liu & Gray, (2006), Nature Chem Biol. 7, 358- 364). Compounds which preferentially bind to the inactive form or that utilise sites other than0 the purine site can prevent a kinase from adopting the optimum conformation required for the catalytic machinery to be appropriately positioned to facilitate the transfer of phosphate. These compounds can therefore inhibit catalysis via a different mechanism: inhibition, or prevention, of kinase activation. Prevention of kinase activation is understood to mean inhibition of up-regulation from a basal level of kinase activity which would otherwise be expected in response to regulatory factors. These mechanisms of inhibition are not mutually exclusive since compounds can interact with multiple binding sites, and as such a single compound may act via multiple mechanisms of inhibition.

Currently, the majority of known kinase inhibitors predominantly bind within the purine site. This is perhaps unsurprising, as most inhibitors have been discovered using a typical biochemical assay that uses recombinant, active kinase catalytic domain and measures the phosphorylation of small peptides at very low concentrations of ATP in the presence of candidate inhibitors. Such assays are heavily biased towards the identification of inhibitors of catalysis.

In order to identify a greater diversity of compounds, it is necessary to generate assays that recapitulate the whole activation process of a kinase so that no particular mechanism of inhibition is excluded. A cell assay should be able to identify inhibitors operating via multiple mechanisms of inhibition since the kinase would be expressed in its usual cellular environment and undergoing its physiological activation process. However, cell assays are frequently unsuitable for high throughput screening since they can suffer from some of the following issues:

• difficulty in identifying weak compound leads, either due to issues with lack of cell permeability or the requirement for potent compounds to achieve a measurable change in signal

• cellular toxicity either due to compounds or DMSO concentrations

• a small and variable assay window resulting in reduced certainty in compound data

• complicated assay protocols resulting in a reduced compound throughput. Since biochemical assays tend to be more robust, higher throughput and more sensitive than cell assays it is advantageous to use a biochemical kinase assay that can identify compounds that are inhibitors of catalysis and/or inhibitors of kinase activation.

There is no known biochemical kinase assay that can simultaneously identify compounds that are inhibitors of catalysis and/or inhibitors of kinase activation of an autophosphorylating kinase. Thus there is a need for a biochemical assay that can simultaneously identify compounds that are inhibitors of catalysis and/or inhibitors of kinase activation of an autophosphorylating kinase.

Accordingly, the present invention provides a biochemical assay method for simultaneously identifying compounds that are inhibitors of catalysis and/or inhibitors of kinase activation of an autophosphorylating kinase. In particular there is provided a method of screening simultaneously for compounds that are inhibitors of catalysis and/or inhibitors of kinase activation of an autophosphorylating kinase. More particularly, there is provided a method of screening simultaneously for compounds that are inhibitors of catalysis and/or inhibitors of activation of the autophosphorylating receptor tyrosine kinase c-Met.

According to a first aspect of the invention there is provided a method of screening simultaneously for an inhibitor of catalysis and/or an inhibitor of activation of an autophosphorylating kinase comprising: mixing a test compound with active and inactive forms of the autophosphorylating kinase in the presence of ATP, and measuring phosphorylation of the inactive autophosphorylating kinase, wherein a decrease in phosphorylation of the inactive autophosphorylating kinase in the presence of the test compound compared with phosphorylation in the absence of the test compound indicates that the test compound is an inhibitor of catalysis and/or an inhibitor of activation of the autophosphorylating kinase.

According to another aspect of the invention there is provided a method of screening for an inhibitor of an autophosphorylating kinase comprising: mixing a test compound with active and inactive forms of the autophosphorylating kinase in the presence of ATP, and measuring phosphorylation of the inactive autophosphorylating kinase, wherein a decrease in phosphorylation of the inactive autophosphorylating kinase in the presence of the test compound compared with phosphorylation in the absence of the test compound indicates that the test compound is an inhibitor of the autophosphorylating kinase.

In one embodiment there is provided a method of screening for an inhibitor of an autophosphorylating kinase as described hereinabove wherein the inactive autophosphorylating kinase is catalytically dead. The catalytically dead inactive autophosphorylating kinase may comprise one or more amino acid mutations. In another embodiment there is provided a method of screening for an inhibitor of an autophosphorylating kinase as described hereinabove wherein activation of the inactive autophosphorylating kinase is measured by measuring phosphorylation of the inactive autophosphorylating kinase at a specific phosphorylation site. In one embodiment, activation 5 of the inactive autophosphorylating kinase is measured by measuring phosphorylation at a specific phosphorylation site on the activation loop of the inactive autophosphorylating kinase.

In another embodiment there is provided a method of screening for an inhibitor of an autophosphorylating kinase as described hereinabove wherein the active autophosphorylating

I₀ kinase and inactive autophosphorylating kinase comprise the kinase domain of c-Met.

In another embodiment there is provided a method of screening for an inhibitor of an autophosphorylating kinase as described hereinabove wherein the autophosphorylating kinase is c-Met. In a further embodiment there is provided a method of screening for an inhibitor of c-Met as described hereinabove wherein the c-Met is catalytically dead. In one embodiment is the catalytically dead c-Met may comprise one or more amino acid mutations. In another embodiment catalytically dead c-Met comprises the amino acid mutations D1204N and/or R1208Q.

In another embodiment there is provided a method of screening for an inhibitor of an autophosphorylating kinase as described hereinabove wherein phosphorylation of the inactive o autophosphorylating kinase is measured using any one of an anti-phosphotyrosine antibody, an anti-phosphothreonine antibody or an anti-phosphoserine antibody. In one embodiment phosphorylation of the inactive autophosphorylating kinase is measured using a site-specific anti-phospho antibody, for example a site-specific anti-phosphotyrosine antibody, a site- specific anti-phosphothreonine antibody or a site-specific anti-phosphoserine antibody. In

25 another embodiment phosphorylation of the inactive autophosphorylating kinase is measured using a c-Met activation loop-specific anti-phosphotyrosine antibody. In another embodiment phosphorylation of the inactive autophosphorylating kinase is measured using an anti- p Yp Y 1234/1235c-Met monoclonal antibody.

In another embodiment there is provided a method of screening for an inhibitor of an

30 autophosphorylating kinase as described hereinabove wherein the method for measuring phosphorylation of the inactive autophosphorylating kinase is selected from any technique capable of measuring site-specific phosphorylation. In one embodiment the method for measuring phosphorylation of the inactive autophosphorylating kinase is selected from ELISA, AlphaScreen (Perkin Elmer, Bosse R., Illy C, Elands J. and Chelsky D. (2000) Drug Discovery Today. Jun: 1(1): 42-7; Seethala R. and Prabhavathi F. (2001) Homogeneous Assays: AlphaScreen. Handbook of Drug Screening. Marcel Dekker Pub., pp. 106-110), TR- FRET (Lanthascreen, LANCE, DELFIA), FRET (Z-lyte), Fluorescence (Omnia), HTRF, Caliper, SPA, Flashplate, ATP depletion, ADP accumulation, IMAP, FP (PolarScreen), PK- LDH, filter capture or mass spectrophotometry. The detection method for measuring phosphorylation selected is not crucial to the practice of the invention, and other possible methods for measuring phosphorylation are contemplated. In one embodiment the method for measuring phosphorylation of the inactive autophosphorylating kinase is selected from ELISA or AlphaScreen.

In another embodiment there is provided a method of screening for an inhibitor of an autophosphorylating kinase as described hereinabove wherein the inactive form of the autophosphorylating kinase is in excess in comparison with the active form of the autophosphorylating kinase.

In another embodiment there is provided a method of screening for an inhibitor of an autophosphorylating kinase as described hereinabove wherein the inhibitor inhibits site specific phosphorylation and consequent activation of an autophosphorylating kinase. In one embodiment the inhibitor inhibits catalysis and/or inhibits kinase activation of an autophosphorylating kinase.

According to another aspect of the invention there is provided an inhibitor of c-Met identified by the method as described hereinabove. In one embodiment the inhibitor inhibits site specific phosphorylation and consequent activation of c-Met. Preferably the inhibitor inhibits catalysis and/or inhibits kinase activation of c-Met. In one embodiment there is provided use of an inhibitor as described hereinabove in the manufacture of a medicament for the treatment or prevention of cancer.

In one embodiment there is provided an inhibitor as described hereinabove for use in the treatment or prevention of cancer.

In one embodiment there is provided use of an inhibitor as described hereinabove for the treatment or prevention of cancer.

In one embodiment there is provided a method of treating or preventing cancer in a patient comprising administering an inhibitor as described hereinabove. According to another aspect of the invention there is provided a pharmaceutical composition comprising an inhibitor of c-Met identified by the method as described hereinabove.

According to another aspect of the invention there is provided a method of preparing the pharmaceutical composition comprising determining whether the test compound is an inhibitor of c-Met, and incorporating the inhibitor, or a derivative thereof, with a pharmaceutically acceptable carrier.

In one embodiment there is provided a method of treating or preventing cancer in a patient comprising administering a pharmaceutical composition as described hereinabove.

In one embodiment there is provided use of a pharmaceutical composition as described hereinabove for the treatment or prevention of cancer.

In one embodiment there is provided use of a pharmaceutical composition as described hereinabove in the manufacture of a medicament for the treatment or prevention of cancer.

According to another aspect of the invention there is provided a method of screening simultaneously for an inhibitor of a target kinase comprising mixing a test compound with the target kinase in inactive form and a kinase activator, and measuring activation of the inactive target kinase, wherein a decrease in activation of the inactive target kinase in the presence of the test compound compared with activation in the absence of the test compound indicates that the test compound is an inhibitor of the kinase.

According to another aspect of the invention there is provided a method of screening for an inhibitor of a target kinase comprising mixing a test compound with the target kinase in inactive form and a kinase activator, and measuring activation of the inactive target kinase, wherein a decrease in activation of the inactive target kinase in the presence of the test compound compared with activation in the absence of the test compound indicates that the test compound is an inhibitor of the kinase. In one embodiment the kinase activator is a kinase capable of activating the target kinase, but is not the same kinase as the target kinase. In an alternative embodiment the kinase activator comprises the target kinase in active form.

In another embodiment activation of the inactive target kinase is measured directly, preferably not by measurement of phosphorylation of a substrate of the target kinase.

The following terms, unless otherwise indicated, shall be understood to have the following meanings:

With respect to an autophosphorylating kinase 'active' means phosphorylated in a region of interest, in particular in the activation loop and 'inactive' means not phosphorylated in the region of interest, in particular in the activation loop.

With respect to c-Met, 'active' means phosphorylated in the activation loop and 'inactive' means not phosphorylated in the activation loop.

A compound may be a polypeptide, nucleic acid, carbohydrate, lipid, small molecular weight compound (for example those having a molecular weight of less than 2000 Daltons), an oligonucleotide, an oligopeptide, RNA interference (RNAi), siRNA, antisense, a recombinant protein, an antibody, or conjugates or fusion proteins thereof. For a review of RNAi see Milhavet O, Gary DS, Mattson MP. (Pharmacol Rev. 2003 Dec;55(4):629-48. Review.) and for antisense see Opalinska JB, Gewirtz AM. (Sci STKE. 2003 Oct 28;2003(206):pe47.). The invention will now be illustrated by the following non-limiting examples, which are provided for illustrative purposes only and are not to be construed as limiting upon the teachings herein, in which

Figure 1. Shows a method of screening for both inhibitors of catalysis and compounds that prevent the activation of the autophosphorylating receptor tyrosine kinase c-Met using AlphaScreen, in which wild type activated c-Met phosphorylates a mutant form of c-Met lacking catalytic activity but retaining the ability to be phosphorylated on the activating residues. Figure 1 labels are as follows: 1 is the phosphorylation stage; 2 is the detection stage; plates are excited at 680nm and emission read at 520-620nm; D = Donor; S = Streptavidin; B = Biotin; O = Singlet oxygen; A = Protein A AlphaScreen acceptor beads; C = active cMet; K = Kinase dead cMet; Ab = anti-pYpY^1234/1235c-Met antibody. Figure 2. Shows an IC50 curve for a reference compound in the AlphaScreen c-Met assay, with response on the Y axis against concentration in nM on the X axis. Data shows the compound to inhibit catalysis of active c-Met or activation of inactive c-Met, as increasing concentrations of compound show a decrease in phosphorylation of catalytically dead c-Met comprising the amino acid mutations D1204N and R1208Q. IC50 = 87.54nM, 95% CIR =1.28.

EXAMPLE 1 : KINASE ASSAYS

To determine inhibition of enzyme activity, kinase assays were conducted using AlphaScreen or ELISA technology.

AlphaScreen Assay

Kinase activity assays were performed in 384-well low- volume white plates (Greiner, 784075) with a total volume of 12 μL in each well. Each kinase reaction contained picogram- nanogram amounts of active kinase, nanogram amounts of a tagged-catalytically dead protein as substrate, suitable buffer at physiological pH; containing reducing agents, phosphatase inhibitors, detergent, cofactors and ATP.

Various concentrations of test compounds were each added in 6% (v/v) DMSO to yield a final assay DMSO concentration of 1% (v/v). The kinase reactions were incubated at room temperature for 60 minutes and stopped by adding 5 μL containing nanogram quantities of phosphotyrosine or site-specific phospho-antibody, with nanogram quantities of appropriate AlphaScreen acceptor beads (Perkin Elmer) and nanogram quantities of appropriate donor beads (Perkin Elmer) in buffer at physiological pH 7.4 containing EDTA and BSA under low-level light conditions. Plates were sealed under low-level light conditions and incubated in the dark for 20 hours. Plates were read using an Envision (Perkin Elmer) with excitation at 680nm, emission 520-620nm. The mean data values for each test compound concentration, untreated control wells and 100% inhibition control wells were used to determine the test compounds IC50 value. IC50 value is the concentration of test compound that inhibits 50% of kinase activity.

ELISA Assay Black high binding 384 well ELISA plates (Greiner bio-one, 781077) were coated with 80 μL per well appropriate buffer, containing microgram quantities of an antibody which binds to a tag on the substrate protein, covered with an adhesive seal and incubated at 4⁰C for 24h. ELISA plates were washed 3 times with 40 μL per well appropriate buffer, containing 0.05-1% detergent, 0.1% (w/v) blocking reagent. The ELISA plates were then blocked with 50 μL per well blocking reagent in appropriate buffer at room temperature for up to 4h. Kinase activity assays were performed in 384 well polypropylene plates (Matrix, #4314) with a total reaction volume of 24 μL in each well. Each kinase reaction contained picogram-nanogram quantities of active kinase, nanogram amounts of a tagged-catalytically dead protein as substrate, suitable buffer at physiological pH; containing reducing agents, phosphatase inhibitors, detergent, cofactors and ATP. Various concentrations of test compounds were each added in 6% (v/v) DMSO to yield a final assay DMSO concentration of 1% (v/v). The kinase reactions were incubated at room temperature for up to 60 minutes and stopped by addition of 24 μL stop buffer per well containing millimolar concentrations of EDTA in appropriate buffer containing detergents, reducing agent and phosphatase inhibitors. ELISA plates were washed 3 times with 40 μL per well appropriate buffer, containing 0.05-1% detergent, 0.1% (w/v) blocking reagent to remove blocking buffer. The ELISA plates were then prepared for transfer from the kinase reaction by addition of 30 μL per well of buffer containing millimolar concentrations of EDTA in appropriate buffer containing detergents, reducing agent and phosphatase inhibitors. A 10 μL aliquot from each stopped kinase assay reaction was transferred to the corresponding ELISA plate well. ELISA plates were then covered with an adhesive seal and incubated overnight at 4⁰C. The plates were washed 3 times with 40 μL per well appropriate buffer, containing 0.05-1% detergent, 0.1% (w/v) blocking reagent before addition of a 40 μL aliquot of the primary phosphospecifϊc or phosphotyrosine antibody diluted in appropriate buffer, containing 0.05-1% detergent, 0.1% (w/v) blocking reagent. ELISA plates were covered with an adhesive seal and incubated at room temperature for up to 4h. Plates were washed 3 times with 40 μL per well appropriate buffer, containing 0.05-1% detergent, 0.1% (w/v) blocking reagent and the secondary antibody (against the host that the primary was raised in) coupled to an enzyme was added in 40 μL aliquots per well, diluted in appropriate buffer, containing 0.05-1% detergent, 0.1% (w/v) blocking reagent. The ELISA plates were covered with an adhesive seal and incubated at room temperature for up to 2h. Plates were washed a further 3 times with 40μl per well appropriate buffer, containing 0.05-1% detergent, 0.1% (w/v) blocking reagent. To detect phosphorylated substrate added 40 μL chromogen or substrate, which changes colour or fluoresces when cleaved by the enzyme attached to the secondary antibody. Plates are incubated at room temperature for sufficient time for a signal to develop, then ELISA plates are read using a suitable detection system, such as a spectrophotometer or fluorescence plate reader.

The data values for each test compound concentration, untreated control wells and 100% inhibition control wells are fitted to biologically relevant equations using non-linear regression in order to determine the test compounds IC50 value. The IC50 value is the concentration of test compound that inhibits 50% of the kinase reaction control rate.

EXAMPLE 2: C-MET ASSAYS

To determine inhibition of c-Met activity, assays can be conducted using AlphaScreen or ELISA technology.

AlphaScreen Assay

Kinase activity assays were performed in 384- well low- volume white plates (Greiner, 784075) with a total volume of 12 μL in each well. Each kinase reaction contained 40pg (10OpM) pY¹²³⁴pY¹²³⁵c-Met(1074-1366) kinase domain, 44ng (10OnM) cMyc-

[D1204N,R1208Q]c-Met(1069-1366)-biotin, 25mM HEPES (pH7.4), O. lmM Na₃VO₄, ImM DTT, 0.01% (v/v) Tween-20, 1OmM MgCl₂, 0.1% BSA, 5OuM ATP.

Various concentrations of test compounds were each added in 6% (v/v) DMSO to yield a final assay DMSO concentration of 1% (v/v). The kinase reactions were incubated at room temperature for 60 minutes and stopped by adding 5 μL containing 0.5ng anti- pYpY^1234/1235c-Met rabbit polyclonal antibody (AstraZeneca Pharmaceuticals) with 200ng rabbit IgG Protein A AlphaScreen acceptor beads (Perkin Elmer 6760617R) & 200ng streptavidin donor beads (Perkin Elmer 6760617R) in 25mM HEPES (pH 7.4), 84.5mM EDTA, 0.3% BSA under low-level light conditions. Plates were sealed under low-level light conditions and incubated in the dark for 20 hours. Plates were read using an Envision (Perkin Elmer) with excitation at 680nm, emission 520-620nm. The mean data values for each test compound concentration, untreated control wells and 100% inhibition control wells were used to determine the test compounds IC5₀ value. IC₅0 value is the concentration of test compound that inhibits 50% of c-Met kinase activity.

ELISA Assay Black high binding 384 well ELISA plates (Greiner bio-one, 781077) were coated with 80 μL per well TBS, containing 0.8 μg 9E10 antibody (lOμg/mL, 66.7nM, AstraZeneca Pharmaceuticals, deposit number ECACC 85102202), covered with an adhesive seal and incubated at 4⁰C for 24h. ELISA plates were washed 3 times with 40 μL per well TBS, 0.05% (v/v) Tween 20, 0.1% (w/v) BSA. The ELISA Plates were then blocked with 50 μL per well SuperBlock blocking buffer in TBS (Pierce Biotechnology, #37535) at room temperature for 4h.

Kinase activity assays were performed in 384 well polypropylene plates (Matrix, #4314) with a total reaction volume of 24 μL in each well. Each kinase reaction contained 0.77ng (InM) _PY¹²³⁴pY¹²³⁵c-Met(1074-1366) kinase domain, 88.8ng (10OnM) cMyc- [D1204N,R1208Q]c-Met(1069-1366)-biotin, 25mM HEPES (pH7.4), 1OmM MgCl₂ , 0.ImM Na₃VO₄, ImM DTT, 0.002% (v/v) Tween-20, 50μM ATP.

Various concentrations of test compounds were each added in 6% (v/v) DMSO to yield a final assay DMSO concentration of 1% (v/v). The kinase reactions were incubated at room temperature for 60 minutes and stopped by addition of 24 μL stop buffer per well containing 4OmM EDTA, 25mM HEPES (pH 7.4), 1OmM MgCl₂ , 0. ImM Na₃VO₄, ImM DTT, 0.002% (v/v) Tween-20.

ELISA plates were washed 3 times with 40 μL per well TBS, 0.05% (v/v) Tween 20, 0.1% (w/v) BSA, to remove SuperBlock blocking buffer. The ELISA plates were then prepared for transfer from the kinase reaction by addition of 30 μL per well of buffer containing 2OmM EDTA, 25mM HEPES (pH7.4), 1OmM MgCl₂ , 0. ImM Na₃VO₄, ImM DTT, 0.002% (v/v) Tween-20. A 10 μL aliquot from each stopped kinase assay reaction (containing 18.5ng cMyc-[D1204N,R1208Q]c Met( 1069-1366)-biotin) was transferred to the corresponding ELISA plate well to a final concentration of 12.5nM. ELISA plates were then covered with an adhesive seal and incubated overnight at 4⁰C. The plates were washed 3 times with 40 μL per well TBS, 0.05% (v/v) Tween 20, 0.1% (w/v) BSA before addition of a 40 μL aliquot of the primary rabbit (polyclonal) anti-c-Met (pYpYpY^{1230/1234/1235}) phosphospecific antibody (Biosource, 44-888G) used at a dilution of 1 : 1000 in TBS, 0.05% (v/v) Tween 20, 0.1% (w/v) BSA. ELISA plates were covered with an adhesive seal and incubated at room temperature for 4h. Plates were washed 3 times with 40 μL per well TBS, 0.05% (v/v) Tween 20, 0.1% (w/v) BSA and the secondary anti-rabbit IgG HRP-linked antibody (Cell Signalling Technology, #7074) was added in 40 μL aliquots per well, at a dilution of 1 :5000 in TBS, 0.05% (v/v) Tween 20, 0.1% (w/v) BSA. The ELISA plates were covered with an adhesive seal and incubated at room temperature for 2h. Plates were washed a further 3 times with 40 μL per well TBS, 0.05% (v/v) Tween 20, 0.1% (w/v) BSA before addition of the QuantaBlu NS/K Fluorogenic Peroxidase Substrate (Pierce Biotechnology, #15162). The fluorogenic substrate is mixed 9 parts substrate to 1 part stable peroxidase, 40 μL is added to each well and the plates incubated for 7min at room temperature. ELISA plates were read using a Tecan Safire plate reader (Magellan version 4.00 software) with excitation at 325nm and emission at 420nm. The data values for each test compound concentration, untreated control wells and 100% inhibition control wells were fitted to biologically relevant equations using non-linear regression (GraFit, version 5.0.5, from

Erithacus Software Limited) in order to determine the test compounds IC₅₀ value. The IC₅₀ value is the concentration of test compound that inhibits 50% of the c-Met kinase reaction control rate.

EXAMPLE 3: PURIFICATION OF C-MET

Purification of wt pY¹²³⁴pY^123S c Metf 1074- 1366)

Cell pellets from an expression grow of the construct in Sf9 insect cells, harvested 48hr post infection, were thawed and resuspended in buffer A+ Complete EDTA free tablets (Roche). The cells were disrupted by Dounce homogenisation (see chapter 2 part 3, page 71, Purification and Analysis of Recombinant Proteins, Edited by Ramnath Seetharam, Satish K. Sharma, published by Marcel Dekker, New York 1991) and the crude lysate clarified by centrifugation (34,50Og for lhr). The lysate supernatant was passed over a glutathione sepharose column (GE healthcare) pre-equilibrated in buffer A at 4°C. The column was washed to baseline with buffer A. The column was then washed with 10 column volumes (cv) of buffer B and bound protein was finally eluted with lOcv of buffer B+ 2OmM reduced glutathione. Fractions containing the c-Met were pooled. Tev protease was added to the pool and the mixture left at room temperature to allow cleavage of the GST tag to occur. Once cleavage was complete the protein mixture was loaded onto a Heparin Sepharose column pre- equilibrated in buffer B. After loading, the column was washed to baseline with buffer B and bound proteins were eluted with buffer B + IM NaCl. Eluted fractions containing the c-Met were pooled, concentrated and passed down a Superdex 75 column, pre-equilibrated in buffer C. Eluted fractions containing the c-Met were pooled and the concentration adjusted to 0.5mg/ml. The c-Met was phosphorylated by the addition OfMnCl₂, MgCl_2, ATP and incubating the mixture at RT for lhr. After phosphorylation had been confirmed by mass spectrometry the phosphorylated c-Met was buffer exchanged into buffer A+0. ImM sodium ortho vanadate, 10% glycerol using a desalt column. The final pool of c-Met was aliquoted and snap frozen and stored at -8O⁰C prior to use.

Buffer A: 2OmM HEPES pH 7.4, 15OmM NaCl, 5mM DTT, 10% Glycerol Buffer B: 2OmM MES pH 6.5, 5OmM NaCl, 5mM DTT, 10% Glycerol Buffer C: 5OmM MOPS pH7, 5OmM NaCl, 5mM DTT

Purification of Unphosphorylated cMvc-rD1204N.R1208O1c-Met(1069-1366Vbiotin

Purification and subsequent single biotinylation of unphosphorylated cMyc- [D1204N,R1208Q]c-Met(1069-1366)-Avi was undertaken using the following procedure. Cell pellets from an expression grow of the construct in Sf21, harvested 48hr post infection were thawed and resuspended in buffer A. The cells were disrupted by Dounce homogenisation and the crude lysate clarified by centrifugation (34,500g for lhr). The lysate supernatant was passed over a glutathione sepharose column pre-equilibrated in buffer A at 4⁰C at a flow rate of 0.2ml/min. After washing the column back to baseline with buffer A the bound protein was eluted in buffer A + 2OmM reduced glutathione. Fractions were collected and those containing the protein were pooled. Tev protease was added to the pool and the mixture left at room temperature to allow cleavage of the GST tag to occur. Once cleavage was complete the protein mixture was buffer exchanged into buffer B by passing down desalting columns equilibrated in buffer B at 5ml/min. The desalted pool was loaded on to a Mono Q HRl 0/10 anion exchange column pre-equilibrated in buffer B and bound protein eluted using a 0-25% buffer B+ IM NaCl gradient over 25 column volumes. The peak containing the cMET construct was determined and the appropriate elution fractions pooled. The pool was passed over a glutathione sepharose column pre-equilibrated in buffer B and the non-binding fraction containing the c-Met was collected. This non-binding fraction was desalted into buffer C by passing aliquots of the pool over pre-equilibrated PD-IO desalting columns. The fractions containing the c-Met were pooled from all the PD-10 columns. Biotinylation of the purified c-Met was achieved by addition of the appropriate volumes of biotin ligase and biomix-A and biomix-B solution (Avidity) and incubating the reaction mixture at 31⁰C for 2hrs. Biotinylation success was measured by mass spectrometry of samples pre and post reaction. Addition of a single biotin being equivalent to an increase in mass of 226Da. The biotinylated c-Met was concentrated and passed an S75 26/60 column pre-equilibrated in buffer D. Fractions from the column containing the biotinylated c-Met were pooled and aliquots of the pool were stored at -80⁰C prior to use.

Buffer A: 2OmM HEPES pH 7.4, 15OmM NaCl, 5mM DTT, 10% Glycerol

Buffer B: 2OmM TRlS, pH8.0, 2OmM NaCl, 5mM DTT, 10% Glycerol

Buffer C: 2OmM TRIS, pH8.0 Buffer D: 5OmM MOPS pH7.0, 15OmM NaCl, 5mM DTT, 10% Glycerol

Claims

1. A method of screening simultaneously for an inhibitor of catalysis and/or an inhibitor 5 of activation of an autophosphorylating kinase comprising: mixing a test compound with active and inactive forms of the autophosphorylating kinase in the presence of ATP, and measuring phosphorylation of the inactive autophosphorylating kinase, wherein a decrease in phosphorylation of the inactive autophosphorylating kinase in io the presence of the test compound compared with phosphorylation in the absence of the test compound indicates that the test compound is an inhibitor of the autophosphorylating kinase.

2. A method of screening according to claim 1 wherein the inactive autophosphorylating i₅ kinase is catalytically dead.

3. A method of screening according to any preceding claim wherein activation of the inactive autophosphorylating kinase is measured by measuring phosphorylation of the inactive autophosphorylating kinase at a specific phosphorylation site.

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4. A method of screening according to claim 3 wherein activation of the inactive autophosphorylating kinase is measured by measuring phosphorylation at a specific phosphorylation site on the activation loop of the inactive autophosphorylating kinase.

25 5. A method of screening according to any preceding claim wherein phosphorylation of the inactive autophosphorylating kinase is measured using any one of an anti- phosphotyrosine antibody, an anti-phosphothreonine antibody or an anti- phosphoserine antibody.

30 6. A method of screening according to any preceding claim wherein phosphorylation of the inactive autophosphorylating kinase is measured using a site-specific anti-phospho antibody.

7. A method of screening according to any preceding claim wherein the method for measuring phosphorylation of the inactive autophosphorylating kinase is selected from ELISA or AlphaScreen.

8. A method of screening according to any preceding claim wherein the inactive form of the autophosphorylating kinase is in excess in comparison with the active form of the autophosphorylating kinase.

9. A method of screening according to any preceding claim wherein the autophosphorylating kinase is c-Met.

10. A method of screening according to any preceding claim wherein the active autophosphorylating kinase and inactive autophosphorylating kinase comprise the kinase domain of c-Met.

11. An inhibitor of c-Met identified by the method of any one of claims 9 or 10.

12. An inhibitor of c-Met according to claim 11 wherein the inhibitor inhibits catalysis and/or inhibits kinase activation of c-Met.

13. Use of an inhibitor according to claim 11 or 12 for the treatment or prevention of cancer.

14. A pharmaceutical composition comprising an inhibitor according to claim 11 or 12.

15. A method of preparing a pharmaceutical composition according to claim 14 comprising determining whether the test compound is an inhibitor of c-Met, and incorporating the inhibitor, or a derivative thereof, with a pharmaceutically acceptable carrier.

16. A method of treating or preventing cancer in a patient comprising administering the pharmaceutical composition of claim 15.