WO2008078085A1 - Procédé - Google Patents

Procédé Download PDF

Info

Publication number
WO2008078085A1
WO2008078085A1 PCT/GB2007/004924 GB2007004924W WO2008078085A1 WO 2008078085 A1 WO2008078085 A1 WO 2008078085A1 GB 2007004924 W GB2007004924 W GB 2007004924W WO 2008078085 A1 WO2008078085 A1 WO 2008078085A1
Authority
WO
WIPO (PCT)
Prior art keywords
kinase
autophosphorylating
inactive
met
inhibitor
Prior art date
Application number
PCT/GB2007/004924
Other languages
English (en)
Inventor
Andrew Paul Garner
Geoffrey Allan Holdgate
Original Assignee
Astrazeneca Ab
Astrazeneca Uk Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Astrazeneca Ab, Astrazeneca Uk Limited filed Critical Astrazeneca Ab
Publication of WO2008078085A1 publication Critical patent/WO2008078085A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • This invention relates to a method of screening for a compound which inhibits an autophosphorylating kinase.
  • the invention relates to a method of screening simultaneously for both inhibitors of catalysis and compounds that prevent the activation of an autophosphorylating kinase.
  • 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.
  • 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 ⁇ -chain and a 145 kDa ⁇ -chain.
  • HGF/ scatter factor hepatocyte growth factor
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • cell assays are frequently unsuitable for high throughput screening since they can suffer from some of the following issues:
  • 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.
  • biochemical kinase assay that can simultaneously identify compounds that are inhibitors of catalysis and/or inhibitors of kinase activation of an autophosphorylating kinase.
  • biochemical assay that can simultaneously identify compounds that are inhibitors of catalysis and/or inhibitors of kinase activation of an autophosphorylating kinase.
  • 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.
  • 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.
  • 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.
  • 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.
  • the catalytically dead inactive autophosphorylating kinase may comprise one or more amino acid mutations.
  • 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.
  • I 0 kinase and inactive autophosphorylating kinase comprise the kinase domain of c-Met.
  • 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.
  • 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.
  • 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.
  • 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.
  • the method for measuring phosphorylation of the inactive autophosphorylating kinase is selected from any technique capable of measuring site-specific phosphorylation.
  • 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.
  • TR- FRET Lanthascreen, LANCE, DELFIA
  • FRET Z-lyte
  • Fluorescence Omnia
  • HTRF Caliper
  • SPA Flashplate
  • ATP depletion ADP accumulation
  • IMAP IMAP
  • FP PolyScreen
  • 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.
  • the method for measuring phosphorylation of the inactive autophosphorylating kinase is selected from ELISA or AlphaScreen.
  • the inhibitor inhibits catalysis and/or inhibits kinase activation of an autophosphorylating kinase.
  • an inhibitor of c-Met identified by the method as described hereinabove.
  • the inhibitor inhibits site specific phosphorylation and consequent activation of c-Met.
  • the inhibitor inhibits catalysis and/or inhibits kinase activation of c-Met.
  • an inhibitor as described hereinabove for use in the treatment or prevention of cancer.
  • a method of treating or preventing cancer in a patient comprising administering an inhibitor as described hereinabove.
  • a pharmaceutical composition comprising an inhibitor of c-Met identified by the method as described hereinabove.
  • 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.
  • a method of treating or preventing cancer in a patient comprising administering a pharmaceutical composition as described hereinabove.
  • 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.
  • 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.
  • the kinase activator is a kinase capable of activating the target kinase, but is not the same kinase as the target kinase.
  • the kinase activator comprises the target kinase in active form.
  • activation of the inactive target kinase is measured directly, preferably not by measurement of phosphorylation of a substrate of the target kinase.
  • 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.
  • '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.
  • RNAi RNA interference
  • siRNA siRNA
  • antisense see Opalinska JB, Gewirtz AM. (Sci STKE. 2003 Oct 28;2003(206):pe47.).
  • 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 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.
  • kinase assays were conducted using AlphaScreen or ELISA technology.
  • kinase activity assays were performed in 384-well low- volume white plates (Greiner, 784075) with a total volume of 12 ⁇ L 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.
  • 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 ⁇ L 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.
  • 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 ⁇ L 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 4 0 C for 24h. ELISA plates were washed 3 times with 40 ⁇ L per well appropriate buffer, containing 0.05-1% detergent, 0.1% (w/v) blocking reagent. The ELISA plates were then blocked with 50 ⁇ L 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 ⁇ L 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 ⁇ L 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 ⁇ L 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 ⁇ L per well of buffer containing millimolar concentrations of EDTA in appropriate buffer containing detergents, reducing agent and phosphatase inhibitors. A 10 ⁇ L 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 4 0 C.
  • the plates were washed 3 times with 40 ⁇ L per well appropriate buffer, containing 0.05-1% detergent, 0.1% (w/v) blocking reagent before addition of a 40 ⁇ L aliquot of the primary phosphospecif ⁇ c 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 ⁇ L 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 ⁇ L 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 40 ⁇ l per well appropriate buffer, containing 0.05-1% detergent, 0.1% (w/v) blocking reagent.
  • 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.
  • assays can be conducted using AlphaScreen or ELISA technology.
  • kinase activity assays were performed in 384- well low- volume white plates (Greiner, 784075) with a total volume of 12 ⁇ L in each well.
  • Each kinase reaction contained 40pg (10OpM) pY 1234 pY 1235 c-Met(1074-1366) kinase domain, 44ng (10OnM) cMyc-
  • kinase reactions were incubated at room temperature for 60 minutes and stopped by adding 5 ⁇ L containing 0.5ng anti- pYpY 1234/1235 c-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.
  • IC5 0 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 ⁇ L per well TBS, containing 0.8 ⁇ g 9E10 antibody (lO ⁇ g/mL, 66.7nM, AstraZeneca Pharmaceuticals, deposit number ECACC 85102202), covered with an adhesive seal and incubated at 4 0 C for 24h. ELISA plates were washed 3 times with 40 ⁇ L per well TBS, 0.05% (v/v) Tween 20, 0.1% (w/v) BSA. The ELISA Plates were then blocked with 50 ⁇ L 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 ⁇ L in each well.
  • Each kinase reaction contained 0.77ng (InM) P Y 1234 pY 1235 c-Met(1074-1366) kinase domain, 88.8ng (10OnM) cMyc- [D1204N,R1208Q]c-Met(1069-1366)-biotin, 25mM HEPES (pH7.4), 1OmM MgCl 2 , 0.ImM Na 3 VO 4 , ImM DTT, 0.002% (v/v) Tween-20, 50 ⁇ M ATP.
  • 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 ⁇ L stop buffer per well containing 4OmM EDTA, 25mM HEPES (pH 7.4), 1OmM MgCl 2 , 0. ImM Na 3 VO 4 , ImM DTT, 0.002% (v/v) Tween-20.
  • ELISA plates were washed 3 times with 40 ⁇ L 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 ⁇ L per well of buffer containing 2OmM EDTA, 25mM HEPES (pH7.4), 1OmM MgCl 2 , 0. ImM Na 3 VO 4 , ImM DTT, 0.002% (v/v) Tween-20.
  • the plates were washed 3 times with 40 ⁇ L per well TBS, 0.05% (v/v) Tween 20, 0.1% (w/v) BSA before addition of a 40 ⁇ L 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.
  • the IC 50 value is the concentration of test compound that inhibits 50% of the c-Met kinase reaction control rate.
  • 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 OfMnCl 2 , 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 -8O 0 C 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
  • 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 31 0 C 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 -80 0 C 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

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Food Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biophysics (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne un procédé de criblage permettant de déterminer un composé qui inhibe une kinase d'autophosphorylation. L'invention concerne en particulier un procédé de criblage permettant de déterminer simultanément tant des inhibiteurs de catalyse que des composés susceptibles d'empêcher l'activation d'une kinase d'autophosphorylation. L'invention concerne plus particulièrement un procédé de criblage permettant de déterminer simultanément tant des inhibiteurs de catalyse que des composés susceptibles d'empêcher l'activation du c-Met, un récepteur à activité tyrosine kinase capable d'autophosphorylation.
PCT/GB2007/004924 2006-12-22 2007-12-20 Procédé WO2008078085A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US87146806P 2006-12-22 2006-12-22
US60/871,468 2006-12-22

Publications (1)

Publication Number Publication Date
WO2008078085A1 true WO2008078085A1 (fr) 2008-07-03

Family

ID=39047796

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2007/004924 WO2008078085A1 (fr) 2006-12-22 2007-12-20 Procédé

Country Status (1)

Country Link
WO (1) WO2008078085A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011145035A1 (fr) 2010-05-17 2011-11-24 Indian Incozen Therapeutics Pvt. Ltd. Nouveau composés de 3,5-disubstitués-3h-imidazo[4,5-b]pyridine et 3,5- disubstitués -3h-[1,2,3]triazolo[4,5-b] pyridine utilisés comme modulateurs des protéines kinases
WO2013144737A2 (fr) 2012-03-30 2013-10-03 Rhizen Pharmaceuticals Sa Nouveaux composés 3,5-disubstitués-3h-imidazo[4,5-b]pyridines et 3,5-disubstitués-3h-[1,2,3]triazolo[4,5-b]pyridines en tant que modulateurs des protéines kinases c-met

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5861266A (en) * 1994-02-28 1999-01-19 New York University Treatment of diabetes mellitus and insulin receptor signal transduction
WO2003104479A2 (fr) * 2002-06-11 2003-12-18 The General Hospital Corporation Identification d'inhibiteurs de la mitose
US20040102622A1 (en) * 2002-11-23 2004-05-27 Isis Pharmaceuticals Inc. Modulation of hepatocyte growth factor receptor expression
WO2006108048A1 (fr) * 2005-04-05 2006-10-12 The General Hospital Corporation Methode de prediction de la sensibilite a des medicaments

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5861266A (en) * 1994-02-28 1999-01-19 New York University Treatment of diabetes mellitus and insulin receptor signal transduction
WO2003104479A2 (fr) * 2002-06-11 2003-12-18 The General Hospital Corporation Identification d'inhibiteurs de la mitose
US20040102622A1 (en) * 2002-11-23 2004-05-27 Isis Pharmaceuticals Inc. Modulation of hepatocyte growth factor receptor expression
WO2006108048A1 (fr) * 2005-04-05 2006-10-12 The General Hospital Corporation Methode de prediction de la sensibilite a des medicaments

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011145035A1 (fr) 2010-05-17 2011-11-24 Indian Incozen Therapeutics Pvt. Ltd. Nouveau composés de 3,5-disubstitués-3h-imidazo[4,5-b]pyridine et 3,5- disubstitués -3h-[1,2,3]triazolo[4,5-b] pyridine utilisés comme modulateurs des protéines kinases
US8481739B2 (en) 2010-05-17 2013-07-09 Incozen Therapeutics Pvt. Ltd. 3,5-Disubstituted-3H-imidazo[4,5-b]pyridine and 3,5-disubstituted-3H[1,2,3]triazolo [4,5-b] Pyridine Compounds as Modulators of protein kinases
US8912331B2 (en) 2010-05-17 2014-12-16 Rhizen Pharmaceuticals Sa 3,5-disubstituted-3H-imidazo[4,5-B]pyridine and 3,5-disubstituted-3H-[1,2,3]triazolo[4,5-B] pyridine compounds as modulators of protein kinases
US10087182B2 (en) 2010-05-17 2018-10-02 Incozen Therapeutics Pvt. Ltd. 3,5-disubstituted-3H-imidazo[4,5-B]pyridine and 3,5-disubstituted-3H-[1,2,3]triazolo[4,5-B] pyridine compounds as modulators of protein kinases
EP3450432A1 (fr) 2010-05-17 2019-03-06 Incozen Therapeutics Pvt. Ltd. Nouveaux composés 3,5 disubstitués-3h-imidazo[4,5-b] pyridine et 3,5-disubstitués-3h-[1,2,3]triazolo[4,5-b] pyridine utilisés comme modulateurs de kinases de protéine
US10590129B2 (en) 2010-05-17 2020-03-17 Rhizen Pharmaceuticals Sa 3,5-disubstituted-3H-imidazo[4,5-B]pyridine and 3,5-disubstituted-3H-[1,2,3]triazolo[4,5-B] pyridine compounds as modulators of protein kinases
WO2013144737A2 (fr) 2012-03-30 2013-10-03 Rhizen Pharmaceuticals Sa Nouveaux composés 3,5-disubstitués-3h-imidazo[4,5-b]pyridines et 3,5-disubstitués-3h-[1,2,3]triazolo[4,5-b]pyridines en tant que modulateurs des protéines kinases c-met
US9815831B2 (en) 2012-03-30 2017-11-14 Rhizen Pharmaceuticals Sa 3,5-disubstituted-3H-imidazo[4,5-B]pyridine and 3,5-disubstituted-3H-[1,2,3]triazolo[4,5-B] pyridine compounds as modulators of c-Met protein kinases
US11066402B2 (en) 2012-03-30 2021-07-20 Rhizen Pharmaceuticals Sa 3,5-disubstituted-3H-imidazo[4,5-b]pyridine and 3,5-disubstituted-3H-[1,2,3]triazolo[4,5-B] pyridine compounds as modulators of c-Met protein kinases

Similar Documents

Publication Publication Date Title
Ha et al. Mechanism of phosphorylation-dependent binding of APC to β-catenin and its role in β-catenin degradation
Thastrup et al. SPAK/OSR1 regulate NKCC1 and WNK activity: analysis of WNK isoform interactions and activation by T-loop trans-autophosphorylation
Hsu et al. Defining interactions between DNA-PK and ligase IV/XRCC4
Levinson et al. Structural basis for the recognition of c-Src by its inactivator Csk
Murányi et al. Myotonic dystrophy protein kinase phosphorylates the myosin phosphatase targeting subunit and inhibits myosin phosphatase activity
Spassov et al. A dimerization function in the intrinsically disordered N-terminal region of Src
US20070015231A1 (en) Assay for protein tyrosine phosphatases
Chan et al. Down-regulation of active ACK1 is mediated by association with the E3 ubiquitin ligase Nedd4-2
Klevernic et al. Characterization of the reversible phosphorylation and activation of ERK8
Horvath et al. 14-3-3 proteins inactivate DAPK2 by promoting its dimerization and protecting key regulatory phosphosites
Vacratsis et al. Zipper-mediated oligomerization of the mixed lineage kinase SPRK/MLK-3 is not required for its activation by the GTPase cdc 42 but is necessary for its activation of the JNK pathway: monomeric SPRK L410P does not catalyze the activating phosphorylation of Thr258 of murine mitogen-activated protein kinase kinase 4
Wang et al. Structural and mechanistic insights into Mps1 kinase activation
EP2087355B1 (fr) Procédé pour la détection de modifications post-traductionnelles
King et al. The adaptor protein Grb14 regulates the localization of 3-phosphoinositide-dependent kinase-1
Igarashi et al. Autophosphorylation of heme‐regulated eukaryotic initiation factor 2α kinase and the role of the modification in catalysis
Gao et al. Role of the PH domain in regulating in vitro autophosphorylation events required for reconstitution of PDK1 catalytic activity
Ng et al. Why an A-loop phospho-mimetic fails to activate PAK1: understanding an inaccessible kinase state by molecular dynamics simulations
WO2008078085A1 (fr) Procédé
WO2006091701A2 (fr) Methodes et compositions permettant de moduler la mort cellulaire a l'aide de kinases ou de phosphatases controlant la survie cellulaire ou l'apoptose
Fouda et al. An Affinity‐Based, Cysteine‐Specific ATP Analog for Kinase‐Catalyzed Crosslinking
Mallon et al. An enzyme-linked immunosorbent assay for the Raf/MEK1/MAPK signaling cascade
Thompson et al. The regulatory α and β subunits of phosphorylase kinase directly interact with its substrate, glycogen phosphorylase
Yong et al. Discovery of Nedd4 auto-ubiquitination inhibitors
Smolko et al. An ultrasensitive fiveplex activity assay for cellular kinases
Szafranska et al. Kinetic mechanism for p38 MAP kinase α: A partial rapid‐equilibrium random‐order ternary‐complex mechanism for the phosphorylation of a protein substrate

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07848652

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 07848652

Country of ref document: EP

Kind code of ref document: A1