Methods for Measuring Real Time Kinase Activity

Abstract

The present invention relates to methods for detecting and/or measuring the activity of a specific kinase, with the methods comprising contacting one or more kinases with a binding agent to isolate a specific kinase of interest. The isolated kinase is then contacted with a kinase activity sensor, where the kinase activity sensor is comprised of a kinase recognition motif that is capable of being recognized by the isolated kinase, and at least one phosphorylation site. The isolated kinase phosphorylates the amino acid target of the kinase activity sensor and levels of the phosphorylated target amino acid can then be quantified.

1 . A method of measuring the activity of a specific kinase, said method comprising: 
 providing a solid or semi-solid support comprising an immobilized binding agent;    contacting the immobilized binding agent with a sample comprising the specific kinase to form an immobilized specific kinase;    contacting the immobilized specific kinase with a kinase activity sensor to form a contacted specific kinase, wherein the kinase activity sensor comprises at least one peptide capable of being phosphorylated and a chelator;    incubating the contacted specific kinase for a sufficient amount of time for the kinase to phosphorylate the peptide in the presence of a phosphate source and a metal ion, wherein the kinase activity sensor forms a ternary complex with the metal ion and phosphorylated peptide to generate a detectable signal; and    detecting the signal whereby the activity of the specific kinase is measured.    
     
     
         2 . The method of  claim 1 , wherein the binding agent comprises an antibody or a functional fragment thereof.  
     
     
         3 . The method of  claim 1 , wherein the support is glass, plastic, metal, polymeric particle, polymeric gel or a polymeric membrane.  
     
     
         4 . The method according to  claim 1 , wherein the kinase activity sensor has an increased fluorescence signal when complexed with the metal ion.  
     
     
         5 . The method of  claim 1 , wherein the chelator comprises a signaling moiety.  
     
     
         6 . The method according to  claim 5 , wherein the signaling moiety comprises a coumarin, cyanine, benzofuran, a quinoline, a quinazolinone, an indole, a benzazole, a borapolyazaindacene or a xanthene.  
     
     
         7 . The method according to  claim 1 , wherein the chelator comprises a quinoline or a derivative thereof, phenanthrolines or derivatives thereof, BAPTA, IDA, DTPA and derivatives thereof.  
     
     
         8 . The method according to  claim 1 , wherein the kinase activity sensor further comprises a signaling moiety that is separate from the chelator.  
     
     
         9 . The method according to  claim 8 , wherein the signaling moiety comprises a coumarin, cyanine, benzofuran, a quinoline, a quinazolinone, an indole, a benzazole, a borapolyazaindacene or a xanthene.  
     
     
         10 . The method according to  claim 1 , wherein the method further comprises contacting the immobilized specific kinase with ATP.  
     
     
         11 . The method according to  claim 1 , wherein the metal ion is Ca 2+ , Zn 2+ , Mg 2+ , Ga 3+ , Tb 3− , La 3+ , Pb 2+ , Hg 2+ , Cd 2+ , Cu 2+ , Ni 2+ , Co 2+ , Fe 2+ , Mn 2+ , Ba 2+ , or Sr 2+ .  
     
     
         12 . The method of  claim 1 , wherein the peptide comprises an amino acid that when phosphorylated complexes magnesium.  
     
     
         13 . The method according to  claim 1 , wherein the kinase activity sensor comprises the formula:  
       
         
           
           
               
               
           
         
         where at least one R group is —SO 2 X, where X is —OR″ or —NR″R′″;  
         R′ is hydroxy, amino, or thiol;  
         R″ is C 1-6  alkyl;  
         R′″ is hydrogen or alkyl; and  
         n is 1, 2 or 3.  
       
     
     
         14 . The method of  claim 1 , wherein said kinase activity sensor has the formula:  
       
         
           
           
               
               
           
         
         wherein R 1 , is H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, halo, hydroxy, nitro, SO 3 H, sulfonyl, substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthio, substituted alkylthio, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl;  
         R 2  is a fluorophore, H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, halo, hydroxy, nitro, SO 3 H, sulfonyl, substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthio, substituted alkylthio, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl;  
         R 3  is a fluorophore, H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, halo, hydroxy, nitro, SO 3 H, sulfonyl, substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthio, substituted alkylthio, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl;  
         R 4  is a fluorophore, H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, halo, hydroxy, nitro, SO 3 H, sulfonyl, substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthio, substituted alkylthio, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl;  
         R 5  is a fluorophore, H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, halo, hydroxy, nitro, SO 3 H, sulfonyl, substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthio, substituted alkylthio, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl; and  
         R 6  is a fluorophore, H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, halo, hydroxy, nitro, SO 3 H, sulfonyl, substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthio, substituted alkylthio, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, or substituted heterocyclyl;  
         R 7  is H, alkyl, substituted alkyl, carbonyl, or a protecting group; and  
         R 8  is H, alkyl, substituted alkyl, carbonyl, or a protecting group;  
         L is a linker; and  
         Y is a peptide;  
         or a tautomer, stereoisomer, or salt thereof.  
       
     
     
         15 . The method of  claim 13 , wherein at least one of R 2 , R 3 , R 4 , R 5  and R 6  is a fluorophore.  
     
     
         16 . The method according to  claim 1 , wherein the peptide is at least one of SEQ ID NO. 1 through SEQ ID NO. 133.  
     
     
         17 . A composition comprising: 
 (a) a kinase immobilized by a binding agent; and    (b) a kinase activity sensor comprising at least one peptide capable of being phosphorylated and a chelator.    
     
     
         18 . A kit for detecting the activity of a specific kinase, comprising a binding agent that binds to the specific kinase and a kinase activity sensor, wherein said kinase activity sensor comprises at least one peptide capable of being phosphorylated and a chelator.  
     
     
         19 . The kit according to  claim 18 , further comprising ATP and a metal ion that has affinity for both the chelator and the phosphorylated peptide.