A Generic,Homogenous Method for Measuring Kinase and Inhibitor Activity via Adenosine 5′-Diphosphate Accumulation

Abstract

The authors describe an assay to measure the generation of adenosine 5′-diphosphate (ADP) resulting from phosphorylation of a substrate by a kinase. ADP accumulation is detected by conversion to a fluorescent signal via a coupled enzyme system. The technology has potential applications for the assessment of inhibitor potency and mode of action as well as kinetic analysis of enzyme activity. The assay has a wide dynamic range (0.25-75 μM) and has been validated with several kinases including the highly active cyclic adenosine monophosphate-dependent protein kinase (PKAα), casein kinase 1 (CK1), and the weakly active kinase Jun N-terminal kinase 2 (Jnk2α2). Kinase activity can be measured either in an end point or continuous mode. Assay performance in end point mode was compared with an adenosine 5′-triphosphate (ATP) depletion assay and in continuous mode with a pyruvate kinase/lactate dehydrogenase coupled assay. The ability to characterize kinase kinetics was demonstrated by deriving ATP/substrate affinity (Michaelis-Menten constant; K_m) values for PKAα, CK1, and Jnk2α2. The assay readily measured activity with kinase reactions using protein substrates, indicating the suitability for use with large macromolecules. A wide range of inhibitor activities could be determined even in the presence of high ATP concentrations, making the assay highly suitable to characterize the mode of action of the inhibitor in question. Collectively, this assay provides a homogenous, generic method for a number of applications in kinase drug discovery.

Keywords

ADP accumulation coupled-enzyme assay serine/threonine protein kinase tyrosine protein kinase phosphorylation inhibitor mode of action fluorescent detection

References

Manning G , Whyte DB , Martinez R , Hunter T , Sudarsanam S : The protein kinase complement of the human genome. Science 2002;298: 1912-1934.

Levitzki A : Protein kinase inhibitors as a therapeutic modality. Acc Chem Res 2003;36:462-469.

Vieth M , Sutherland JJ , Robertson DH , Campbell RM : Kinomics: characterizing the therapeutically validated kinase space. Drug Discov Today 2005;10:839-846.

Druker BJ , Tamura S , Buchdunger E , Ohno S , Segal GM , Fanning S , et al : Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med 1996;2:561-566.

Druker BJ , Sawyers CL , Kantarjian H , Resta DJ , Reese SF , Ford JM , et al : Activity of a specific inhibitor of the BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome. N Engl J Med 2001;344:1038-1042.

Druker BJ , Talpaz M , Resta DJ , Peng B , Buchdunger E , Ford JM , et al : Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med 2001;344:1031-1037.

Barker AJ , Gibson KH , Grundy W , Godfrey AA , Barlow JJ , Healy MP , et al : Studies leading to the identification of ZD1839 (IRESSA): an orally active, selective epidermal growth factor receptor tyrosine kinase inhibitor targeted to the treatment of cancer. Bioorg Med Chem Lett 2001;11:1911-1914.

Hidalgo M , Eckhardt SG : Matrix metalloproteinase inhibitors: how can we optimize their development? Ann Oncol 2001;12:285-287.

Noble ME , Endicott JA , Johnson LN : Protein kinase inhibitors: insights into drug design from structure. Science 2004;303:1800-1805.

10.

von Ahsen O , Bomer U : High-throughput screening for kinase inhibitors. Chembiochem 2005;6:481-490.

11.

Wu JJ : Comparison of SPA, FRET, and FP for kinase assays. Methods Mol Biol 2002;190:65-85.

12.

Park YW , Cummings RT , Wu L , Zheng S , Cameron PM , Woods A , et al : Homogeneous proximity tyrosine kinase assays: scintillation proximity assay versus homogeneous time-resolved fluorescence. Anal Biochem 1999;269:94-104.

13.

Zaman GJ , Garritsen A , de Boer T , van Boeckel CA : Fluorescence assays for high-throughput screening of protein kinases. Comb Chem High Throughput Screen 2003;6:313-320.

14.

Fowler A , Swift D , Longman E , Acornley A , Hemsley P , Murray D , et al : An evaluation of fluorescence polarization and lifetime discriminated polarization for high throughput screening of serine/threonine kinases. Anal Biochem 2002;308:223-231.

15.

Turek-Etienne TC , Lei M , Terracciano JS , Langsdorf EF , Bryant RW , Hart RF , et al : Use of red-shifted dyes in a fluorescence polarization AKT kinase assay for detection of biological activity in natural product extracts. J Biomol Screen 2004;9:52-61.

16.

Eglen RM : Enzyme fragment complementation: a flexible high throughput screening assay technology. Assay Drug Dev Technol 2002;1:97-104.

17.

Li Y , Cummings RT , Cunningham BR , Chen Y , Zhou G : Homogeneous assays for adenosine 5′-monophosphate-activated protein kinase. Anal Biochem 2003;321:151-156.

18.

Loomans EE , van Doornmalen AM , Wat JW , Zaman GJ : High-throughput screening with immobilized metal ion affinity-based fluorescence polarization detection, a homogeneous assay for protein kinases. Assay Drug Dev Technol 2003;1:445-453.

19.

Beasley JR , Dunn DA , Walker TL , Parlato SM , Lehrach JM , Auld DS : Evaluation of compound interference in immobilized metal ion affinity-based fluorescence polarization detection with a four million member compound collection. Assay Drug Dev Technol 2003;1:455-459.

20.

Rodems SM , Hamman BD , Lin C , Zhao J , Shah S , Heidary D , et al : A FRET-based assay platform for ultra-high density drug screening of protein kinases and phosphatases. Assay Drug Dev Technol 2002;1:9-19.

21.

Burke TJ , Loniello KR , Beebe JA , Ervin KM : Development and application of fluorescence polarization assays in drug discovery. Comb Chem High Throughput Screen 2003;6:183-194.

22.

Koresawa M , Okabe T : High-throughput screening with quantitation of ATP consumption: a universal non-radioisotope, homogeneous assay for protein kinase. Assay Drug Dev Technol 2004;2:153-160.

23.

Singh P , Harden BJ , Lillywhite BJ , Broad PM : Identification of kinase inhibitors by an ATP depletion method. Assay Drug Dev Technol 2004;2:161-169.

24.

Barker SC , Kassel DB , Weigl D , Huang X , Luther MA , Knight WB : Characterization of pp60c-src tyrosine kinase activities using a continuous assay: autoactivation of the enzyme is an intermolecular autophosphorylation process. Biochemistry 1995;34:14843-14851.

25.

Kiianitsa K , Solinger JA , Heyer WD : NADH-coupled microplate photometric assay for kinetic studies of ATP-hydrolyzing enzymes with low and high specific activities. Anal Biochem 2003;321: 266-271.

26.

Dominguez J-M , Rodriguez B , Zapatero C , Vazquez M-J , Magan C , Vela L , et al : A new fluorogenic method for the detection of ADP. Abstract presented at the Society for Biomolecular Screening Conference, The Hague, the Netherlands, September 2002.

27.

Nakajima N, Sakanishi S, Nonaka U, inventors: Enzymatic determination of ADP. Japanese patent JP 61092598. May 10, 1986.

28.

Kosower NS , Kosower EM , Wertheim B , Correa WS : Diamide, a new reagent for the intracellular oxidation of glutathione to the disulfide. Biochem Biophys Res Commun 1969;37:593-596.

29.

Moore MJ , Adams JA , Taylor SS : Structural basis for peptide binding in protein kinase A: role of glutamic acid 203 and tyrosine 204 in the peptide-positioning loop. J Biol Chem 2003;278:10613-10618.

30.

Grant BD , Tsigelny I , Adams JA , Taylor SS : Examination of an active-site electrostatic node in the cAMP-dependent protein kinase catalytic subunit. Protein Sci 1996;5:1316-1324.

31.

Kemp BE , Graves DJ , Benjamini E , Krebs EG : Role of multiple basic residues in determining the substrate specificity of cyclic AMP-dependent protein kinase. J Biol Chem 1977;252:4888-4894.

32.

Agostinis P , Pinna LA , Meggio F , Marin O , Goris J , Vandenheede JR , et al : A synthetic peptide substrate specific for casein kinase I. FEBS Lett 1989;259:75-78.

33.

Estrada E , Agostinis P , Vandenheede JR , Goris J , Merlevede W , Francois J , et al : Phosphorylation of yeast plasma membrane H+-ATPase by casein kinase I. J Biol Chem 1996;271:32064-32072.

34.

Meggio F , Donella Deana A , Ruzzene M , Brunati AM , Cesaro L , Guerra B , et al : Different susceptibility of protein kinases to staurosporine inhibition: kinetic studies and molecular bases for the resistance of protein kinase CK2. Eur J Biochem 1995;234:317-322.

35.

Fabian MA , Biggs WH III , Treiber DK , Atteridge CE , Azimioara MD , Benedetti MG , et al : A small molecule-kinase interaction map for clinical kinase inhibitors. Nat Biotechnol 2005;23:329-336.

36.

Copeland RA : Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis. 2nd ed. New York: Wiley, 2000.

37.

Zhang B , Senator D , Wilson CJ , Ng SC : Development of a high-throughput robotic fluorescence-based assay for HsEg5 inhibitor screening. Anal Biochem 2005;345:326-335.