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

DiscoveRx Corporation, Fremont, California

Lindy Kauffman

DiscoveRx Corporation, Fremont, California

Raj Singh

DiscoveRx Corporation, Fremont, California

Richard M. Eglen

DiscoveRx Corporation, Fremont, California

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 (PKA), casein kinase 1 (CK1), and the weakly active kinase Jun N-terminal kinase 2 (Jnk22). 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 PKA, CK1, and Jnk22. 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.

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

This version was published on June 1, 2006

Journal of Biomolecular Screening, Vol. 11, No. 4, 390-399 (2006)
DOI: 10.1177/1087057106286829


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