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•Application Note: ADP Hunter Assay For HTS Of Kinase Inhibitors Using The PHERAstar

As mediators of eukaryotic signal transduction, controlling multiple cellular processes such as gene transcription, cell cycling, migration, apoptosis and differentiation, protein kinases are considered important targets in drug discovery. Kinase HTS screens ideally require kinase assay platforms with broad applicability in the inhibitor drug discovery process and are thus generically applicable to both the kinase target and substrate. Most non-radioactive kinase assay formats are limited in this respect in that they require either specific antibodies, or affinity capture reagents, to detect generation of the phosphorylated substrate. Often specific antibodies to the phosphorylated substrate are unavailable while affinity capture methods have limitations in the kinase assay conditions that can be used. During substrate phosphorylation, all kinases consume ATP and assays have been developed to measure ATP depletion occurring during the kinase reaction. However, this approach is limited by the ATP concentration required by the kinase, resulting in a high assay background. Consequently, small signal decreases, as occurs with weakly active kinases are difficult to detect.

An optimal approach is to measure the accumulation of a generic product of the kinase reaction i.e. ADP. This technique results in an increase in assay signal directly proportional to kinase activity and has the marked advantage that the assay is performed at a range of ATP concentrations, including those at the ATP Km value. Consequently, the inhibitory potency of novel compounds, when evaluated at the ATP Km, is a robust measure of activity at the ATP binding site and is easily compared to similar measurements at other kinases. Moreover, measurement of ADP accumulation allows flexibility in choice of the kinase substrate, so that both peptides and endogenous substrates (including during autophosphorylation) the kinase may be utilized.

DiscoveRx has developed a homogeneuos fluorescence based assay to measure the generation of ADP, a universal product of kinase activity. The assay uses an enzyme-coupled reaction that produces a red-shifted fluorescence signal that is directly proportional to the amount of ADP in the solution.

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•Application Note: ADP Hunter Assay For HTS Of Kinase Inhibitors Using The PHERAstar

SOURCE: BMG LABTECH, GmbH