ADP Hunter™: A Generic Homogeneous Assay for High Throughput Kinase Inhibitor Screening

Abstract

The DiscoveRx ADP Hunter™ assay measures the generation of ADP resulting from kinase phosphorylation of a substrate, in a format specifically designed for the high throughput screening laboratory. ADP is measured through a coupled enzyme system, which is linked to a positive, red-shifted fluorescent readout. The assay has a wide dynamic range (1.2 – 120 µM), and can be used with a wide range of ATP, from less than 5 µM up to 300 µM. No substrate labeling is required, and the assay can accommodate either peptide or whole protein substrates.

The simple assay format, with a single sixty minute incubation, provides robust and reproducible results with signal to background ratios > 10 and typical Z’ values > 0.7. The assay has been validated with several kinases, and against the LOPAC library with excellent results. Compound interference rates are low, and known kinase inhibitor IC₅₀ values are consistent with literature references. In all, the ADP Hunter provides a useful, generic tool for screening novel inhibitors of kinase activity.