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 specifi c antibodies, or affi nity capture reagents, to detect generation of the phosphorylated substrate. Often specifi c antibodies to the phosphorylated substrate are unavailable while affi nity 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 diffi cult 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 fl exibility 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 fl uorescence 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 fl uorescence signal that is directly proportional to the amount of ADP in the solution. ADP Hunter is a biochemical assay to measure the accumulation of ADP (fi gure 1), a universal product of kinase enzyme activity.
ADP Hunter is specifi cally designed for high throughput screening of kinase inhibitors. The assay has been designed for use in fullvolume 384-well microplates, but can also be run in additional microplate formats. To allow for automation, a Stop Solution is also provided for added signal and background stabilization. Unlike alternative generic approaches that monitor the depletion of ATP from a kinase reaction, this method follows the product of the reaction, and offers a convenient gain-of-signal assay format. This kinase assay is a generic, non-radioactive method that doesn’t require use of antibody. This application note describes the use of the ADP Hunter assay measured on BMG LABTECH’s PHERAstar multimode HTS plate reader.
This novel technology recently developed and commercialized by DiscoveRx Corp provides generic approaches to kinases research and inhibitor. This assay is a useful tool for those customers who do not have access to a modified substrate, a phosphorylation-state specific antibody, or the ability or desire to use radioactivity. These reagents are designed to be robust and applicable to high throughput fluid dispensing systems using simple plate readers. The PHERAstar with its innovative and user friendly , intuitive software allows for the greatest flexibility and ease of use.
For more information on DiscoveRx assays please refer to the web site: www.discoverx.com ADP Hunter, DiscoveRx and the DiscoveRx logo are registered trademarks of the Disc
Text taken from www.bmglabtech.com