In this way a protease mediated FRET read-out (Z-lite™, Invitrogen) can be used as an assay for kinase activity (Rodems et al., 2002). Protein kinase assays can be set up a variety of ways depending on the desired mode of action.

DAPT purchase For example, screening the inactive state of a protein kinase to find type II inhibitors can be achieved by incorporating a kinase activation step within the assay system or activity independent systems such as binding methods (Lu et al., 2004, Newbatt et al., 2006 and Vainshtein et al., 2002). Competition-binding assays have been developed using ATP competitive compounds which have been applied to profile selectivity of inhibitors against 442 members (80%) of the human kinome (Davis et al., 2011). Modern protein kinase assays attempt to use technologies where the native substrate can be incorporated into the assay to improve the physiological relevance of the assay. Both protein tyrosine (EC 3.1.3.48) and serine (EC 3.1.3.3) phosphatases are of interest for drug discovery. The fluorogenic tyrosine phosphatase substrate difluoromethyl umbelliferyl phosphate (DiFMUP) is one of the most commonly employed substrates

for tyrosine phosphatase assays (Gee et al., 1999). Fluorogenic substrates based on fluorinated umbelliferones remains the substrate of choice for continuous assays of phosphatases. Enzyme-catalyzed hydrolysis of the phosphate group liberates the blue-fluorescent difluoromethyl umbelliferone which is conveniently BCKDHB followed by its emission at 450 nm (λex=360 nm). Due to potential issues with compound interference this system is ideally performed in a kinetic mode and should learn more include a pre-read of the fluorescence following compound addition but before addition of phosphatase enzyme. Bioluminescent ATP/ADP detection methods used for protein kinases are also useful for other classes such as lipid kinases (Vidugiriene et al., 2009) and metabolic enzymes such as hexokinase (EC 2.7.1.1) and pyruvate kinase (EC 2.7.1.40). For pyruvate kinase, ATP is the

product of the kinase reaction rather than the substrate (Inglese et al., 2006). Pyruvate kinase, the M2 isoform being an important target in cancer, has been screened using this approach where potent activators and inhibitors of the enzyme have been identified (Boxer et al., 2010 and Jiang et al., 2010). Many of the glycololytic enzymes can be assayed alone or in combination using bioluminescent ATP or ADP detection. ATP and ADP formation methods should be adaptable to other enzyme classes such as phosphodiesterases, and ATPases such as heat-shock proteins or polymerases. The red shifted FP based system used for protein kinases has also been expanded to an analogous antibody-based system for detecting UDP which is useful for UDP-dependent glycosyltransferases (EC 2.4), AMP/GMP for PDEs and ligases (EC 6). EFC has also been applied to a wide variety of targets (Eglen, 2002 and Eglen and Singh, 2003).