The advantages of the Transcreener platform for detection of diverse members of the kinase superfamily are illustrated in Figure 2. Most kinase assay methods rely on detection of a specific phosphorylated molecule that is produced by one or a small subset of enzymes, thus specific detection reagents and/or conditions are required for individual enzymes or subsets of enzymes.
For example, antibody recognition of phosphoserine in peptides or proteins is dependent on the flanking amino acids, so many different antibodies would be required to detect the phosphorylated products of the hundreds of ser-thr kinases in humans. In contrast, the Transcreener Kinase Assay relies on detection of ADP, the invariant product of all kinase reactions, so the same reagents can be used for any kinase, regardless of the acceptor substrate. Figure 2 demonstrates the generic nature of the assay. Serine-threonine and tyrosine protein kinases are readily detected using either peptide or native protein acceptors as are lipid and carbohydrate kinases with their physiological substrates.
The Transcreener ADP detection method provides flexibility for development and optimization of kinase screening parameters, providing opportunities for increased efficiencies in primary screening and lead optimization. For instance, a number of different peptide and/or protein substrates can be easily tested to identify the optimal acceptor for a target protein kinase.
Profiling hits from a primary screen across a diverse panel of kinases to assess selectivity is also greatly simplified by the generic nature of the method. The Transcreener Kinase Assay does not have the ATP concentration limitations inherent in some other kinase assay methods, so ATP can be matched to the Km of the enzyme of interest.
By providing a single method and output for all primary and secondary kinase screening functions the Transcreener platform has the potential to enable a more integrated and iterative approach to kinase drug discovery.
Like kinases, ATPases catalyze a phosphoryl transfer reaction using ATP as a donor, resulting in the production of ADP. ATPases differ in that they transfer the gamma phosphate of ATP to water rather than a biomolecule. ADP detection with the Transcreener reagents provide a homogenous, fluorescent assay method for ATPases, shown in Figure 3 for the chaperonins HSC70 and HSP90.
The method is perhaps even more enabling for screening ATPases than for kinases, because there are far fewer alternative assay methods. Colorimetric phosphate detection is the default method, and it is highly susceptible to interference from colored compounds common in screening libraries.