The central role of protein kinases in signal transduction and recent clinical success with small molecule kinase inhibitor drugs, most notably Gleevec (Novartis), have generated intense interest in kinase enzymes as therapeutic targets.1 Next to G-protein coupled receptors, protein kinases have become the most intensively screened target class.2
However, the ability to validate and pursue new kinases for drug discovery is being hampered by a lack of screening assays capable of accommodating diverse kinase isoforms and their corresponding acceptor substrates. Most kinase assay methods rely on detection of a tagged phosphopeptide, and significant development or optimization is required to accommodate each new peptide sequence recognized by a particular kinase.
Though there is overlap in substrate specificity among related kinases, there is no consensus sequence that is phosphorylated by a large number of kinases.3 The time and money required to develop assays for kinases with novel recognition sequences is slowing the identification of selective inhibitors for validating new kinase targets or as potential lead molecules (Figure 1).
Moreover, new therapeutic strategies are resulting in increased demand for kinase screening assays that can accommodate nonpeptide acceptors. Examples include:
Non protein substrates (lipid kinases)
The assay development bottleneck extends further downstream in the discovery process as well.
At some point following a primary screen, secondary screens are often run against a panel of 10100 kinases to obtain selectivity profiles for the most promising hits. These profiling studies generally include potency determinations for compounds where significant off-target activity is observed. The available nonradioactive HTS assay methods are not suitable for quantitative assays with a broad spectrum or kinases, and studies have shown that there are significant differences in the identity and potency of hits determined with different HTS assays methods.4
For these reasons, the bulk of selectivity profiling is done using the traditional 33P-radioassays. Most pharmas contract this work, at considerable expense and inconvenience, to service providers who are willing to assume the regulatory and disposal requirements.