Over the past decade, small molecule kinase inhibitors have emerged as promising therapeutics, particularly in oncology. Through data that has emerged during this period, it has become increasingly apparent that small molecule inhibitors interact differently with the various conformational and activation states of a protein kinase.
For example, the FDA-approved kinase inhibitors Gleevec®, Nexavar®, and Tykerb® preferentially interact with nonactive kinase conformations, and such modes of binding can lead to increased specificity due to compound interactions with less-conserved residues that are accessible only in nonactive forms of the kinase. In other cases, the preferential binding of small molecule inhibitors to nonactive forms of a kinase can be accompanied by slow compound “off rates”. Slower off rates can lead to prolonged biological effects, which in turn can boost the overall efficacy of the inhibitor (an observation reported for Tykerb).
Challenge of Traditional Assays
An understanding of the diverse binding modes of kinase inhibitors and their effects on biological systems can provide researchers with important clues regarding the effective design of new drugs.
At this time, however, efficient, broadly applicable high-throughput screening (HTS) analysis of kinase inhibitors is primarily achieved through kinase activity assays. Activity assays, by their very nature, are only useful for measuring inhibition of active kinase preparations, which means that analysis is limited to active targets for which suitable in vitro substrates have been identified.
An alternative to the activity assay involves directly measuring the binding of small molecules to the kinase under investigation. Such binding assays can be used to study kinases prepared both in active and nonactive states. This allows the researcher the freedom to choose the specific kinase state to be targeted in HTS campaigns. Moreover, the mechanisms of newly identified inhibitors can be evaluated by measuring binding characteristics to multiple states of the kinase target.