Allosteric kinase inhibitors are highly sought after as cancer drugs to override resistance to conventional kinase inhibitors, a major clinical problem that severely limits the duration of response to many front-line cancer therapies. Unfortunately, most existing high-throughput screening assays used in kinase drug discovery cannot distinguish allosteric from conventional inhibitors, and are in fact biased towards identifying conventional ATP- competitive inhibitors due to the reliance on phosphorylated and catalytically active kinases. Consequently, allosteric inhibitors are not available for most kinase drug targets, and new technologies are sorely needed to streamline the development of allosteric inhibitors and bring this important new class of drugs to the clinic. In this proposal we develop a cutting-edge high-throughput screening technology for allosteric inhibitor discovery. The technology combines an allostery biosensor based on time-resolved FRET, which tracks allosteric structural changes in the kinase drug target with angstrom-level precision, with new state-of-the-art fluorescence lifetime plate readers, which enable lifetime measurements in 384- and 1536-well format with exceptional speed and precision. Together these capabilities allow for the direct identification of compounds in large chemical libraries that induce specific allosteric changes in the drug target. Here we apply this assay technology to the protein kinase Aurora A (AurA), an important driver of mitosis and a major cancer drug target for which allosteric inhibitors are badly needed but are not currently available. We use time-resolved FRET data obtained in high-throughput format to construct a comprehensive structural map of inhibitor- and activator-induced allosteric movements in AurA, calibrating the FRET readout and setting the stage for the identification of compounds with specific allosteric modes of action. We then translate the assay into a high-throughput platform for identifying genuine allosteric inhibitors that bind outside the conserved ATP site. The successful completion of this R21 project will provide the technology platform for a subsequent R33 project to identify genuine allosteric inhibitors of AurA that disrupt the oncogenic c-Myc:AurA complex or selectively target other specific cellular subpopulations of AurA implicated in cancer. Because our assay technology tracks allosteric movements of the activation loop, a central regulatory element in all protein kinases, the technology is readily adaptable to other kinase drug targets. It is thus likely that the success of this project would lead to rapid implementation for numerous other systems and could have a broad long-term impact on allosteric drug discovery and cancer treatment.
Protein kinase inhibitors are important cancer drugs, but current inhibitors suffer from poor selectivity and rapid emergence of clinical resistance. The goal of this project is to provide the technology to streamline the development of more sophisticated inhibitors that work by inducing specific structural changes that block activity. Such allosteric inhibitors are much more selective that conventional kinase inhibitors, can be used as combination therapies to override clinical resistance, and would be invaluable for treating the many forms of cancer that are driven by unrestrained kinase activity.
| Levinson, Nicholas Mark (2018) The multifaceted allosteric regulation of Aurora kinase A. Biochem J 475:2025-2042 |
| Lake, Eric W; Muretta, Joseph M; Thompson, Andrew R et al. (2018) Quantitative conformational profiling of kinase inhibitors reveals origins of selectivity for Aurora kinase activation states. Proc Natl Acad Sci U S A 115:E11894-E11903 |
| Ruff, Emily F; Muretta, Joseph M; Thompson, Andrew R et al. (2018) A dynamic mechanism for allosteric activation of Aurora kinase A by activation loop phosphorylation. Elife 7: |
