The protein kinases are the top class of drug targets for the development of new cancer therapeutics. Existing kinase inhibitors, which target the highly-conserved active sites of kinases, have major limitations including poor selectivity and a high incidence of clinical resistance leading to treatment failure. New allosteric inhibitors, which bind in other pockets outside the kinase active site and trigger structural changes that block kinase activity, are far more selective and are highly effective at overriding clinical resistance to conventional kinase inhibitors. However, allosteric kinase inhibitors have proven extremely challenging to identify with existing drug screening technologies, and are only available for a small handful of kinases. A major reason for this failure of existing drug screening technologies is that they cannot detect the atomic- scale structural changes that define the mode of action of allosteric kinase inhibitors. We have developed a game-changing high-throughput screening technology, based on nanosecond time-resolved fluorescence, that can identify allosteric inhibitors by tracking with atomic resolution the structural changes they trigger in the kinase drug target. Applying this technology to the mitotic protein kinase Aurora A, we have shown that it can simultaneously track inhibitor binding affinity and allosteric effects on the kinase, can classify inhibitors into different allosteric subtypes, and is sufficiently accurate, rapid and scalable to handle high-throughput screening projects. To maximize the impact of the technology on the drug discovery pipeline, several technical barriers need to be surmounted to expand the scope of the technology beyond the current single drug target Aurora A. Our current technology is based on a chemical labeling procedure for incorporating fluorescent probes, cysteine labeling, that is not readily applicable to many important kinase drug targets due to the presence of cysteine residues important for structural integrity and catalytic function. In this proposal, we broaden the scope of the technology to make it applicable to the majority of the ~500 human kinases by developing a series of new tools for site-specific probe incorporation and by expanding the range and type of small molecules that can be identified in screening. Finally, we benchmark the suitability of the technology for real-world drug discovery efforts by performing a high-throughput screening project to identify novel allosteric inhibitors of at least one protein kinase for which no allosteric inhibitors are currently available. The success of this project will bring an entirely-new allosteric drug discovery technology into being, with unique capabilities that no existing technology can provide. Employment of this approach could jumpstart the discovery of allosteric kinase inhibitors for a large number of important cancer drug targets, broadening the range of therapeutic options for cancer patients and providing a much-needed new approach for combating the high prevalence of clinical resistance to first-line kinase inhibitor therapies.

Public Health Relevance

Protein kinases are the targets of the majority of new cancer drug development, but existing ATP-competitive inhibitors have major limitations, and next-generation allosteric inhibitors are challenging to identify with existing screening technologies. This project develops a cutting-edge new technology for identifying allosteric inhibitors by tracking the structural changes they trigger in the target kinase with atomic resolution, and broadens the scope of this new technology to make it applicable to the majority of human kinases.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Exploratory/Developmental Grants Phase II (R33)
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Special Emphasis Panel (ZCA1)
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Mckee, Tawnya C
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University of Minnesota Twin Cities
Schools of Medicine
United States
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