Quantitative conformational profiling of kinase inhibitors reveals origins of selectivity for Aurora kinase activation states

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 115; no. 51; pp. E11894 - E11903
• Main Authors: Lake, Eric W., Muretta, Joseph M., Thompson, Andrew R., Rasmussen, Damien M., Majumdar, Abir, Faber, Erik B., Ruff, Emily F., Thomas, David D., Levinson, Nicholas M.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 18.12.2018
• Series: PNAS Plus
• Subjects: Activation; Allosteric properties; Aurora kinase; Binding; Biological Sciences; Fluorescence; Inhibitor drugs; Inhibitors; Kinases; Melanoma; Neuroblastoma; Phosphorylation; Physical Sciences; PNAS Plus; Prostate cancer; Proteins; Selectivity; Tracks (paths); protein kinases; Aurora inhibitors; conformational selectivity; DFG motif
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1811158115

Protein kinases undergo large-scale structural changes that tightly regulate function and control recognition by small-molecule inhibitors. Methods for quantifying the conformational effects of inhibitors and linking them to an understanding of selectivity patterns have long been elusive. We have developed an ultrafast time-resolved fluorescence methodology that tracks structural movements of the kinase activation loop in solution with angstrom-level precision, and can resolve multiple structural states and quantify conformational shifts between states. Profiling a panel of clinically relevant Aurora kinase inhibitors against the mitotic kinase Aurora A revealed a wide range of conformational preferences, with all inhibitors promoting either the active DFG-in state or the inactive DFG-out state, but to widely differing extents. Remarkably, these conformational preferences explain broad patterns of inhibitor selectivity across different activation states of Aurora A, with DFG-out inhibitors preferentially binding Aurora A activated by phosphorylation on the activation loop, which dynamically samples the DFG-out state, and DFG-in inhibitors binding preferentially to Aurora A constrained in the DFG-in state by its allosteric activator Tpx2. The results suggest that many inhibitors currently in clinical development may be capable of differentiating between Aurora A signaling pathways implicated in normal mitotic control and in melanoma, neuroblastoma, and prostate cancer. The technology is applicable to a wide range of clinically important kinases and could provide a wealth of valuable structure–activity information for the development of inhibitors that exploit differences in conformational dynamics to achieve enhanced selectivity.