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， w... More
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.
