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

Significance Many drugs trigger changes to the structure of their target receptor upon binding. These conformational effects are thought to be an essential part of molecular recognition but have proven challenging to quantify. Using a high-throughput method for tracking structural changes in a protein kinase in solution, we discovered that many clinically important cancer drugs trigger substantial structural changes to their target protein kinase Aurora A, and that these effects systematically account for the ability of the drugs to differentiate between different biochemical forms of Aurora A. The results provide insight into mechanisms of drug selectivity and suggest strategies for tailoring inhibitors to target certain cancers in which Aurora A has been dysregulated in different ways.
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.