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

Proc Natl Acad Sci U S A. 2018 Dec 18;115(51):E11894-E11903. doi: 10.1073/pnas.1811158115. Epub 2018 Dec 5.


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.

Keywords: Aurora inhibitors; DFG motif; conformational selectivity; protein kinases.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Allosteric Regulation
  • Amino Acid Motifs
  • Aurora Kinase A / drug effects*
  • Aurora Kinase A / metabolism*
  • Binding Sites
  • Cell Cycle Proteins / metabolism
  • Cell Division
  • Crystallography, X-Ray
  • Humans
  • Microtubule-Associated Proteins / metabolism
  • Models, Molecular
  • Molecular Docking Simulation
  • Molecular Dynamics Simulation
  • Nuclear Proteins / metabolism
  • Oligopeptides
  • Phosphorylation
  • Protein Binding
  • Protein Conformation*
  • Protein Interaction Domains and Motifs*
  • Protein Kinase Inhibitors / pharmacology*


  • Cell Cycle Proteins
  • DFG peptide
  • Microtubule-Associated Proteins
  • Nuclear Proteins
  • Oligopeptides
  • Protein Kinase Inhibitors
  • TPX2 protein, human
  • Aurora Kinase A