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Both first-generation αC-out and newer αC-in RAF inhibitors paradoxically activate BRAF kinase at subsaturating concentrations. Paradoxical activation by αC-in inhibitors is linked to the formation of BRAF dimers, but why activation rather than inhibition occurs remains unclear. We used biophysical methods tracking BRAF conformation and dimerization combined with thermodynamic modeling to define the allosteric coupling mechanism underlying paradoxical activation. Allosteric coupling between αC-in inhibitors and BRAF dimerization is both extremely strong and highly asymmetric, with the first inhibitor contributing the bulk of dimer promotion. This asymmetric allosteric coupling mechanism results in the induction of dimers in which only one protomer is inhibited while the other is activated. The type II class of RAF inhibitors currently in clinical trials are more asymmetrically coupled and possess greater activation potential than older type I inhibitors.19F NMR data demonstrate that the BRAF dimer displays dynamic conformational asymmetry, with only a subset of protomers locked in the αC-in state, explaining how the conformational effects of drug binding can efficiently drive BRAF dimerization and activation at substoichiometric concentrations.

Cyclin-dependent kinases (CDKs) are the master regulators of the eukaryotic cell cycle. To become activated, CDKs require both regulatory phosphorylation and binding of a cognate cyclin subunit. We studied the activation process of the G1/S kinase Cdk2 in solution and developed a thermodynamic model that describes the allosteric coupling between regulatory phosphorylation, cyclin binding, and inhibitor binding. The results explain why monomeric Cdk2 lacks activity despite sampling an active-like state, reveal that regulatory phosphorylation enhances allosteric coupling with the cyclin subunit, and show that this coupling underlies differential recognition of Cdk2 and Cdk4 inhibitors. We identify an allosteric hub that has diverged between Cdk2 and Cdk4 and show that this hub controls the strength of allosteric coupling. The altered allosteric wiring of Cdk4 leads to compromised activity toward generic peptide substrates, and comparative specialization toward its primary substrate Retinoblastoma (RB).

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.