Clayton, J.; Romany, A.; Matenoglou, E.; Gavathiotis, E.; Poulikakos, P. I.; Shen, J.

Aberrant signaling of BRAF(V600E) is a major cancer driver. Current FDA-approved RAF inhibitors selectively inhibit the monomeric BRAF(V600E) and suffer from tumor resistance. Recently, dimer-selective and equipotent RAF inhibitors have been developed; however, the mechanism of dimer selectivity is poorly understood. Here, we report extensive molecular dynamics (MD) simulations of the monomeric and dimeric BRAF(V600E) in the apo form or in complex with one or two dimer-selective (PHI1) or equipotent (LY3009120) inhibitor(s). The simulations uncovered the unprecedented details of the remarkable allostery in BRAF(V600E) dimerization and inhibitor binding. Specifically, dimerization retrains and shifts the alphaC helix inward and increases the flexibility of the DFG motif; dimer compatibility is due to the promotion of the alphaC-in conformation, which is stabilized by a hydrogen bond formation between the inhibitor and the alphaC Glu501. A more stable hydrogen bond further restrains and shifts the alphaC helix inward, which incurs a larger entropic penalty that disfavors monomer binding. This mechanism led us to propose an empirical way based on the co-crystal structure to assess the dimer selectivity of a BRAF(V600E) inhibitor. Simulations also revealed that the positive cooperativity of PHI1 is due to its ability to preorganize the alphaC and DFG conformation in the opposite protomer, priming it for binding the second inhibitor. The atomically detailed view of the interplay between BRAF dimerization and inhibitor allostery as well as cooperativity has implications for understanding kinase signaling and contributes to the design of protomer selective RAF inhibitors.