BRAF inhibitors promote intermediate BRAF(V600E) conformations and binary interactions with activated RAS

Oncogenic BRaf mutations initiate tumor formation by unleashing the autoinhibited kinase conformation and promoting RAS-decoupled proliferative RAF-MEK-ERK signaling. We have engineered luciferase-based biosensors to systematically track full-length BRaf conformations and interactions affected by tumorigenic kinase mutations and GTP loading of Ras. Binding of structurally diverse αC-helix-OUT BRaf inhibitors (BRAFi) showed differences in specificity and efficacy by shifting patient mutation-containing BRaf reporters from the definitive opened to more closed conformations. Unexpectedly, BRAFi engagement with the catalytic pocket of V600E-mutated BRaf stabilized an intermediate and inactive kinase conformation that enhanced binary RAS:RAF interactions, also independently of Raf dimerization in melanoma cells. We present evidence that the interference with Ras interactions and nanoclustering antagonizes the sequential formation of drug-induced RAS:RAF tetramers. This suggests a previously unappreciated allosteric effect of Anticancer drug-driven intramolecular communication between the kinase and RAS-binding domains of mutated BRaf, which may further promote paradoxical kinase activation and drug resistance mechanisms.