Fig. 6

Mode of action of activating RAF inhibitors and proposed characteristics of next-generation RAF inhibitors. a Model depicting the biological mode of action of RAF inhibitors in untreated (1 and 2; left) or inhibitor-treated cells (3 and 4; right). (1) In quiescent cells where RAS activity is low, RAF proteins are autoinhibited through an interaction between their N-terminal regulatory region (NTR) and their kinase domain (KD). Upon physiological RAS activation or in RAS-activated cancer cells (2), RAF proteins shuttle between an autoinhibited monomeric state in the cytoplasm and an activated dimeric state bound to RAS−GTP at the plasma membrane. At steady-state, ERK signaling is moderated by this ON/OFF equilibrium. (3) ON-state RAF inhibitors disrupt the normal NTR−KD interaction. In cells with low-RAS activity, derepressed RAF can dimerize but this process is not stabilized by active RAS and therefore does not lead to paradoxical pathway activation. (4) RAS activity and RAF inhibitors co-operatively promote RAS−RAF complex formation and RAF dimerization. On the one hand, NTR−KD disruption is triggered by two separate means (RAS-binding to the RBD and compound binding to the catalytic cleft) resulting in dissociation. On the other hand, the increase in RAF effective concentration when bound to RAS nanoclusters coupled with the closed/active-like kinase domain conformation stabilized by the inhibitors leads to increased side-to-side RAF dimerization. In turn, RAF dimerization further augments the population of RAS nanoclusters. Together, these events co-operatively drive RAS−RAF complex formation and RAF dimerization. At sub-saturating compound concentrations, this phenomenon leads to catalytic transactivation of the compound-free protomer. b Unbiased clustering of RAF inhibitors based on their effects on dimerization, NTR−KD interaction and RAS−RAF association (BRET EC₅₀s and YMAXs values served as clustering parameters). The EC₅₀s and YMAXs used for clustering were the average of three independent repeats (Supplementary Data 1). c Each class of RAF inhibitors is positioned along an axis from ON-state (red) to OFF-state compounds (blue). Ideal (OFF-state) RAF inhibitors would not allow kinase domain dimerization, but would stabilize the NTR−KD interaction, thereby maintaining RAF proteins in an autoinhibited state irrespective of the status of RAS activity