Mechanism and inhibition of BRAF kinase

Abstract

The role of BRAF in tumor initiation has been established, however, the precise mechanism of autoinhibition has only been illustrated recently by several structural studies. These structures uncovered the basis by which the regulatory domains engage in regulating the activity of BRAF kinase domain, which lead to a more complete picture of the regulation cycle of RAF kinases. Small molecule BRAF inhibitors developed specifically to target BRAF^V600E have proven effective at inhibiting the most dominant BRAF mutant in melanomas, but are less potent against other BRAF mutants in RAS-driven diseases due to paradoxical activation of the MAPK pathway. A variety of new generation inhibitors that do not show paradoxical activation have been developed. Alternatively, efforts have begun to develop inhibitors targeting the dimer interface of BRAF. A deeper understanding of BRAF regulation together with more diverse BRAF inhibitors will be beneficial for drug development in RAF or RASdriven cancers.

Keywords: 14-3-3; Allosteric kinase inhibitor; Autoinhibition; Paradoxical activation; RAF kinase; RAS; RAS-RAF interaction.

Figure 1. Conserved regions of the RAF family.

The RAF isoforms indicating the conserved regions (CR): CR1 (orange), CR2 (grey), and CR3 (blue). Labeled within the three regions are RAS-binding domain (RBD)/cysteine-rich domain (CRD), serine/threonine rich domain (S/T Rich), and kinase domain contained in CR1/CR2/CR3, respectively. On BRAF, there is a BRAF-specific region (red) located at the N-terminus. The amino acid numbers of the beginning and end of the RAF isoform and CR are shown.

Figure 2. Cryo-EM structures of BRAF.

(a) The cryo-EM structure of a dimeric B-Raf:14-3-3 complex (PDB ID: 6UAN); (b) the cryo-EM structure of autoinhibited BRAF:14:3:3:MEK1 complex (PDB ID: 6NYB); (c) the cryo-EM structure of autoinhibited BRAF:14-3-3:MEK1 complex with RBD visible (PDB ID: 6MFD).

Figure 3. RAF activation mechanism.

(a) Monomeric inactive state. When RAS exists in the GDP-bound inactive form (light gray), RAF is found in the cytosol as an autoinhibited inactive monomer with the CRD (green) binding to the CR3 (gray, kinase-domain). GDP-bound RAS (light gray) is inactive. (b) Dimeric active state. GTP-bound active RAS (magenta) binds to the RBD (orange) and CRD and recruits the RAF to plasma membrane, leading to the release of CRD from the dimer interface of CR3. RAS nano-clustering brings RAF molecules into close proximity for dimerization and activation of CR3 (blue). Both the autoinhibitory interaction and kinase domain dimerization are facilitated by 14-3-3 binding to the CR2 and the C-terminal tail of RAF, respectively. For clarity, 14-3-3 proteins and the C-terminal tail are not included to the schematic diagram.

Figure 4

Chemical structures of representative RAF inhibitors.