Development of Allosteric BRAF Peptide Inhibitors Targeting the Dimer Interface of BRAF

Abstract

BRAF is the most frequently mutated kinase in human cancers and is one of the major effectors of oncogenic RAS, making BRAF a target of considerable interest for anti-cancer drug development. Wild-type BRAF and a variety of oncogenic BRAF mutants are dependent on dimerization of the kinase domain, which also emerges as a culprit of drug resistance and side effects of current BRAF therapies. Thus, allosteric BRAF inhibitors capable of disrupting BRAF dimers could abrogate hyperactivated MAPK (mitogen-activated protein kinase) signaling driven by oncogenic BRAF or RAS and overcome the major limitations of current BRAF inhibitors. To establish this, we applied an in silico approach to design a series of peptide inhibitors targeting the dimer interface of BRAF. One resulting inhibitor was found to potently inhibit the kinase activity of BRAF homo- and heterodimers, including oncogenic BRAFG469A mutant. Moreover, this inhibitor synergizes with FDA-approved, ATP-competitive BRAF inhibitors against dimeric BRAF, suggesting that allosteric BRAF inhibitors have great potential to extend the application of current BRAF therapies. Additionally, targeting the dimer interface of BRAF kinase leads to protein degradation of both RAF and MEK, uncovering a novel scaffolding function of RAF in protecting large MAPK complexes from protein degradation. In conclusion, we have developed a potent lead peptide inhibitor for targeting the dimer interface of BRAF in cancer cells. The dual function of this peptide inhibitor validates the strategy for developing allosteric BRAF inhibitors that specifically dissociate RAF dimers and destabilize the MAPK signaling complex.

Drug resistance in targeted cancer therapies with RAF inhibitors

“…In particular, given the unique activating and catalytic mechanisms of RAF family kinases, targeting RAF/RAF and RAF/MEK interactions has become an attractive strategy for designing novel RAF inhibitors [76] . Although no small molecular inhibitors that target RAF/RAF dimer interface have been developed at present, several groups have demonstrated that disrupting RAF/RAF dimers by using peptide inhibitors can effectively block hyperactive ERK signaling and thereby inhibit the growth of cancer cells harboring active RAF or RAS mutations [177][178][179] , indicating that the RAF/RAF dimer interface is indeed a valid target for drug development. As for RAF/MEK interaction, it is dynamically altered in the process of MEK phosphorylation by RAF, as described above.…”

RAS Dimers: The Novice Couple at the RAS-ERK Pathway Ball

“…Structural analyses have unveiled that RAF dimerizes through its catalytic domain, forming "side to side" dimers, and mutations in critical residues therein impede RAFdependent MEK phosphorylation [29]. Likewise, peptides targeting the dimerization interface induced cell death in RAS-mutant tumor cells [30]. As such, RAF dimerization emerged as a promising target for therapeutic intervention-more so when it was found that such a process underlies the acquisition of resistance to BRAF kinase inhibitors in tumors harboring wild-type BRAF [31,32].…”

“…Structurally, RAS proteins consist of identical N-terminal (residues 1-86) and C-terminal lobes (residues 87-171) with an 80% overall homology. The N-terminal lobe is considered to be the effector lobe, since it accommodates the P-loop (residues 10-14), switch I (residues [30][31][32][33][34][35][36][37][38], and switch II (residues 60-76) regions, which are responsible for effectors, GAPs, and GEFs binding. Both switches I and II are close to the GTP binding site, and the activation status of the protein implies a conformational change that determines the binding to effectors and regulatory proteins and also implies reorientation relative to the membrane [14][15][16].…”

Discovery of a novel lactate dehydrogenase tetramerization domain using epitope mapping and peptides

“…16 Moreover, disruptors of protein self-assembly can induce protein misfolding and degradation. 38,39 Therefore, targeting the LDH oligomeric state could reduce its intracellular concentration, leading to sub-stoichiometric inhibition, hence higher efficacy.…”