Abstract
Braftide, originally designed as a potent allosteric RAF kinase dimer disruptor, was intended to inhibit RAF dimerization by targeting the conserved RAF dimer interface. Intriguingly, Braftide has also been observed to trigger proteasome-mediated protein degradation with an unclear mechanism of action. This study elucidates the mechanism underlying Braftide's dual functionality and assesses its potential as a chemical probe to target kinase-chaperone interaction. CDC37, a selectivity co-chaperone in the HSP90 chaperone machinery, plays a crucial role in facilitating the recognition of client kinase. The RAF dimer interface overlaps with the CDC37-kinase client recognition motif, known as the αC helix-β4 loop. Using co-immunoprecipitation and NanoBiT assays, we confirmed Braftide's ability to selectively disrupt the CDC37-client kinase interaction while sparing HSP90. Through deuterium exchange mass spectrometry, molecular dynamic simulations, and in vitro crosslinking analyses, we mapped Braftide's binding region within the BRAF kinase domain, as well as the CDC37 region implicated in the association of CDC37-client kinase complex. Consequently, this disruption destabilizes RAF kinase clients, resulting in proteasomal degradation, reduced cellular proliferation, and increased apoptosis in cancer cell lines. Furthermore, Braftide exhibits synergy with HSP90 inhibitors, jointly destabilizing both the CDC37-RAF complex and HSP90. Our work demonstrates the feasibility of disrupting the CDC37-client kinase interaction as an innovative therapeutic strategy and identifies the αC helix-β4 loop as a novel allosteric site with significant potential for the development of next-generation therapeutics.