Targeting the protein–protein interaction between the CDC37 cochaperone and client kinases by an allosteric RAF dimer breaker

CDC37, a selectivity cochaperone in the heat shock protein (HSP90) chaperone machinery, plays a crucial role in facilitating recognition of client kinases and aiding their folding and maturation. RAF kinases, central components of the mitogen-activated protein kinase signaling pathway, rely on their interaction with CDC37 for stability and function. The RAF dimer interface, a key determinant of RAF kinase activity, overlaps with the CDC37-kinase client recognition motif known as the αC helix-β4 loop region. Here, we report that braftide, a peptide originally designed as a potent allosteric RAF kinase dimer disruptor, also triggers proteasome-mediated degradation of RAF kinases through a previously unclear mechanism. This study elucidates the mechanism underlying braftide's dual functionality and evaluates the potential of targeting kinase-chaperone interactions in cancer cell lines. Using coimmunoprecipitation 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 dynamics simulations, and in vitro crosslinking analyses, we mapped braftide's binding region within the BRAF kinase domain and identified the CDC37 region implicated in client kinase association. Disruption of this interaction 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 CDC37-RAF complexes and HSP90. Our work identifies the αC helix-β4 loop as a novel allosteric site for targeting kinase-chaperone interactions and demonstrates the feasibility of disrupting the CDC37-client kinase interaction as an innovative therapeutic strategy.