Fig 2: Braftide disrupts RAF dimers and the CDC37–RAF client kinase complex.A, braftide binds to the dimer interface of RAF to dissociate RAF dimers. B, braftide binds to CDC37 to disrupt the cochaperone CDC37 with client kinases. Braftide treatment increases the ubiquitination of RAF, which leads to proteasomal-mediated degradation.

Fig 3: Braftide binds to CDC37 and RAF kinases and induces proteasomal degradation of the client kinase via the ubiquitin–proteasome pathway.A and B, cartoon schematic of photo-activated crosslinking (XLINK) strategy of biotinylated (BTN)–braftide (B) with BRAF. Crosslinked BTN–braftide complexes are coimmunoprecipitated via streptavidin magnetic resin for further analysis. C, representative immunoblot of overexpressed BRAF-V5 and CDC37-FLAG in HEK293 cells in the absence and presence of 10 μM BTN–braftide and then immunoprecipitated and probed for individual proteins. D and E, representative immunoblot (D) and densitometry (E) of overexpressed MBP-CRAF-FLAG immunoprecipitated and probed for ubiquitination in HEK293 cells in the absence and presence of 50 μM braftide. At least three independent biological replicates were performed for each experiment. F and G, braftide-mediated CDC37 disruption triggers apoptosis in HEK293 (F) cells and HCT116 (G) cells treated with indicated concentrations of braftide for 4 h. Cells were stained with Annexin V, an apoptosis marker, and sorted via flow cytometry (n = 3). HEK293, human embryonic kidney 293 cell line.

Fig 4: Braftide disrupts the interaction between the client RAF kinase and the CDC37 cochaperone.A–D, BRAF-FLAG (A and B)/MBP-CRAF-FLAG (C and D) exogenously expressed in HEK293 cells in the absence and presence of 50 μM braftide treatment (4 h). Representative immunoblot of immunoprecipitated (IP) B/CRAF-FLAG for the coimmunoprecipitated (co-IP) CDC37 and HSP90 chaperone complex. B and D, densitometry analysis of CDC37 and HSP90 normalized to immunoprecipitated BRAF (B)/CRAF (D) across three biological replicates. E–H, CDC37-FLAG coexpressed with BRAF-V5 (E and F) or CRAF-HA (G and H). Representative blot of three biological replicates of immunoprecipitated CDC37-FLAG for co-IP BRAF-V5 (E) or CRAF-HA (G) and HSP90 in the absence and presence of braftide treatment (25 μM) in HEK293 cells. I, schematic illustrating NanoBiT luminescence assay. J and L, NanoBiT assay in the absence and presence of braftide (25 μM, 4 h) in HEK293 cells expressing NanoBiT constructs of BRAFSmBiT–CDC37LgBiT (J) and CRAFSmBiT–CDC37LgBiT (L). K and M, representative immunoblot of HEK239 cells expressing BRAFSmBiT–CDC37LgBiT (K) and CRAFSmBiT–CDC37LgBiT (M) in the absence and presence of braftide. K–M, densitometry analysis of CDC37LgBiT and BRAFSmBiT (K) or CRAFSmBIT (M) normalized to no treatment control across three biological replicates. At least three independent biological replicates were performed for each experiment. HEK293, human embryonic kidney 293 cell line; HSP90, heat shock protein 90; LgBiT, large BiT; SmBiT, small BiT.