Evangelia Matenoglou, MD
Albert Einstein College of Medicine
Hinge-Targeting BRAF Inhibitors with Prolonged Residence Time for Durable Suppression of MAPK Signaling
Resistance to current RAF inhibitors limits their efficacy in tumors driven by RAF dimers, such as p61BRAFV600E and non-V600E BRAF mutants. We developed a hinge-targeting hybrid inhibitor strategy to enhance drug–target residence time and improve selectivity for dimeric RAF species. Guided by structural insights of established RAF inhibitors, we engineered K-tail hybrid inhibitors by fusing BRAF hinge region–binding elements to distinct ATP-competitive kinase scaffolds. K-tail hybrid inhibitors showed markedly prolonged residence time, sustained ERK suppression, and superior potency compared to approved RAF inhibitors in biochemical kinase assays, cellular washout experiments, and antiproliferative studies. Notably, these inhibitors demonstrated enhanced activity in cancer cell models driven by RAF dimer signaling, including BRAF class II and III mutant cell lines as well as RAS-mutant/RAF wild-type contexts. Co-crystal structures and molecular dynamics simulations revealed that hinge anchoring stabilizes asymmetric inhibitor conformations through direct and water-mediated hydrogen-bond networks, providing a mechanistic basis for the observed kinetic advantage. In BRAFV600E melanoma xenografts, a representative K-tail inhibitor, DABK, achieved durable target engagement, extended MAPK pathway inhibition, and enhanced anti-tumor activity relative to FDA-approved Dabrafenib. These findings establish hinge-targeting hybrid RAF inhibitors as a rational chemical strategy to increase drug–target residence time and broaden therapeutic opportunities for tumors driven by non-V600E BRAF mutations or RAS-dependent RAF signaling.
Resistance to current RAF inhibitors limits their efficacy in tumors driven by RAF dimers, such as p61BRAFV600E and non-V600E BRAF mutants. We developed a hinge-targeting hybrid inhibitor strategy to enhance drug–target residence time and improve selectivity for dimeric RAF species. Guided by structural insights of established RAF inhibitors, we engineered K-tail hybrid inhibitors by fusing BRAF hinge region–binding elements to distinct ATP-competitive kinase scaffolds. K-tail hybrid inhibitors showed markedly prolonged residence time, sustained ERK suppression, and superior potency compared to approved RAF inhibitors in biochemical kinase assays, cellular washout experiments, and antiproliferative studies. Notably, these inhibitors demonstrated enhanced activity in cancer cell models driven by RAF dimer signaling, including BRAF class II and III mutant cell lines as well as RAS-mutant/RAF wild-type contexts. Co-crystal structures and molecular dynamics simulations revealed that hinge anchoring stabilizes asymmetric inhibitor conformations through direct and water-mediated hydrogen-bond networks, providing a mechanistic basis for the observed kinetic advantage. In BRAFV600E melanoma xenografts, a representative K-tail inhibitor, DABK, achieved durable target engagement, extended MAPK pathway inhibition, and enhanced anti-tumor activity relative to FDA-approved Dabrafenib. These findings establish hinge-targeting hybrid RAF inhibitors as a rational chemical strategy to increase drug–target residence time and broaden therapeutic opportunities for tumors driven by non-V600E BRAF mutations or RAS-dependent RAF signaling.