Abstract A057: Hinge-targeting BRAF inhibitors with prolonged residence time for durable suppression of MAPK signaling

Abstract Resistance to RAF inhibitors remains a major limitation in the treatment of RAF-driven malignancies, particularly those involving dimeric RAF species such as p61BRAFV600E and non-V600E BRAF mutants. While current ATP-competitive inhibitors like Dabrafenib and Vemurafenib are effective against BRAFV600E monomers, their inability to inhibit RAF dimers contribute to therapeutic failure. We have developed and validated a novel chemical strategy to enhance drug-target residence time via hinge-targeting hybrid kinase inhibitors, leading to improved efficacy and kinetic selectivity. Using Encorafenib, a BRAFV600E inhibitor with known durable MAPK suppression, as a starting point, we elucidated the structural and kinetic basis of its prolonged on-target engagement. Co-crystal structures of Encorafenib bound to BRAFV600E revealed a dynamic inhibitor conformation forming water-mediated interactions with hinge residues. Guided by this insight, we engineered a new class of hybrid RAF inhibitors by rational fusion of hinge-binding groups to the ATP-binding core of Dabrafenib, generating the lead compound DAB-K1. DAB-K1 displayed dramatically prolonged residence time on both monomeric and dimeric BRAFV600E, as determined by TR-FRET–based kinase assays and cellular washout experiments and maintained longer ERK suppression compared to Dabrafenib and Vemurafenib. Notably, DAB-K1 analogs demonstrated improved potency in cancer cell lines harboring BRAF class II and III mutations and in RAF WT cells dependent on RAF dimer signaling. Co-crystal structures of the BRAFV600E–DAB-K1 complex confirmed that the hybrid inhibitor preserves canonical ATP-pocket engagement while introducing asymmetric conformations of hinge-anchoring groups stabilized through a network of direct and water-mediated hydrogen bonds. Molecular dynamics simulations further highlighted hinge flexibility and dynamic interactions that likely underlie the prolonged residence time. In A375 BRAFV600E melanoma xenograft models, pharmacodynamic studies demonstrated sustained ERK pathway inhibition in tumors up to 24 hours post-dose, with superior target occupancy compared to Dabrafenib, supported by favorable pharmacokinetic properties. DAB-K1 also exhibited enhanced anti-tumor activity, as reflected by delayed tumor progression and extended suppression of MAPK signaling markers. Importantly, structural modeling and docking studies showed that hinge-anchoring inhibitors effectively engage hinge residues by clinically resistance-associated mutations in ARAFG387D and CRAFG426D dimers, suggesting a rational strategy to overcome resistance to current RAF-targeted therapies. Overall, our findings provide a mechanistic and translational foundation for the development of next-generation RAF inhibitors with hinge-targeting properties to overcome dimer-driven resistance mechanisms commonly observed in non-V600E BRAF mutant and RAF wild-type tumors. Citation Format: Evangelia Matenoglou, Bogos Agianian, Joanna Elise. Luo, Nadege Gitego, Andrea Lopez, Lars Ulrik. Nordstrøm, Evripidis Gavathiotis. Hinge-targeting BRAF inhibitors with prolonged residence time for durable suppression of MAPK signaling [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr A057.