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Sci Adv. 2019 Aug 14;5(8):eaav8463. doi: 10.1126/sciadv.aav8463. eCollection 2019 Aug.

BRAF inhibitors promote intermediate BRAF(V600E) conformations and binary interactions with activated RAS.

Author information

1
Institute of Biochemistry and Center for Molecular Biosciences, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria.
2
Bioinformatics Institute (BII), Agency for Science Technology and Research (ASTAR), Singapore 138671, Singapore.
3
Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine and Laura and Isaac Perlmutter Cancer Center, New York University Langone Medical Center, New York, NY 10016, USA.
4
Department of Pharmacology, Department of Chemistry and Biochemistry, and Howard Hughes Medical Institute, University of California, San Diego, San Diego, CA 92093, USA.
5
Daniel Swarovski Research Laboratory, Department of Visceral, Transplant and Thoracic Surgery, Innsbruck Medical University, Innrain 66, 6020 Innsbruck, Austria.

Abstract

Oncogenic BRAF mutations initiate tumor formation by unleashing the autoinhibited kinase conformation and promoting RAS-decoupled proliferative RAF-MEK-ERK signaling. We have engineered luciferase-based biosensors to systematically track full-length BRAF conformations and interactions affected by tumorigenic kinase mutations and GTP loading of RAS. Binding of structurally diverse αC-helix-OUT BRAF inhibitors (BRAFi) showed differences in specificity and efficacy by shifting patient mutation-containing BRAF reporters from the definitive opened to more closed conformations. Unexpectedly, BRAFi engagement with the catalytic pocket of V600E-mutated BRAF stabilized an intermediate and inactive kinase conformation that enhanced binary RAS:RAF interactions, also independently of RAF dimerization in melanoma cells. We present evidence that the interference with RAS interactions and nanoclustering antagonizes the sequential formation of drug-induced RAS:RAF tetramers. This suggests a previously unappreciated allosteric effect of anticancer drug-driven intramolecular communication between the kinase and RAS-binding domains of mutated BRAF, which may further promote paradoxical kinase activation and drug resistance mechanisms.

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