RAF isoform selectivity of MEK inhibitors and rational design of a covalent ARAF-MEK inhibitor

Aberrant activation of the RAS/RAF/MEK/ERK pathway is a frequent cause of cancer. Allosteric MEK inhibitors block this pathway by binding RAF-MEK complexes to prevent activation of MEK by RAF. However, how MEK inhibitor potency varies across the three RAF isoforms remains poorly understood. We profiled seven allosteric MEK inhibitors and observed a striking hierarchy of sensitivity: all most potently inhibited CRAF-driven MEK activation while relatively sparing ARAF-driven activation. We identified point mutations in ARAF and CRAF proximate to the MEK inhibitor binding site that markedly altered inhibitor sensitivity. Using a rational design approach, we developed a more potent inhibitor of ARAF-driven MEK signaling, TWG-07-148. Our cryo-EM structure shows how this acrylamide-containing analog of MEK inhibitor trametinib covalently targets Cys514, a residue unique to ARAF. Our studies highlight the importance of the activating RAF isoform as a determinant of MEK inhibitor sensitivity and provide proof-of-concept for development of MEK inhibitors that more effectively block ARAF-driven MEK signaling via covalent targeting of Cys514.

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Publication Details

Journal
bioRxiv (Cold Spring Harbor Laboratory)
Published
2025-12-17
DOI
https://doi.org/10.64898/2025.12.15.693503
Primary Topic
Melanoma and MAPK Pathways
Type
article
Field-Weighted Citation Impact
0.00

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article

RAF isoform selectivity of MEK inhibitors and rational design of a covalent ARAF-MEK inhibitor

Byung Hak Ha, David A. Scott, Jie Jiang, Sayan Chakraborty et al.
bioRxiv (Cold Spring Harbor Laboratory)
Melanoma and MAPK Pathways
article

RAF isoform selectivity of MEK inhibitors and rational design of a covalent ARAF-MEK inhibitor

Byung Hak Ha, David A. Scott, Jie Jiang, Sayan Chakraborty, Maurício T. Tavares, Dong Man Jang, Michael J. Eck, Pasi Antero Janne, Rebecca J. Metivier, Emre Tkacik, Talya S. Levitz, Thomas W. Gero, Hyesung Jeon, Anna M. Schmoker, Felix H. Gottlieb, Georgia Dittemore, Dimitris Gazgalis, Javier Vinals Camallonga
article en

Abstract

Aberrant activation of the RAS/RAF/MEK/ERK pathway is a frequent cause of cancer. Allosteric MEK inhibitors block this pathway by binding RAF-MEK complexes to prevent activation of MEK by RAF. However, how MEK inhibitor potency varies across the three RAF isoforms remains poorly understood. We profiled seven allosteric MEK inhibitors and observed a striking hierarchy of sensitivity: all most potently inhibited CRAF-driven MEK activation while relatively sparing ARAF-driven activation. We identified point mutations in ARAF and CRAF proximate to the MEK inhibitor binding site that markedly altered inhibitor sensitivity. Using a rational design approach, we developed a more potent inhibitor of ARAF-driven MEK signaling, TWG-07-148. Our cryo-EM structure shows how this acrylamide-containing analog of MEK inhibitor trametinib covalently targets Cys514, a residue unique to ARAF. Our studies highlight the importance of the activating RAF isoform as a determinant of MEK inhibitor sensitivity and provide proof-of-concept for development of MEK inhibitors that more effectively block ARAF-driven MEK signaling via covalent targeting of Cys514.

bioRxiv (Cold Spring Harbor Laboratory)
Harvard University (US), Dana-Farber Cancer Institute (US)
Pediatric Brain Tumor Foundation, National Institutes of Health
Openalex Percentile: Top 63%
Melanoma and MAPK Pathways
DOIOpen Access PDFOpenAlex
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