Locking Out RAS

Reflecting work in the Therrien Lab

Published here June 11, 2026

Targeting the H/KRAS α4-β6-α5 Allosteric Lobe with Macrocyclic Peptides

Kien Tran, Hugo Lavoie, Amal Wahhab, Damien Garrido, Chang Hwa Jo, Marc-André Poupart, Tarun Arya, Alexandre Beautrait, Ryan Killoran, Cédric Dicaire-Leduc, Éric Bonneil, Michael Osborne, Doris A. Schuetz, Faraz Shaikh, Pierre Thibault, Matthew J. Smith, Anne Marinier, and Marc Therrien

ACS Med. Chem. Lett. 2026, 17, 1154–1162. https://doi.org/10.1021/acsmedchemlett.6c00078

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Mutations in RAS proteins appear in nearly 19% of cancer patients, and the past decade has delivered therapeutic progress: five approved drugs and more than ten in clinical trials as of 2025. Yet acquired resistance remains a stubborn obstacle. Secondary mutations at residues such as Q61, Y96, H95, and Y64 can disable both Switch-II inhibitors, SW2i, and cyclophilin A molecular glues. The α4-β6-α5 surface of H/KRAS offers a structurally distinct escape route: this allosteric lobe is the binding site of the NS1 monobody, which disrupts RAS nanoclustering and downstream ERK signaling without touching the nucleotide pocket. Because NS1 is roughly 10 kDa and cannot reach intracellular RAS, the open question has been whether smaller molecules can recapitulate its binding logic.

Researchers in the Therrien and Marinier Groups at the Institute for Research in Immunology and Cancer, Université de Montréal, published in ACS Medicinal Chemistry Letters, used structure-based drug design to develop 10-mer macrocyclic peptides mimicking the NS1 FG-loop. Starting from the NS1–HRAS cocrystal structure (PDB 5E95), they applied site-directed mutagenesis to establish Trp75 and Tyr82 of the FG-loop as critical affinity determinants, then engineered conformational restriction through four modifications: cyclization with an Aib or β-amino isovaleric acid linker, inversion of Trp75 to D-Trp, a Gly79-to-D-Pro swap enforcing a type II' β-turn, and His78-to-Lys for solubility. A homogeneous tryptophan-quenching fluorescence assay, exploiting the absence of Trp in H/KRAS, measured dissociation constants across the series.

Conformational restriction proved decisive. The linear FG-loop sequence showed no detectable HRAS binding by NMR, while a macrocyclic analog bearing D-Trp at position 75 produced measurable chemical-shift perturbations. Iterative optimization yielded compound 2, at an HRAS KD of 66 μM, more than a 5-fold gain over the initial hit. The team then placed electrophilic warheads at position AA80 to engage Cys118 of HRAS, whose side chain sits within 4 Å of NS1 Gln80 in the cocrystal. One bromoacetamide analog reached complete labeling of wild-type HRAS by LC/MS against only 24% in a C118S control, and showed no cross-reactivity with RALA and RHOG, two related RAS-family GTPases, confirming Cys118 selectivity. X-ray costructures of three covalent analogs confirmed engagement at the α4-β6-α5 interface and guided warhead and linker geometry.

A pocket adjacent to Cys118, opened by an N5-benzyl-L-glutamine substituent at AA80, then guided a noncovalent campaign. Compound 11 bound HRAS at a KD of 4.4 μM and KRAS at 18 μM, 15-fold and 7.2-fold gains over compound 2. Adding D-Arg at position 79 and a C2-methylated Trp at position 83 delivered lead analog 14, at HRAS KD 1.9 μM and KRAS KD 2.7 μM, confirmed independently by surface plasmon resonance. Across both isoforms, the oncogenic variants G12D, G12V, G13R, and Q61K, and GTP-analog-loaded states, compound 14 held KD values in the 1.4 to 9.0 μM range, the binding resistance-agnostic and nucleotide-state independent. Alanine scanning showed that Trp77 and Tyr82 occupy adjacent sub-pockets on helix α4, which a single chimeric amino acid could satisfy together.

The work establishes the α4-β6-α5 allosteric lobe as a tractable target for macrocyclic inhibitors that hold affinity across the nucleotide states and resistance mutants which defeat current clinical agents. The covalent analog SP129 labeled H/KRAS in cell lysates with submicromolar potency, IC50 0.7 μM, though fluorescence imaging of a TAMRA-labeled probe indicated that endosomal escape, rather than biochemical potency, currently limits activity in live cells. Molecular dynamics simulations suggest the macrocyclic backbone forms stable intramolecular hydrogen bonds, a basis for chameleonicity tuning through backbone N-methylation and reduced side-chain polarity. That two critical pharmacophores can merge into a single chimeric residue offers a path from constrained peptide toward a lower-molecular-weight inhibitor of a resistance-agnostic RAS site.

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Additional Authors

Author

Dr. Hugo Lavoie joined IRIC in 2009 as a postdoctoral intern and is now Assistant Director of the Intracellular Research Unit, where he leads a team of research scientists. In this role, he has conducted and led protein structure–function and drug discovery programs, resulting in several high-impact publications. His strong background in genetics, biology, and biochemistry, combined with leadership skills, talent development, and immense scientific enthusiasm, has enabled him to consistently achieve organizational and research goals.

Author

Dr. Amal Wahhab is a team leader in the drug discovery unite at IRIC. She obtained her Ph.D. from the University of Calgary. Subsequently, she worked as an industrial postdoctoral researcher at Novartis, Horsham, England, then as a research scientist at BioChem Pharma, Montreal, Canada. She started the combinatorial chemistry group and became a director at MethylGene, Montreal. She then moved to Tranzyme Pharma, Sherbrooke, working with macrocyclic libraries. She was a cofounder of the startup Cyclenium Pharma, Montreal.

Author

Dr. Anne Marinier is a Full Professor in the Pharmacology and Physiology Department and Head of the Drug Discovery Unit, DDU, at the Institute of Research in Immunology and Cancer, IRIC, Université de Montréal, Canada. She obtained her Ph.D. in organic chemistry at Université de Sherbrooke, Canada, in 1990 under the supervision of Dr. Pierre Deslongchamps. She then joined Dr. Garland Marshall’s team at Washington University School of Medicine, in St. Louis, for a postdoctoral fellowship. After a distinguished 15-year career at Bristol-Myers Squibb where she served as a Group Leader in drug discovery, Dr. Marinier co-founded the DDU in 2008, building a 60-member translational research unit with strong industrial expertise. The DDU spans the full drug development from target ID to IND-enabling studies, transforming innovations from UdeM/IRIC into value-added therapeutic assets. Since its inception, five small-molecule programs have advanced into clinical development. In addition to this translational work, Dr. Marinier’s research focuses on developing molecular and biological tools, probes and entities aimed at uncovering novel therapeutic targets and exploring uncharted chemical space and screening modalities. Her group is developing a unique small-molecule collection and extensive DNA-encoded library platforms biased towards macrocyclic peptides and peptidomimetics. She is also Co-founder and CEO of RejuvenRx Inc. and Co-founder of ExCellThera Inc.

Author

Marc Therrien, Ph.D., is a Full Professor in the Department of Pathology and Cell Biology at the Université de Montréal and Chief Executive Officer of the Institute for Research in Immunology and Cancer (IRIC). He obtained his Ph.D. in Biochemistry from Université de Montréal in 1993 under the supervision of Jacques Drouin and completed postdoctoral training in molecular genetics with Gerald M. Rubin at the University of California, Berkeley. There, using Drosophila genetics, he discovered several key components of the RAS–ERK signaling pathway, including KSR and CNK. After establishing his research program at the Montreal Clinical Research Institute (IRCM), he joined IRIC in 2003 as a founding member and Director of the Intracellular Signaling Research Unit. His research focuses on the molecular mechanisms that regulate RAS–ERK signaling in development and cancer, combining genetics, biochemistry, structural biology, and drug discovery. His laboratory has made seminal contributions to the understanding of RAF activation, including the discovery that RAF kinases are activated through allosteric transactivation mediated by kinase-domain dimerization, a mechanism that has profoundly influenced both basic research and the development of targeted cancer therapies.

Locking Out RAS

Author Spotlight

Author

Dr. Kien Tran is a postdoctoral scholar specializing in medicinal chemistry and chemical biology. He earned his Ph.D. from the Université de Sherbrooke, Canada, in 2021, co-supervised by Profs. Éric Marsault and Philippe Sarret. Dr. Tran then completed postdoctoral training, 2021–2024, at the Institute for Research in Immunology and Cancer, IRIC, Université de Montréal, in the laboratories of Drs. Anne Marinier and Marc Therrien. His work centers on the design and synthesis of macrocyclic peptides for challenging therapeutic targets, such as H/K-Ras and GPCRs, which play key roles in cancer and cardiometabolic diseases.