Blocking C-terminal processing of KRAS4b via a direct covalent attack on the CaaX-box cysteine

• 成果类型: Article
• 署名作者: Maciag, Anna E.; Yang, Yue; Sharma, Alok K.; Turner, David M.; Dehart, Caroline J.; Abdelkarim, Hazem; Fan, Lixin; Smith, Brian P.; Kumari, Vandana; Dyba, Marcin; Rigby, Megan; Badillo, Jean A. Castillo; Adams, Lauren; Fornelli, Luca; Fox, Stephen; Brafman, Alla; Turbyville, Thomas; Gillette, William; Messing, Simon; Agamasu, Constance; Wolfe, Andrew L.; Gysin, Stephan; Chan, Albert H.; Simanshu, Dhirendra K.; Esposito, Dominic; Chertov, Oleg; Stephen, Andrew G.; Arkin, Michelle; Renslo, Adam; Kelleher, Neil L.; Gaponenko, Vadim; Lightstone, Felice C.; Nissley, Dwight, V; McCormick, Frank
• 署名单位: National Institutes of Health (NIH) - USA; NIH National Cancer Institute (NCI); Frederick National Laboratory for Cancer Research; United States Department of Energy (DOE); Lawrence Livermore National Laboratory; University of Illinois System; University of Illinois Chicago; University of Illinois Chicago Hospital; National Institutes of Health (NIH) - USA; NIH National Cancer Institute (NCI); Frederick National Laboratory for Cancer Research; Northwestern University; Northwestern University; University of California System; University of California San Francisco; UCSF Medical Center; UCSF Helen Diller Family Comprehensive Cancer Center; University of California System; University of California San Francisco; Northwestern University; Northwestern University; Northwestern University
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-11483
• DOI: 10.1073/pnas.2410766122
• 发表日期: 2025-05-13
• 关键词: k-ras; plasma-membrane; proteins; mutations; gtpases; p21ras; cancer; domain; motif

摘要

RAS is the most frequently mutated oncogene in cancer. RAS proteins show high sequence similarities in their G-domains but are significantly different in their C-terminal hyper-variable regions (HVR). These regions interact with the cell membrane via lipid anchors that result from posttranslational modifications (PTM) of cysteine residues. KRAS4b is unique as it has only one cysteine that undergoes PTM, C185. Small molecule covalent modification of C185 would block any form of prenylation and subsequently inhibit attachment of KRAS4b to the cell membrane, blocking its biological activity. We translated this concept to the discovery and development of disulfide tethering screen hits into irreversible covalent modifiers of C185. These compounds inhibited proliferation of KRAS4b-driven mouse embryonic fibroblasts, but not cells driven by N-myristoylated KRAS4b that harbor a C185S mutation and are not dependent on C185 prenylation. Top-down proteomics was used to confirm target engagement in cells. These compounds bind in a pocket formed when the HVR folds back between helix 3 and 4 in the G-domain (HVR-alpha 3-alpha 4). This interaction can happen in the absence of small molecules as predicted by molecular dynamics simulations and is stabilized in the presence of C185 binders as confirmed by small-angle X-ray scattering and solution NMR. NOESY-HSQC, an NMR approach that measures internuclear distances of 6 & Aring; or less, and structure analysis identified the critical residues and interactions that define the HVR-alpha 3-alpha 4 pocket. Further development of compounds that bind to this pocket could be the basis of a new approach to targeting KRAS cancers.