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

<i>RAS</i> 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 hypervariable 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-α3-α4). 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 Å or less, and structure analysis identified the critical residues and interactions that define the HVR-α3-α4 pocket. Further development of compounds that bind to this pocket could be the basis of a new approach to targeting KRAS cancers.

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Publication Details

Journal
Proceedings of the National Academy of Sciences
Published
2025-05-09
DOI
https://doi.org/10.1073/pnas.2410766122
Citations
3
Primary Topic
Cellular transport and secretion
Type
article
Field-Weighted Citation Impact
3.45

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article

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

Simon A. J. Messing, Marcin D. Dyba, Felice C. Lightstone, David M. Turner et al.
3 citations
Proceedings of the National Academy of Sciences
Cellular transport and secretion
3.45
article

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

Simon A. J. Messing, Marcin D. Dyba, Felice C. Lightstone, David M. Turner, Dhirendra K. Simanshu, William Gillette, Thomas J. Turbyville, Luca Fornelli, Vandana Kumari, Michelle R. Arkin, Dwight V. Nissley, Adam R. Renslo, Andrew L. Wolfe, Yue Yang, Hazem Abdelkarim, Alla Brafman, Brian P. Smith, Lauren Adams, Constance Agamasu, Caroline J. DeHart, Andrew G. Stephen, Dominic Esposito, Stephan Gysin, Neil L. Kelleher, Oleg Chertov, Vadim Gaponenko, Frank P. McCormick, Megan Rigby, Albert H. Chan, Lixin Fan, Stephen Fox, Asha Sharma, Anna E. Maciag, Jean A. Castillo Badillo
article en
3 citations

Abstract

<i>RAS</i> 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 hypervariable 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-α3-α4). 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 Å or less, and structure analysis identified the critical residues and interactions that define the HVR-α3-α4 pocket. Further development of compounds that bind to this pocket could be the basis of a new approach to targeting KRAS cancers.

Proceedings of the National Academy of SciencesVol. 122(19)
Lawrence Livermore National Laboratory (US), University of Illinois Chicago (US), Frederick National Laboratory for Cancer Research (US), UCSF Helen Diller Family Comprehensive Cancer Center (US)
U.S. Department of Energy, Northwestern University, Frederick National Laboratory for Cancer Research, National Institutes of Health, Office of Science, National Cancer Institute, Argonne National Laboratory, Lawrence Livermore National Laboratory
Openalex Percentile: Top 9%
Cellular transport and secretion
3.45
DOIOpen Access PDFOpenAlex
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