Blocking C-terminal processing of KRAS4b via a direct covalent attack on the CaaX-box cysteine
Abstract
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 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.
Article Details
Journal Info
Proceedings of the National Academy of Sciences
National Academy of Sciences
Authors (34)
Anna E. Maciag
National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research
Yue Yang
Alok K. Sharma
National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research
David M. Turner
National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research
Caroline J. DeHart
National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc.
Hazem Abdelkarim
Department of Biochemistry and Molecular Genetics, College of Medicine, University of Illinois at Chicago
Lixin Fan
Brian P. Smith
National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research
Vandana Kumari
National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research
Marcin Dyba
Megan Rigby
National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research
Jean A. Castillo Badillo
National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research
Lauren Adams
Department of Molecular Biosciences, Northwestern University
Luca Fornelli
Department of Chemistry, Proteomics Center of Excellence, Northwestern University
Stephen Fox
Alla Brafman
National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research
Thomas Turbyville
National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research
William Gillette
National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research
Simon Messing
National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research
Constance Agamasu
National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research
Andrew L. Wolfe
Department of Cellular and Molecular Pharmacology, Helen Diller Family Comprehensive Cancer Center, University of California
Stephan Gysin
Department of Cellular and Molecular Pharmacology, Helen Diller Family Comprehensive Cancer Center, University of California
Albert H. Chan
Dhirendra K. Simanshu
Dominic Esposito
National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc.
Oleg Chertov
Andrew G. Stephen
Michelle Arkin
Adam Renslo
Neil L. Kelleher
Department of Chemistry
Vadim Gaponenko
Felice C. Lightstone
Dwight V. Nissley
Frank McCormick