title: In situ selectivity profiling and crystal structure of SML-8-73-1, an active site inhibitor of oncogenic K-Ras G12C.
abstract: Directly targeting oncogenic V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-Ras) with small-molecule inhibitors has historically been considered prohibitively challenging. Recent reports of compounds that bind directly to the K-Ras G12C mutant suggest avenues to overcome key obstacles that stand in the way of developing such compounds. We aim to target the guanine nucleotide (GN)-binding pocket because the natural contents of this pocket dictate the signaling state of K-Ras. Here, we characterize the irreversible inhibitor SML-8-73-1 (SML), which targets the GN-binding pocket of K-Ras G12C. We report a high-resolution X-ray crystal structure of G12C K-Ras bound to SML, revealing that the compound binds in a manner similar to GDP, forming a covalent linkage with Cys-12. The resulting conformation renders K-Ras in the open, inactive conformation, which is not predicted to associate productively with or activate downstream effectors. Conservation analysis of the Ras family GN-binding pocket reveals variability in the side chains surrounding the active site and adjacent regions, especially in the switch I region. This variability may enable building specificity into new iterations of Ras and other GTPase inhibitors. High-resolution in situ chemical proteomic profiling of SML confirms that SML effectively discriminates between K-Ras G12C and other cellular GTP-binding proteins. A biochemical assay provides additional evidence that SML is able to compete with millimolar concentrations of GTP and GDP for the GN-binding site.
authors.affiliation: Departments of Biochemistry and Radiation Oncology and.
authors.forename: John C
authors.initials: JC
authors.lastname: Hunter
authors.affiliation: Departments of Biochemistry and Radiation Oncology and.
authors.forename: Deepak
authors.initials: D
authors.lastname: Gurbani
authors.affiliation: Department of Cancer Biology andBlais Proteomics Center, Dana-Farber Cancer Institute, Boston, MA 02115;
authors.forename: Scott B
authors.initials: SB
authors.lastname: Ficarro
authors.affiliation: Departments of Biochemistry and Radiation Oncology and.
authors.forename: Martin A
authors.initials: MA
authors.lastname: Carrasco
authors.affiliation: Department of Cancer Biology andDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115; andDepartment of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138.
authors.forename: Sang Min
authors.initials: SM
authors.lastname: Lim
authors.affiliation: Department of Cancer Biology andDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115; and.
authors.forename: Hwan Geun
authors.initials: HG
authors.lastname: Choi
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138.
authors.forename: Ting
authors.initials: T
authors.lastname: Xie
authors.affiliation: Department of Cancer Biology andBlais Proteomics Center, Dana-Farber Cancer Institute, Boston, MA 02115;
authors.forename: Jarrod A
authors.initials: JA
authors.lastname: Marto
authors.affiliation: Department of Biophysics, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390.
authors.forename: Zhe
authors.initials: Z
authors.lastname: Chen
authors.affiliation: Department of Cancer Biology andDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115; and.
authors.forename: Nathanael S
authors.initials: NS
authors.lastname: Gray
authors.affiliation: Departments of Biochemistry and Radiation Oncology and kenneth.westover@utsouthwestern.edu.
authors.forename: Kenneth D
authors.initials: KD
authors.lastname: Westover
mesh_terms: D000081:Acetamides / Q000737:chemistry*; D001665:Binding Sites; D001710:Biotin / Q000737:chemistry; D020134:Catalytic Domain; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D020558:GTP Phosphohydrolases / Q000737:chemistry; D019204:GTP-Binding Proteins / Q000737:chemistry; D011905:Genes, ras; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives* / Q000737:chemistry; D006160:Guanosine Triphosphate / Q000737:chemistry; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D040901:Proteomics; D015398:Signal Transduction; D018631:ras Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry*
chemical_list: D000081:Acetamides; D008024:Ligands; C000591477:SML-8-73-1; D006153:Guanosine Diphosphate; D001710:Biotin; D006160:Guanosine Triphosphate; D019869:Phosphatidylinositol 3-Kinases; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D018631:ras Proteins
keywords: ActivX; CPM assay