title: Combined rational design and a high throughput screening platform for identifying chemical inhibitors of a Ras-activating enzyme.
abstract: The Ras family small GTPases regulate multiple cellular processes, including cell growth, survival, movement, and gene expression, and are intimately involved in cancer pathogenesis. Activation of these small GTPases is catalyzed by a special class of enzymes, termed guanine nucleotide exchange factors (GEFs). Herein, we developed a small molecule screening platform for identifying lead hits targeting a Ras GEF enzyme, SOS1. We employed an ensemble structure-based virtual screening approach in combination with a multiple tier high throughput experimental screen utilizing two complementary fluorescent guanine nucleotide exchange assays to identify small molecule inhibitors of GEF catalytic activity toward Ras. From a library of 350,000 compounds, we selected a set of 418 candidate compounds predicted to disrupt the GEF-Ras interaction, of which dual wavelength GDP dissociation and GTP-loading experimental screening identified two chemically distinct small molecule inhibitors. Subsequent biochemical validations indicate that they are capable of dose-dependently inhibiting GEF catalytic activity, binding to SOS1 with micromolar affinity, and disrupting GEF-Ras interaction. Mutagenesis studies in conjunction with structure-activity relationship studies mapped both compounds to different sites in the catalytic pocket, and both inhibited Ras signaling in cells. The unique screening platform established here for targeting Ras GEF enzymes could be broadly useful for identifying lead inhibitors for a variety of small GTPase-activating GEF reactions.
authors.affiliation: From the Division of Experimental Hematology and Cancer Biology.
authors.forename: Chris R
authors.initials: CR
authors.lastname: Evelyn
authors.affiliation: Division of Biomedical Informatics.
authors.forename: Jacek
authors.initials: J
authors.lastname: Biesiada
authors.affiliation: From the Division of Experimental Hematology and Cancer Biology.
authors.forename: Xin
authors.initials: X
authors.lastname: Duan
authors.affiliation: Division of Immunobiology, and the Drug Discovery Center and.
authors.forename: Hong
authors.initials: H
authors.lastname: Tang
authors.affiliation: From the Division of Experimental Hematology and Cancer Biology.
authors.forename: Xun
authors.initials: X
authors.lastname: Shang
authors.affiliation: the Drug Discovery Center and Departments of Neurology and.
authors.forename: Ruben
authors.initials: R
authors.lastname: Papoian
authors.affiliation: the Drug Discovery Center and Division of Oncology, Children's Hospital Research Foundation, Cincinnati, Ohio 45229 and.
authors.forename: William L
authors.initials: WL
authors.lastname: Seibel
authors.affiliation: the Drug Discovery Center and.
authors.forename: Sandra
authors.initials: S
authors.lastname: Nelson
authors.affiliation: Division of Biomedical Informatics, Environmental Health, University of Cincinnati, Cincinnati, Ohio 45267.
authors.forename: Jaroslaw
authors.initials: J
authors.lastname: Meller
authors.affiliation: From the Division of Experimental Hematology and Cancer Biology, Yi.Zheng@cchmc.org.
authors.forename: Yi
authors.initials: Y
authors.lastname: Zheng
mesh_terms: D004305:Dose-Response Relationship, Drug; D016503:Drug Delivery Systems / Q000379:methods; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics / Q000378:metabolism; D020703:ras GTPase-Activating Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D020663:ras Guanine Nucleotide Exchange Factors / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D020703:ras GTPase-Activating Proteins; D020663:ras Guanine Nucleotide Exchange Factors; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)
keywords: chemical biology; drug discovery; guanine nucleotide exchange factor (GEF); signal transduction; small GTPase
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