Combined Rational Design and a High Throughput Screening Platform for Identifying Chemical Inhibitors of a Ras-activating Enzyme

• DOI: 10.1074/jbc.m114.634493
• Publication Date: 2015-04-01T09:35:14Z
• Authors: Chris R. Evelyn; Jacek Biesiada; Xin Duan; Hong Tang; Xun Shang; Ruben Papoian; William L. Seibel; Sandra Nelson; Jaroslaw Meller; Yi Zheng
• Keywords: Small GTPase; High-Throughput Screening

The Ras family small GTPases regulate multiple cellular processes, including cell growth, survival, movement, and gene expression, are intimately involved in cancer pathogenesis. Activation of these is catalyzed by a special class enzymes, termed guanine nucleotide exchange factors (GEFs). Herein, we developed molecule screening platform for identifying lead hits targeting GEF enzyme, SOS1. We employed an ensemble structure-based virtual approach combination with tier high throughput experimental screen utilizing two complementary fluorescent assays to identify inhibitors catalytic activity toward Ras. From library 350,000 compounds, selected set 418 candidate compounds predicted disrupt the GEF-Ras interaction, which dual wavelength GDP dissociation GTP-loading identified chemically distinct inhibitors. Subsequent biochemical validations indicate that they capable dose-dependently inhibiting activity, binding SOS1 micromolar affinity, disrupting interaction. Mutagenesis studies conjunction structure-activity relationship mapped both different sites pocket, inhibited signaling cells. unique established here enzymes could be broadly useful variety GTPase-activating reactions.