Combined Rational Design and a High-Throughput Screening Platform for Identifying Chemical Inhibitors of a Ras Activating Enzyme [Enzymology]
March 30th, 2015 by Evelyn, C. R., Biesiada, J., Duan, X., Tang, H., Shang, X., Papoian, R., Seibel, W. L., Nelson, S., Meller, J., Zheng, Y.
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 are 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 a unique ensemble structure-based virtual screening approach in combination with a multi-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 that satisfied minimized energy docking criteria to the GEF molecule, of which dual wave-length 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.