Identification and Characterization of Small‐Molecule Ras Modulators and their Impact in Cancer Drug Discovery

Abstract

Ras is a small GTPase enzyme that binds guanine diphosphate (GDP), a nucleotide substrate, to its guanine‐nucleotide binding pocket. Ras activation occurs by the coordination of SOS, a guanine nucleotide exchange factor, which catalyzes the substitution of GDP for guanine triphosphate (GTP), rendering the Ras protein in an active state. Oncogenic Ras mutants are constitutively active, propagating proliferative cell signaling that leads to aberrant cell growth and metastatic tumors that are present in 30% of all human cancers. Discovery of a potent, small molecule Ras inhibitor has been challenging. We have characterized a novel class of Ras modulators that exhibit both Ras activation. These small molecules were discovered using Ras‐SOS molecular modeling and computational docking studies focused on identifying potential compound binding to the Ras‐SOS complex interface. Several in‐vitro biochemical assays were performed to elucidate the structural significance, binding kinetics, and nucleotide exchange effects of these small molecule binders. A fluorescence nucleotide exchange assay was optimized to discover compounds that had an atypical nucleotide exchange rate (off‐state to on‐state) in a kinetic assay. Compounds discovered in the assay were then analyzed by Surface Plasmon Resonance (SPR) to further elucidate details on the binding kinetics between Ras and compound as well as between Ras and SOS in the presence of the compound. Of the examined compounds, several exhibited activation of the Ras‐SOS catalytic reaction. An ongoing structure activity relationship (SAR) study has identified essential components that facilitate binding to the Ras:SOS interface. This presumably allosteric interaction may have uncovered an unprobed regulatory site that can be manipulated to create Ras inhibitors. This discovery of activator compounds provides a promising new direction for the development of potent Ras modulators.Support or Funding InformationThe Center for Healthcare Innovation (CHI), Stevens Institute of Technology