Using a Modified Yeast Two-Hybrid System to Screen for Chemical GEF Inhibitors

Abstract

GTPases of the Ras superfamily act as signaling switches, active when bound to GTP and inactive when bound to GDP. There is now considerable evidence that over-activation of Ras-like pathways participates in the development of many cancer types. In particular, GTPases of the Rho family control cell adhesion, survival, motility, and invasion, cell properties dysregulated in most cancer types. Rho activation is triggered by RhoGEFs, most of which form complexes with growth-factor receptors and initiate downstream Rho signaling pathways in response to extracellular clues. As such, RhoGEFs represent attractive targets to inhibit Rho pathways and may have interesting druggability for cancer therapeutics. Here we describe a procedure derived from the yeast two-hybrid system, in which activation of a mammalian Rho GTPase by its cognate RhoGEF is converted into variation in the yeast growth. The experimental design is thus suitable for identiying RhoGEF inhibitors and has been optimized for medium-throughput screening. The major advantages of this method lie in the direct monitoring of GEF activity in a living organism and the rapid detection of false positive hits.