See our latest publication, “A High-Throughput Assay for Rho Guanine Nucleotide Exchange Factors Based on the Transcreener GDP Assay” in the Journal of Biomolecular Screening
BellBrook Labs scientists Tom Zielinski and Meera Kumar collaborated with investigators in Mel Reichman’s laboratory at the Lankenau Institute for Medical Research to develop a robust, HTS-compatible assay for Rho guanine nucleotide exchange factors (GEFs). The concept behind the assay is a little counterintuitive, as it measures GEF-dependent stimulation of steady state Rho GTPase activity, which would seem more like a GTPase-activating protein (GAP) effect. However, GDP dissociation is the rate-limiting step in the Rho GTPase catalytic cycle, so any acceleration in its release—which is what GEFs do—will increase the rate of GTP hydrolysis.
We demonstrated this Rho GEF assay method with the GEF Dbs and three Rho GTPases, and have since used it for the PI3K-dependent Rac exchange factor, P-Rex1 and Rac1. The assay is homogenous, uses a far-red FP readout and should be broadly applicable to the more than 70 members of the Rho GEF family, as GTP dissociation is generally rate-limiting.
Current GEF assay methods are based on fluor-GTP binding or protein-protein interaction, both of which suffer from a number of drawbacks for HTS, but most importantly, they are mechanistically biased. Our goal was to develop an HTS-friendly assay that is agnostic with respect to the mechanism of inhibition. The GTPase acceleration assay meets this criterium, as it should enable identification of inhibitors regardless of whether they bind to the GEF, the GTPase or the complex, whether they are competitive with GTP or act allosterically, and whether they prevent formation of the complex. The catalytic nature of the assay also makes it more sensitive than fluor-GTP binding assays, which reduces the amounts of recombinant Rho and GEF proteins required.