Poster: Profiling GTPase Inhibitors for Effects on GTP Hydrolysis Using the Transcreener GDP Assay

Small GTPases have been shown to be frequently mutated or dysregulated in many human diseases including cancers, neurodegenerative disorders and cardiomyopathy. Current strategies for biochemical screening and analysis of small GTPase inhibitors rely on the use of fluorescent GTP and GDP analogs to detect effects on GDP release and GTP binding. Though robust and widely used, these methods do not provide information on GTP hydrolysis, a critical part of the GTPase signal transduction process. We have developed methods for detecting GTP hydrolysis by small GTPases using the Transcreener GDP assay, a homogenous, competitive immunoassay for GDP with far-red fluorescence readout.

Because GDP release is generally rate limiting, stimulation by GEFs can also be measured by monitoring GDP formation. We have optimized these assays for initial velocity detection of GTPase activity for Rho and Ras family members with robustness suitable for high throughput screening and tested a number of compounds reported to inhibit GTP and/or GDP binding.

In this poster the following targets will be discussed: