A High-Throughput Assay for Rho Guanine Nucleotide Exchange Factors Based on the Transcreener GDP Assay.

Ras homologous (Rho) family GTPases act as molecular switches controlling cell growth, movement, and gene expression by cycling between inactive guanosine diphosphate (GDP)- and active guanosine triphosphate (GTP)-bound conformations. Guanine nucleotide exchange factors (GEFs) positively regulate Rho GTPases by accelerating GDP dissociation to allow formation of the active, GTP-bound complex. Rho proteins are directly involved in cancer pathways, especially cell migration and invasion, and inhibiting GEFs holds potential as a therapeutic strategy to diminish Rho-dependent oncogenesis. Methods for measuring GEF activity suitable for high-throughput screening (HTS) are limited. We developed a simple, generic biochemical assay method for measuring GEF activity based on the fact that GDP dissociation is generally the rate-limiting step in the Rho GTPase catalytic cycle, and thus addition of a GEF causes an increase in steady-state GTPase activity. We used the Transcreener GDP Assay, which relies on selective immunodetection of GDP, to measure the GEF-dependent stimulation of steady-state GTP hydrolysis by small GTPases using Dbs (Dbl's big sister) as a GEF for Cdc42, RhoA, and RhoB. The assay is well suited for HTS, with a homogenous format and far red fluorescence polarization (FP) readout, and it should be broadly applicable to diverse Rho GEF/GTPase pairs.

New informatics and automated infrastructure to accelerate new leads discovery by high throughput screening (HTS).

The Lankenau Institute for Medical Research Chemical Genomics Center, Inc. has developed a new (patents issued and pending) Nanotube Automated Repository System (NARS) for dynamic storage of millions of 'single-shot' samples stored in a new monolithic microtiter-storage tube plate of our own design we call 'nanotubes.' We have integrated the NARS with customized software to efficiently access up to 10,000,000 samples stored continuously frozen (-20°C) in a dehumidified enclosure and sealed in a new microtiter NARS plate that is SBS compliant. Additional software was developed to analyze HTS data from orthogonally pooled compound libraries. Following 'de-convolution' of pooled HTS data, the software designates confirmatory retest samples to be 'cherry-picked' using the NARS. The application of a new, fully-integrated infrastructure for new leads discovery is described in detail. Other applications for our technologies and new infrastructure are discussed.