RESUMO
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.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Imunoensaio/métodos , Fatores de Troca de Nucleotídeo Guanina Rho/análiseRESUMO
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.