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Methods Enzymol

Title:		Yeast-based screening for inhibitors of RGS proteins
Author(s):		Young KH; Wang Y; Bender C; Ajit S; Ramirez F; Gilbert A; Nieuwenhuijsen BW;
Address:		"Neuroscience Discovery Research, Wyeth Research, Princeton, New Jersey 08543-8000, USA"
Journal Title:		Methods Enzymol
Year:		2004
Volume:		389
Issue:
Page Number:		277 - 301
DOI:		10.1016/S0076-6879(04)89017-7
ISSN/ISBN:		0076-6879 (Print) 0076-6879 (Linking)
Abstract:		"This article provides information on two screening platforms for the identification of regulators of G-protein signaling (RGS) protein modulators. Utilization of the yeast pheromone response pathway enabled the creation of a functional screen for RGS4 modulators. The RGSZ1-focused screen employs advances in yeast two-hybrid screening technologies and targets the protein-protein interface of the RGS domain/Galpha interaction. Moreover, the RGSZ1 screen provides the opportunity to multiplex the screening of two targets of interest, given the development of two different luciferase reporter genes that enabled sequential determination and intraassay controls. The screen formats were validated, implemented, and conducted as automated 384-well, liquid-based, high-throughput small molecule screens. Primary 'hits' were confirmed using benchtop 96-well formats of these assays and advanced to in vitro functional evaluation assays. The yeast-based assay platforms provide robust cellular assays that result in the identification of small molecule modulators for both RGS targets. These molecules can serve both as tools with which to probe biological implications of RGS proteins and as potential starting points toward the development of novel modulators of G-protein signaling pathways. Such modulators may show potential for controlling and treating diseases resulting from inappropriate activity of G-protein signaling pathways"
Keywords:		"Amino Acid Sequence *Drug Evaluation, Preclinical GTP-Binding Protein alpha Subunits, Gi-Go/metabolism Genes, Reporter Inhibitory Concentration 50 Luciferases/*analysis/genetics Pheromones/metabolism Promoter Regions, Genetic Protein Isoforms/*antagonists;Neuroscience;"
Notes:		"MedlineYoung, Kathleen H Wang, Yuren Bender, Corey Ajit, Seena Ramirez, Fernando Gilbert, Adam Nieuwenhuijsen, Bart W eng 2004/08/18 Methods Enzymol. 2004; 389:277-301. doi: 10.1016/S0076-6879(04)89017-7"