%0 Journal Article
%T A High Throughput Screening Assay System for the Identification of Small Molecule Inhibitors of gsp
%A Nisan Bhattacharyya
%A Xin Hu
%A Catherine Z. Chen
%A Lesley A. Mathews Griner
%A Wei Zheng
%A James Inglese
%A Christopher P. Austin
%A Juan J. Marugan
%A Noel Southall
%A Susanne Neumann
%A John K. Northup
%A Marc Ferrer
%A Michael T. Collins
%J PLOS ONE
%D 2014
%I Public Library of Science (PLoS)
%R 10.1371/journal.pone.0090766
%X Mis-sense mutations in the ¦Á-subunit of the G-protein, Gs¦Á, cause fibrous dysplasia of bone/McCune-Albright syndrome. The biochemical outcome of these mutations is constitutively active Gs¦Á and increased levels of cAMP. The aim of this study was to develop an assay system that would allow the identification of small molecule inhibitors specific for the mutant Gs¦Á protein, the so-called gsp oncogene. Commercially available Chinese hamster ovary cells were stably transfected with either wild-type (WT) or mutant Gs¦Á proteins (R201C and R201H). Stable cell lines with equivalent transfected Gs¦Á protein expression that had relatively lower (WT) or higher (R201C and R201H) cAMP levels were generated. These cell lines were used to develop a fluorescence resonance energy transfer (FRET)¨Cbased cAMP assay in 1536-well microplate format for high throughput screening of small molecule libraries. A small molecule library of 343,768 compounds was screened to identify modulators of gsp activity. A total of 1,356 compounds with inhibitory activity were initially identified and reconfirmed when tested in concentration dose responses. Six hundred eighty-six molecules were selected for further analysis after removing cytotoxic compounds and those that were active in forskolin-induced WT cells. These molecules were grouped by potency, efficacy, and structural similarities to yield 22 clusters with more than 5 of structurally similar members and 144 singleton molecules. Seven chemotypes of the major clusters were identified for further testing and analyses.
%U http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0090766