A High Throughput Screening Assay System for the Identification of Small Molecule Inhibitors of <i>gsp</i>

<div><p>Mis-sense mutations in the α-subunit of the G-protein, G_sα, cause fibrous dysplasia of bone/McCune-Albright syndrome. The biochemical outcome of these mutations is constitutively active G_sα 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 G_sα protein, the so-called <i>gsp</i> oncogene. Commercially available Chinese hamster ovary cells were stably transfected with either wild-type (WT) or mutant G_sα proteins (R201C and R201H). Stable cell lines with equivalent transfected G_sα 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)–based 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 <i>gsp</i> 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.</p>