title: Development of a bimolecular luminescence complementation assay for RGS: G protein interactions in cells.
abstract: Cell based assessment tools and screening platforms are the preferred paradigm for small molecule identification and validation due to selectively identifying molecules with cellular activity and validation of compound activity against target proteins in their native environment. With respect to Regulator of G Protein Signaling (RGS) proteins, current cell based methodologies are either low throughput or monitor downstream signaling consequences. The increasing number of reports indicating RGS function in various disease pathogeneses highlights the need for a robust RGS inhibitor discovery and characterization paradigm. Promega's NanoBit Protein Complementation Assay utilizes NanoLuc, an engineered luciferase with enhanced luminescence characteristics which allow for both robust and kinetic assessment of protein interaction formation and disruption. Here we characterized 15 separate RGS: G protein interactions using this system. The binding profile of RGS: Gα interactions correlates to prior published biochemical binding profiles of these proteins. Additionally, we demonstrated this system is suitable for high throughput screening efforts via calculation of Z-factors for three of the interactions and demonstrated that a known small molecule inhibitor of RGS4 disrupts the RGS4: Gαi1protein-protein interaction. In conclusion, the NanoBit Protein Complementation Assay holds promise as a robust platform for discovery and characterization of RGS inhibitors.
authors.affiliation: Department of Pharmaceutical Sciences and Experimental Therapeutics University of Iowa, 115 S. Grand Avenue S338 PHAR, Iowa City, IA, 52242, USA. Electronic address: christopher-bodle@uiowa.edu.
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Bodle
authors.affiliation: Department of Pharmaceutical Sciences and Experimental Therapeutics University of Iowa, 115 S. Grand Avenue S338 PHAR, Iowa City, IA, 52242, USA. Electronic address: michael-p-hayes@uiowa.edu.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Hayes
authors.affiliation: Department of Pharmaceutical Sciences and Experimental Therapeutics University of Iowa, 115 S. Grand Avenue S338 PHAR, Iowa City, IA, 52242, USA. Electronic address: Joseph-obrien-1@uiowa.edu.
authors.forename: Joseph B
authors.initials: JB
authors.lastname: O'Brien
authors.affiliation: Department of Pharmaceutical Sciences and Experimental Therapeutics University of Iowa, 115 S. Grand Avenue S327 PHAR, Iowa City, IA, 52242, USA; Cancer Signaling and Experimental Therapeutics Program, Holden Comprehensive Cancer Center, University of Iowa Hospitals and Clinics, Iowa City, IA, USA. Electronic address: david-roman@uiowa.edu.
authors.forename: David L
authors.initials: DL
authors.lastname: Roman
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002460:Cell Line; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000235:genetics / Q000378:metabolism*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008163:Luminescent Measurements / Q000379:methods*; D020710:RGS Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats
chemical_list: C554061:GNAQ protein, human; D020710:RGS Proteins; C584114:GNAI1 protein, human; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D043802:GTP-Binding Protein alpha Subunits, Gq-G11
keywords: Gα(i1); Gα(q); High throughput screening; NanoBit; RGS