Journal article
Development of a bimolecular luminescence complementation assay for RGS: G protein interactions in cells
Analytical biochemistry, Vol.522, pp.10-17
04/01/2017
DOI: 10.1016/j.ab.2017.01.013
PMCID: PMC5330260
PMID: 28115169
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: Gαi1 protein-protein interaction. In conclusion, the NanoBit Protein Complementation Assay holds promise as a robust platform for discovery and characterization of RGS inhibitors.
•Characterization of 14 RGS: Gα interactions using NanoBit.•RGS4: Gαi1 interaction disruption with compound 6383479.•Characterization of RGS6Lα2: Gβ5 interaction using NanoBit.•Calculation of Z-factors for RGS6RH: Gαi1, RGS8RH: Gαi1, and RGS6Lα2: Gβ5.
Details
- Title: Subtitle
- Development of a bimolecular luminescence complementation assay for RGS: G protein interactions in cells
- Creators
- Christopher R Bodle - Department of Pharmaceutical Sciences and Experimental Therapeutics University of Iowa, 115 S. Grand Avenue S338 PHAR, Iowa City, IA, 52242, USAMichael P Hayes - Department of Pharmaceutical Sciences and Experimental Therapeutics University of Iowa, 115 S. Grand Avenue S338 PHAR, Iowa City, IA, 52242, USAJoseph B O'Brien - Department of Pharmaceutical Sciences and Experimental Therapeutics University of Iowa, 115 S. Grand Avenue S338 PHAR, Iowa City, IA, 52242, USADavid L Roman - Department of Pharmaceutical Sciences and Experimental Therapeutics University of Iowa, 115 S. Grand Avenue S327 PHAR, Iowa City, IA, 52242, USA
- Resource Type
- Journal article
- Publication Details
- Analytical biochemistry, Vol.522, pp.10-17
- DOI
- 10.1016/j.ab.2017.01.013
- PMID
- 28115169
- PMCID
- PMC5330260
- NLM abbreviation
- Anal Biochem
- ISSN
- 0003-2697
- eISSN
- 1096-0309
- Publisher
- Elsevier BV
- Language
- English
- Date published
- 04/01/2017
- Academic Unit
- Pharmacy; Iowa Neuroscience Institute; Pharmaceutical Sciences and Experimental Therapeutics; Medicinal and Natural Products Chemistry
- Record Identifier
- 9984065498802771
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