qHTS for Inhibitors of Regulator of G Protein Signaling(RGS) 4: Hit Validation
G-protein-coupled receptors (GPCRs) are well-established targets for drug discovery and development. Members of the "regulator of G-protein signaling" (RGS)-protein superfamily have recently emerged as critical modulators of specific GPCR signaling pathways. Via their conserved RGS domain that possesses "GTPase-accelerating protein" (GAP) activity, RGS proteins deactivate heterotrimeric G-protein more ..
BioActive Compounds: 17
G-protein-coupled receptors (GPCRs) are well-established targets for drug discovery and development. Members of the "regulator of G-protein signaling" (RGS)-protein superfamily have recently emerged as critical modulators of specific GPCR signaling pathways. Via their conserved RGS domain that possesses "GTPase-accelerating protein" (GAP) activity, RGS proteins deactivate heterotrimeric G-protein alpha (G) subunits and thus attenuate GPCR signal transduction. Combining specific RGS domain inhibitors with existing GPCR agonists should potentiate cellular responses and could also increase the specificity of action of existing therapeutics. Small molecule modulators of RGS domain activity should also have clinical utility in inhibiting or potentiating the actions of endogenous GPCR agonists. In particular, the diversity of RGS proteins with highly localized and dynamically regulated distributions in the human brain makes this family of proteins attractive targets for pharmacotherapy of central nervous system (CNS) disorders such as Parkinson's disease, schizophrenia, and anxiety.
Hence, in a collaboration between the University of North Caroline Chapel Hill (School of Medicine) and NIH Chemical Genomics Center (NCGC) a novel HTS-compatible enzymatic assay for RGS-accelerated GDP production was developed. This assay was used to screen the NIH Molecular Libraries Small Molecule Repository (MLSMR) in order to identify a small molecule that inhibits RGS4.
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]
MLPCN Grant: DA030555
Assay Submitter (PI): David Siderovski, University of North Carolina Chapel Hill (School of Medicine)
Two microliters of 500nM (2x working concentration) is dispensed into black medium-binding solid-bottom Kalypsys plates. Then with the Kalypsys pintool 23nl of control and compounds are transferred; the control compound is 100mM CCG-4986 (working concentration). After a 10 minute incubation at room temperature 2ul of antibody is added with the Kalypsys dispenser; (-)RGS4/Transcreener GDP solution in columns 3 and 4 which consisted of 10nM (2x working concentration) Alexa633, 20uM GTP, and 20 ug/ml GDP antibody and (+)RGS4/Transcreener GDP solution in columns 1,2, and 5 to 48 which consists of 900nM RGS4 (2x working concentration), 10nM Alexa633, 20 ug/ml GDP antibody. The plate is centrifuged at 1000 RPM for 15 seconds to remove any possible bubbles and then incubated for 5 minutes at ambient room temperature. An initial fluorescent polarization read (620 nm/688 nm) is performed on the Envision before the plate is incubated for 90 minutes at 30 deg C at a relative humidity of 80%. The plate is once more centrifuged at 1000 RPM for 15 seconds and another Envision fluorescent polarization (620 nm/688 nm) read is carried out.
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)