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BioAssay: AID 879

qHTS Assay for Inhibitors of RGS12 GoLoco Motif Activity (Green Fluorophore)

G-protein-coupled receptors (GPCRs) are major targets for drug discovery. The regulator of G-protein signaling (RGS)-protein family has important roles in GPCR signal transduction. RGS proteins contain a conserved RGS-box, which is often accompanied by other signaling regulatory elements. RGS proteins accelerate the deactivation of G proteins to reduce GPCR signaling; however, some also have an more ..
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 Tested Compounds
 Tested Compounds
All(1238)
 
 
Active(12)
 
 
Inactive(1206)
 
 
Inconclusive(20)
 
 
 Tested Substances
 Tested Substances
All(1279)
 
 
Active(12)
 
 
Inactive(1247)
 
 
Inconclusive(20)
 
 
AID: 879
Data Source: NCGC (RGSP681)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2007-12-13
Modify Date: 2008-12-28

Data Table ( Complete ):           View Active Data    View All Data
Targets
BioActive Compounds: 12
Related Experiments
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AIDNameTypeComment
880qHTS Assay for Inhibitors of RGS12 GoLoco Motif Activity (Red Fluorophore)Confirmatorydepositor-specified cross reference
1415Multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions, specifically RGS4-Galphao.Screeningdepositor-specified cross reference
1423Multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions, specifically RGS8-Galphao.Screeningdepositor-specified cross reference
1439Multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions, specifically RGS7-Galphao.Screeningdepositor-specified cross reference
1440Multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions, specifically RGS19-Galphao.Screeningdepositor-specified cross reference
1441Multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions, specifically RGS16-Galphao.Screeningdepositor-specified cross reference
1504Multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions.Summarydepositor-specified cross reference
1836Single point concentration, multiplexed high-throughput screen for confirmation of small molecule regulators of RGS family protein interactions, specifically RGS8-Galphao.Screeningdepositor-specified cross reference
1837Single point concentration, multiplexed high-throughput screen for confirmation of small molecule regulators of RGS family protein interactions, specifically RGS7-Galphao.Screeningdepositor-specified cross reference
1838Single point concentration, multiplexed high-throughput screen for confirmation of small molecule regulators of RGS family protein interactions, specifically RGS16-Galphao.Screeningdepositor-specified cross reference
1840Single point concentration, multiplexed high-throughput screen for confirmation of small molecule regulators of RGS family protein interactions, specifically RGS4-Galphao.Screeningdepositor-specified cross reference
1841Single point concentration, multiplexed high-throughput screen for confirmation of small molecule regulators of RGS family protein interactions, specifically RGS19-Galphao.Screeningdepositor-specified cross reference
1869Dose respone, multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions, specifically RGS8-Galphao.Confirmatorydepositor-specified cross reference
1872Dose response, multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions, specifically RGS4-Galphao.Confirmatorydepositor-specified cross reference
1884Dose response, multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions, specifically RGS19-Galphao.Confirmatorydepositor-specified cross reference
1888Dose respone, multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions, specifically RGS16-Galphao.Confirmatorydepositor-specified cross reference
2295Dose response, multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions, specifically RGS4-Galphao for SAR compoundsConfirmatorydepositor-specified cross reference
2298Dose response, multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions, specifically RGS16-Galphao for SAR CompoundsConfirmatorydepositor-specified cross reference
2307Dose response, multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions, specifically RGS8-Galphao for SAR CompoundsConfirmatorydepositor-specified cross reference
2311Dose response, multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions, specifically RGS19-Galphao for SAR CompoundsConfirmatorydepositor-specified cross reference
2390Confirmation qHTS Assay for Inhibitors of RGS12 GoLoco Motif Activity (Red Fluorophore)Confirmatorydepositor-specified cross reference
2406Probe Development Summary for Inhibitors of RGS12 GoLoco Motif ActivitySummarydepositor-specified cross reference
2827Dose response, multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions, specifically RGS8-Galphao with additional round of SAR compoundsConfirmatorydepositor-specified cross reference
2828Dose response, multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions, specifically RGS16-Galphao with additional round of SAR compoundsConfirmatorydepositor-specified cross reference
2829Dose response, multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions, specifically RGS4-Galphao with additional round of SAR compounds.Confirmatorydepositor-specified cross reference
503Compound Screen Assay, Human RGS18Screeningsame project related to Summary assay
1280In Vitro Kinase Assay Using Purified Enzyme PKC-betaConfirmatorysame project related to Summary assay
1282In Vitro Kinase Assay Using Purified Enzyme IKK-betaConfirmatorysame project related to Summary assay
1775Profiling compound fluorescence on Avidin Beads with 488 nm excitation and 530 nm emissionOthersame project related to Summary assay
1783Counter screen for biotinylated proteins binding to avidin beadsScreeningsame project related to Summary assay
1871Dose response, multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions, specifically RGS7-Galphao.Confirmatorysame project related to Summary assay
1993FPR-induced VLA-4 exposure in U937 cellsScreeningsame project related to Summary assay
2296Dose response, multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions, specifically RGS7-Galphao for SAR CompoundsConfirmatorysame project related to Summary assay
2826Dose response, multiplexed high-throughput screen for small molecule regulators of RGS family protein interactions, specifically RGS7-Galphao with additional round of SAR compounds.Confirmatorysame project related to Summary assay
Description:
Assay Overview:

NIH Molecular Libraries Screening Centers Network [MLSCN]
NIH Chemical Genomics Center [NCGC]
MLSCN Grant: NS053754-01
Assay Provider: Siderovski, David P, University of North Carolina at Chapel Hill

G-protein-coupled receptors (GPCRs) are major targets for drug discovery. The regulator of G-protein signaling (RGS)-protein family has important roles in GPCR signal transduction. RGS proteins contain a conserved RGS-box, which is often accompanied by other signaling regulatory elements. RGS proteins accelerate the deactivation of G proteins to reduce GPCR signaling; however, some also have an effector function and transmit signals. Combining GPCR agonists with RGS inhibitors should potentiate responses, and could markedly increase the agonist's regional specificity. The diversity of RGS proteins with highly localized and dynamically regulated distributions in brain makes them attractive targets for pharmacotherapy of central nervous system disorders. Inhibitors of the RGS:GPCR interaction should prove useful as small molecule tools in this research field. A complex of Gai1 and fluorescently labeled G-alpha-binding peptide (the "GoLoco motif") derived from RGS12 is incubated with library members. Inhibitors of the binding are detected by a decrease in the fluorescence polarization (FP) of the fluorophore. Protein was supplied by Prof. David Siderovski, University of North Carolina at Chapel Hill.
Protocol
Assay Protocol:
Reagents/Controls:
Assay Buffer: 10 mM Tris-HCl, 150 mM NaCl, 0.1 mM GDP (prepared fresh), and 0.05% NP40 at pH 7.5.
Controls: 10 nM FITC labeled peptide dispensed into columns 3 and 4 to generate negative control (full inhibition of binding, -100 % activity).
10 nM FITC labeled peptide/50 nM protein in columns 1, 2, 5 - 48. Titration of unlabeled peptide control ([H]TMGEEDFFDLLAKSQSKRMDDQRVDLAG[NH2], custom synthesized and HPLC-purified by Tufts University Core Facility) from 10 mM, then 1:2 dilution, 16-point in duplicate, pin-transferred to column 2, rows 1 to 32. Column 1 is neutral.
FITC-labeled RGS12-GoLoco peptide was custom-synthesized and HPLC-purified by Michael Berne of Tufts University
Assay Steps.
Four uL of reagents were dispensed to 1536-well Greiner black solid-bottom plates. Compounds and controls (23 nL) were transferred via Kalypsys PinTool. The plates were incubated for 10 min at room temperature, and then read on ViewLux (Perkin-Elmer) High-throughput CCD imager using FITC polarization filter sets for the fluorescein-labeled decapeptide probe.During dispense, reagent bottles were kept submerged into 4 deg C water bath and all liquid lines were covered with aluminum foil to minimize probe and protein degradation. All screening operations were performed on a Kalypsys robotic system (Kalypsys, Inc., San Diego, CA) containing one RX-130 and two RX-90 anthropomorphic robotic arms.
Comment
Keywords: NIH Roadmap, MLSCN, MLI, MLSMR, qHTS, NCGC, rgs, rgs12, regulator of G-protein signalling 12, GTPase accelerating protein
Compound Ranking:
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
From ChEMBL:
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeIndicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive.String
2Potency*Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.FloatμM
3EfficacyMaximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.Float%
4Analysis CommentAnnotation/notes on a particular compound's data or its analysis.String
5Curve_DescriptionA description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically signficant, but below 80% of control.String
6Fit_LogAC50The logarithm of the AC50 from a fit of the ratio data to the Hill equation (calculated based on Molar Units).Float
7Fit_HillSlopeThe Hill slope from a fit of the ratio data to the Hill equation.Float
8Fit_R2R^2 fit value of ratio curve. Closer to 1.0 equates to better Hill equation fit.Float
9Fit_InfiniteActivityThe asymptotic efficacy from a fit of the ratio data to the Hill equation.Float%
10Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the ratio data to the Hill equation.Float%
11Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12Excluded_PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
13Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14Activity at 0.0007320000 uM (0.000732μM**)% Activity at given concentration.Float%
15Activity at 0.00366 uM (0.00365829μM**)% Activity at given concentration.Float%
16Activity at 0.018 uM (0.0182914μM**)% Activity at given concentration.Float%
17Activity at 0.091 uM (0.0914509μM**)% Activity at given concentration.Float%
18Activity at 0.457 uM (0.457226μM**)% Activity at given concentration.Float%
19Activity at 2.286 uM (2.28599μM**)% Activity at given concentration.Float%
20Activity at 11.43 uM (11.4293μM**)% Activity at given concentration.Float%
21Activity at 57.14 uM (57.1429μM**)% Activity at given concentration.Float%
22Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: NS053754-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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