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BioAssay: AID 2390

Confirmation qHTS Assay for Inhibitors of RGS12 GoLoco Motif Activity (Red 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(112)
 
 
Active(59)
 
 
Inactive(24)
 
 
Inconclusive(29)
 
 
 Tested Substances
 Tested Substances
All(112)
 
 
Active(59)
 
 
Inactive(24)
 
 
Inconclusive(29)
 
 
AID: 2390
Data Source: NCGC (RGSP683)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-02-22

Data Table ( Complete ):           View Active Data    View All Data
Target
Sequence: RGS12 [Homo sapiens]
Description ..   
Protein Family: Regulator of G-protein signaling 12 Phosphotyrosine-binding (PTB) PH-like fold

Gene:RGS12     Related Protein 3D Structures     More BioActivity Data..
BioActive Compounds: 59
Related Experiments
AIDNameTypeComment
879qHTS Assay for Inhibitors of RGS12 GoLoco Motif Activity (Green Fluorophore)Confirmatorydepositor-specified cross reference: qHTS Assay for Inhibitors of RGS12 GoLoco Motif Activity (Green Fluorophore)
880qHTS Assay for Inhibitors of RGS12 GoLoco Motif Activity (Red Fluorophore)Confirmatorydepositor-specified cross reference: qHTS Assay for Inhibitors of RGS12 GoLoco Motif Activity (Red Fluorophore)
2406Probe Development Summary for Inhibitors of RGS12 GoLoco Motif ActivitySummarydepositor-specified cross reference
Description:
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.

This BioAssay is a confirmatory run of primary qHTS protocol (AID: 880).

See following reference for more details:
Kimple AJ, et al. Comb Chem High Throughput Screen. 2008 Jun;11(5):396-409.

Assay Submitter: Siderovski, David P, University of North Carolina at Chapel Hill
Screening Center PI: Austin, C.P.
Screening Center: NIH Chemical Genomics Center [NCGC]
NIH Grant: NS053754-01
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: 15 nM Rhodamine labeled peptide dispensed into columns 3 and 4 to generate negative control (full inhibition of binding, -100 % activity).
15 nM Rhodamine labeled peptide/25 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 20 mM, then 1:2 dilution, 16-point in duplicate, pin-transferred to column 2, rows 1 to 32. Column 1 is neutral.

Rhodamine-labeled peptide (Rhodamine-DEAEEFFELISKAQSNRADDQRGLLRKEDLVLPEFLR-NH2) was custom-synthesized and HPLC-purified by Invitrogen (Carlsbad, CA).


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 BODIPY for the rhodamine-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 significant, but below 80% of control.String
6Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
7Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
8Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
9Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
10Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the 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 0.0000068120 uM (6.81196e-06μM**)% Activity at given concentration.Float%
15Activity at 0.0000136239 uM (1.36239e-05μM**)% Activity at given concentration.Float%
16Activity at 0.0000385343 uM (3.85343e-05μM**)% Activity at given concentration.Float%
17Activity at 0.0001089913 uM (0.000108991μM**)% Activity at given concentration.Float%
18Activity at 0.0002179827 uM (0.000217983μM**)% Activity at given concentration.Float%
19Activity at 0.0003970423 uM (0.000397042μM**)% Activity at given concentration.Float%
20Activity at 0.0008719308 uM (0.000871931μM**)% Activity at given concentration.Float%
21Activity at 0.00145 uM (0.00145247μM**)% Activity at given concentration.Float%
22Activity at 0.00329 uM (0.00329464μM**)% Activity at given concentration.Float%
23Activity at 0.00698 uM (0.00697545μM**)% Activity at given concentration.Float%
24Activity at 0.012 uM (0.0120526μM**)% Activity at given concentration.Float%
25Activity at 0.027 uM (0.0273387μM**)% Activity at given concentration.Float%
26Activity at 0.056 uM (0.0558036μM**)% Activity at given concentration.Float%
27Activity at 0.100 uM (0.100011μM**)% Activity at given concentration.Float%
28Activity at 0.223 uM (0.223214μM**)% Activity at given concentration.Float%
29Activity at 0.366 uM (0.365866μM**)% Activity at given concentration.Float%
30Activity at 0.830 uM (0.82989μM**)% Activity at given concentration.Float%
31Activity at 1.786 uM (1.78571μM**)% Activity at given concentration.Float%
32Activity at 3.036 uM (3.03593μM**)% Activity at given concentration.Float%
33Activity at 6.886 uM (6.88638μM**)% Activity at given concentration.Float%
34Activity at 14.29 uM (14.2857μM**)% Activity at given concentration.Float%
35Activity at 25.19 uM (25.192μM**)% Activity at given concentration.Float%
36Activity at 57.14 uM (57.1429μM**)% Activity at given concentration.Float%
37Compound QCSource of compound QCString

* 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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