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BioAssay: AID 489043

qHTS Assay for Agonists of the Relaxin Receptor RXFP1: RXFP2 Hit Validation

The relaxin hormone is involved in the variety of biological functions in normal tissues and diseases. The role of relaxin is well-established in female reproduction and parturition, mammary gland and endometrial development, maintenance of myometrial quiescence during pregnancy. Relaxin signaling through its G protein-coupled receptor (GPCR) RXFP1 results in ECM remodeling through regulation of more ..
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 Tested Compounds
 Tested Compounds
All(467)
 
 
Active(175)
 
 
Inactive(109)
 
 
Inconclusive(182)
 
 
Unspecified(1)
 
 
 Tested Substances
 Tested Substances
All(470)
 
 
Active(176)
 
 
Inactive(111)
 
 
Inconclusive(182)
 
 
Unspecified(1)
 
 
AID: 489043
Data Source: NCGC (RXFP148)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-11-23

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 175
Related Experiments
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AIDNameTypeComment
2676qHTS Assay for Agonists of the Relaxin Receptor RXFP1Confirmatorydepositor-specified cross reference: qHTS assay for agonists of the relaxin receptor RXFP1##
2703qHTS Assay for Agonists of the Relaxin Receptor RXFP1: SummarySummarydepositor-specified cross reference: Summary assay for agonists of the relaxin receptor RXFP1
489012qHTS Assay for Agonists of the Relaxin Receptor RXFP1: RXFP1 Hit ValidationConfirmatorydepositor-specified cross reference: Hit validation assay for agonists of the relaxin receptor RXFP1
492948qHTS Assay for Agonists of the Relaxin Receptor RXFP1: V1B Hit ValidationConfirmatorysame project related to Summary assay
492949qHTS Assay for Agonists of the Relaxin Receptor RXFP1: THP1 Hit ValidationConfirmatorysame project related to Summary assay
602256qHTS Assay for Agonists of the Relaxin Receptor RXFP1: RXFP1 SARConfirmatorysame project related to Summary assay
602258qHTS Assay for Agonists of the Relaxin Receptor RXFP1: RXFP2 SARConfirmatorysame project related to Summary assay
602262qHTS Assay for Agonists of the Relaxin Receptor RXFP1: V1B SARConfirmatorysame project related to Summary assay
602286qHTS Assay for Agonists of the Relaxin Receptor RXFP1: Mouse Liver Microsome StabilityOthersame project related to Summary assay
602287qHTS Assay for Agonists of the Relaxin Receptor RXFP1: PBS SolubilityOthersame project related to Summary assay
602288qHTS Assay for Agonists of the Relaxin Receptor RXFP1: Cytotoxicity SARConfirmatorysame project related to Summary assay
602290qHTS Assay for Agonists of the Relaxin Receptor RXFP1: VEGF expressionOthersame project related to Summary assay
602291qHTS Assay for Agonists of the Relaxin Receptor RXFP1: Cell ImpedanceOthersame project related to Summary assay
Description:
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Production Centers Network [MLPCN]

MLPCN Grant: R03 MH085705-01A1
Assay Submitter (PI): Alexander Agoulnik

NCGC Assay Overview:

The relaxin hormone is involved in the variety of biological functions in normal tissues and diseases. The role of relaxin is well-established in female reproduction and parturition, mammary gland and endometrial development, maintenance of myometrial quiescence during pregnancy. Relaxin signaling through its G protein-coupled receptor (GPCR) RXFP1 results in ECM remodeling through regulation of collagen deposition, cell invasiveness, proliferation and overall tissue homeostasis. Significantly, the therapeutic effects of relaxin in the treatment of renal, cardiac, skin, lung fibrosis, inflammation, and wound healing in animal models are well-established. Recombinant human relaxin (rhRlx) is currently being tested in clinical trials as a protective agent in congestive heart failure, in treating severe preeclampsia, and as an anti-fibrotic agent in systemic sclerosis.

Upon relaxin binding RXFP1 activation leads to the activation of adenylate cyclase (AC) via Gs. cAMP will activate PKA, which phosphorylates many signaling proteins. Thus, detection of cAMP increase is an easy and reliable indication of relaxin receptor activation. To screen for agonists of the relaxin receptor, a HEK293T cell line stably transfected with RXFP1 was used. RXFP1 activation was assayed by changes in cAMP levels as detected with a time-resolved fluorescence energy transfer (TR-FRET) cAMP detection kit.

This assay measures activation of a cell expressing RXFP2, another GPCR targeted by relaxin, to determine the selectivity of compounds for RXFP1.
Protocol
NCGC Assay Protocol Summary:
This is a cell-based assay for cAMP second messenger response to screen for agonists of the RXFP2 receptor. The assay was conducted in the presence of a PDE4 inhibitor (Ro 20174). The cells were lysed and the cAMP levels were measured with a TR-FRET cAMP kit. Presence of cAMP in the cell lysate disrupts binding between the cryptate-labeled anti-cAMP and d2-labeled cAMP pair, leading to a decrease in the TR-FRET signal. Data were normalized to the controls for basal activity (buffer only) and 100% activity (29 nM porcine relaxin). The EC50 values were determined from concentration-response data modeled with the standard Hill equation
1536-well assay protocol for the HEK293 RXFP2 cAMP assay:
(1) Add 3 ul HEK293 RXFP2 cell culture. 2000 cells/well in DMEM/F12, 10% FBS, 1x Pen/Strep (dispensed offline with MultiDrop Combi).
(2) Incubate 16-24 hr at 37C, 5% CO2.
(3) Add 1 ul 400uM Ro20174 in DPBS, 0.05% BSA, 0.005% Tween 20.
(3) Add 23 nl compounds in DMSO solution. Final concentration was 0.5 - 58 uM.
(4) Incubate 30 min at 37C, 5% CO2.
(5) Add 1 ul cAMP-d2 in lysis buffer (HTRF cAMP HiRange kit, CisBio).
(6) Add 1 ul anti-cAMP antibody-cryptate in lysis buffer (HTRF cAMP HiRange kit, CisBio).
(7) Incubate 30 min at room temperature.
(6) Detect the assay plate in a ViewLux plate reader (PerkinElmer) with Ex=320 nm, Em1=615 nm and Em2=665 nm.
Keywords: relaxin, RXFP1, RXFP2, G-protein coupled receptor, cAMP, agonist, high throughput screening, MLSMR, MLSCN, NIH Roadmap, qHTS and NCGC
Comment
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 activators are ranked higher than compounds that showed apparent inhibition.
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 PubChem:
Assay Cell Type: HEK 293T
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 0.0000162214 uM (1.62214e-05μM**)% Activity at given concentration.Float%
15Activity at 0.0000486640 uM (4.8664e-05μM**)% Activity at given concentration.Float%
16Activity at 0.0000648854 uM (6.48854e-05μM**)% Activity at given concentration.Float%
17Activity at 0.0001344756 uM (0.000134476μM**)% Activity at given concentration.Float%
18Activity at 0.0003244270 uM (0.000324427μM**)% Activity at given concentration.Float%
19Activity at 0.0006442756 uM (0.000644276μM**)% Activity at given concentration.Float%
20Activity at 0.0009861444 uM (0.000986144μM**)% Activity at given concentration.Float%
21Activity at 0.00197 uM (0.00197144μM**)% Activity at given concentration.Float%
22Activity at 0.00296 uM (0.00295843μM**)% Activity at given concentration.Float%
23Activity at 0.00681 uM (0.00681308μM**)% Activity at given concentration.Float%
24Activity at 0.011 uM (0.0108925μM**)% Activity at given concentration.Float%
25Activity at 0.021 uM (0.020646μM**)% Activity at given concentration.Float%
26Activity at 0.053 uM (0.0531269μM**)% Activity at given concentration.Float%
27Activity at 0.080 uM (0.0798777μM**)% Activity at given concentration.Float%
28Activity at 0.160 uM (0.159687μM**)% Activity at given concentration.Float%
29Activity at 0.240 uM (0.239633μM**)% Activity at given concentration.Float%
30Activity at 0.552 uM (0.55186μM**)% Activity at given concentration.Float%
31Activity at 1.387 uM (1.38687μM**)% Activity at given concentration.Float%
32Activity at 2.010 uM (2.01μM**)% Activity at given concentration.Float%
33Activity at 4.303 uM (4.30328μM**)% Activity at given concentration.Float%
34Activity at 6.455 uM (6.45537μM**)% Activity at given concentration.Float%
35Activity at 12.93 uM (12.9346μM**)% Activity at given concentration.Float%
36Activity at 19.37 uM (19.3661μM**)% Activity at given concentration.Float%
37Activity at 44.72 uM (44.7183μM**)% Activity at given concentration.Float%
38Activity at 114.9 uM (114.943μM**)% Activity at given concentration.Float%
39Activity at 125.0 uM (125μM**)% Activity at given concentration.Float%
40Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH085705-01A1

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