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BioAssay: AID 2676

qHTS Assay for Agonists of the Relaxin Receptor RXFP1

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(362207)
 
 
Active(1084)
 
 
Inactive(357382)
 
 
Inconclusive(3787)
 
 
Unspecified(23)
 
 
 Tested Substances
 Tested Substances
All(365410)
 
 
Active(1093)
 
 
Inactive(360488)
 
 
Inconclusive(3806)
 
 
Unspecified(23)
 
 
AID: 2676
Data Source: NCGC (RXFP145)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-03-25

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 1084
Depositor Specified Assays
AIDNameTypeComment
492949qHTS Assay for Agonists of the Relaxin Receptor RXFP1: THP1 Hit Validationconfirmatory
602256qHTS Assay for Agonists of the Relaxin Receptor RXFP1: RXFP1 SARconfirmatory
492948qHTS Assay for Agonists of the Relaxin Receptor RXFP1: V1B Hit Validationconfirmatory
489012qHTS Assay for Agonists of the Relaxin Receptor RXFP1: RXFP1 Hit Validationconfirmatory
2703qHTS Assay for Agonists of the Relaxin Receptor RXFP1: Summarysummary
489043qHTS Assay for Agonists of the Relaxin Receptor RXFP1: RXFP2 Hit Validationconfirmatory
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.
Protocol
NCGC Assay Protocol Summary:

This is a cell-based assay for cAMP second messenger response to screen for agonists of the RXFP1 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 RXFP1 cAMP assay:
(1) Add 3 ul HEK293 RXFP1 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.
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 0.018 uM (0.0184μM**)% Activity at given concentration.Float%
15Activity at 0.092 uM (0.092μM**)% Activity at given concentration.Float%
16Activity at 0.460 uM (0.46μM**)% Activity at given concentration.Float%
17Activity at 2.300 uM (2.3μM**)% Activity at given concentration.Float%
18Activity at 11.50 uM (11.5μM**)% Activity at given concentration.Float%
19Activity at 57.50 uM (57.5μM**)% Activity at given concentration.Float%
20Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

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

Data Table (Concise)
Classification
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