qHTS Assay for Agonists of the Relaxin Receptor RXFP1: V1B 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 ..
BioActive Compounds: 201
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 V1B, another GPCR targeted by relaxin, to determine the selectivity of compounds for RXFP1.
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 V1B 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
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
* Activity Concentration. ** Test Concentration.
Data Table (Concise)