AID	Name	Type	Comment
2703	qHTS Assay for Agonists of the Relaxin Receptor RXFP1: Summary	summary	Summary AID

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.

In the primary screen, 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. We conducted this followup assay to measure the effect of the lead candidates on cell health by measuring ATP levels (ATPLite).

NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Production Centers Network [MLPCN]

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

In this assay, the luciferase enzyme catalyzes the conversion of the added substrate D-luciferin to oxyluciferin and light with ATP. Thus, the emitted light is proportional to the ATP concentration. To evaluate the cytotoxic properties of the compounds, HEK293-RXFP1 cells were incubated with compounds for 72 h in growth media (DMEM 10% FBS, 1x Pen/Strep, 0.5 mg/mL G418) in 384-well format. After compound incubation, the levels of ATP in each well were measured with the addition of the ATPLite assay reagent.

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.

Grant Number: MH085705