qHTS Assay for Agonists of the Relaxin Receptor RXFP1: VEGF expression
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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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.
After the primary screen and SAR work, a probe and its analogs were tested in this selectivity assay. THP1 cells (human acute monocytic leukemia cell line) were used to analyze the stimulation of VEGF gene expression after treatment with relaxin or compounds. The VEGF stimulation in these cultured endometrial cells is a well-established property of relaxin.
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Production Centers Network [MLPCN]
MLPCN Grant: R03 MH085705
Assay Submitter (PI): Alexander Agoulnik
400,000 THP1 cells (0.4 mL at 1x106 cells/mL) in test media (RPMI-1640 without phenol red, 0.5% FBS, 1x Pen/Strep, 0.05 mM of 2-mercaptoethanol) were seeded in each well on a 24-well plate. After 24 hours incubation at 37 degrees C, 5% CO2, relaxin or compounds were added for 2 hours. The cells were harvested and RNA was extracted by the Trizol (Invitrogen, Carlsbad, CA) method according to manufacturers' instructions. cDNA was synthesized by using Verso cDNA kit (Thermo Scientific, Waltham, MA) according to manufacturer's protocol. Quantitative real time RT-PCR for VEGF and GAPDH gene expression was done using a Roche LightCycler 480 (Roche Diagnostics, Indianapolis, IN) with the appropriate set of primers and probes spanning different exons. The relative fold change in VEGF mRNA level was calculated by the comparative Ct (2- Ct) method using GAPDH expression for normalization of RNA.
Compounds that showed an increase in relative gene expression are "active"; compounds that showed no increase in relative gene are "inactive"; compounds that are show similar gene expression as the control are "inconclusive".
PubChem Scores are assigned based on the increase of relative gene expression that was achieved. A score of 0 is assigned to inactive compounds.
Data Table (Concise)