Identification of small-molecule agonists of human relaxin family receptor 1 (RXFP1) by using a homogenous cell-based cAMP assay

Abstract

The relaxin hormone is involved in a variety of biological functions, including female reproduction and parturition, as well as regulation of cardiovascular, renal, pulmonary, and hepatic functions. It regulates extracellular matrix remodeling, cell invasiveness, proliferation, differentiation, and overall tissue homeostasis. The G protein-coupled receptor (GPCR) relaxin family receptor 1 (RXFP1) is a cognate relaxin receptor that mainly signals through cyclic AMP second messenger. Although agonists of the receptor could have a wide range of pharmacologic utility, until now there have been no reported small-molecule agonists for relaxin receptors. Here, we report the development of a quantitative high-throughput platform for an RXFP1 agonist screen based on homogenous cell-based HTRF cyclic AMP (cAMP) assay technology. Two small molecules of similar structure were independently identified from a screen of more than 365 677 compounds. Neither compound showed activity in a counterscreen with HEK293T cells transfected with an unrelated GPCR vasopressin 1b receptor. These small-molecule agonists also demonstrated selectivity against the RXFP2 receptor, providing a basis for future medicinal chemistry optimization of selective relaxin receptor agonists.

Keywords: GPCR; RXFP1; agonist; qHTS; relaxin; small molecule.

Figure 1. Assay miniaturization and validation

Concentration responses for (a) relaxin and (b) forskolin in HEK293-RXFP1 cells. Assays were performed in 384-well and 1,536-well formats using the HTRF cAMP assay kit. 100% activity was normalized as 57.7 μM forskolin stimulation and 0% activity was normalized as 0.58% DMSO controls. (c) HTRF signal from 0.57% DMSO, 28.75 nM relaxin and 57.5 μM forskolin treatments in HEK293-RXFP1 cells. Mean and standard deviations were calculated from 12 treatment wells in 384-well assay format. (d) Scatter plot of the ratiometric signal from a 1,536-well DMSO plate to establish assay performance parameters. Column 1 was treated with a relaxin titration (24.9 pM – 1.47 μM), columns 2 and 3 received DMSO, and column 4 received 125 nM relaxin.

Figure 2

Summary of qHTS and follow up assays.

Figure 3. Concentration response of human RXFP1 agonists

HTRF cAMP response in HEK293-RXFP1 (black), HEK293-RXFP2 (blue) and HEK293-V1b (green) cell lines and per cent viability (gray) for (a) relaxin, (b), forskolin, (c) MLS000579461 and (d) MLS000088899. (e) Concentration response of MLS000579461 and MLS000088899 in ELISA cAMP assay. (f) Structures of the two lead compounds. Cyclic AMP activities of compounds were normalized to the maximal response of forskolin (57.5 μM) as 100% activity and DMSO control (0.58% DMSO) as 0% activity.