ML290 is a biased allosteric agonist at the relaxin receptor RXFP1

Abstract

Activation of the relaxin receptor RXFP1 has been associated with improved survival in acute heart failure. ML290 is a small molecule RXFP1 agonist with simple structure, long half-life and high stability. Here we demonstrate that ML290 is a biased agonist in human cells expressing RXFP1 with long-term beneficial actions on markers of fibrosis in human cardiac fibroblasts (HCFs). ML290 did not directly compete with orthosteric relaxin binding and did not affect binding kinetics, but did increase binding to RXFP1. In HEK-RXFP1 cells, ML290 stimulated cAMP accumulation and p38MAPK phosphorylation but not cGMP accumulation or ERK1/2 phosphorylation although prior addition of ML290 increased p-ERK1/2 responses to relaxin. In human primary vascular endothelial and smooth muscle cells that endogenously express RXFP1, ML290 increased both cAMP and cGMP accumulation but not p-ERK1/2. In HCFs, ML290 increased cGMP accumulation but did not affect p-ERK1/2 and given chronically activated MMP-2 expression and inhibited TGF-β1-induced Smad2 and Smad3 phosphorylation. In vascular cells, ML290 was 10x more potent for cGMP accumulation and p-p38MAPK than for cAMP accumulation. ML290 caused strong coupling of RXFP1 to Gαs and GαoB but weak coupling to Gαi3. ML290 exhibited signalling bias at RXFP1 possessing a signalling profile indicative of vasodilator and anti-fibrotic properties.

Real‐time examination of cAMP activity at relaxin family peptide receptors using a BRET‐based biosensor

“… Further increases in cAMP activity have also been shown to occur in a number of cell types, including THP‐1, HEK‐RXFP1, and some human primary vascular cell types downstream of Gα i3 , Gβγ, PI3K, PKCζ, and adenylate cyclase 5 . Coupling of RXFP1 to Gα i3 has also been confirmed by BRET protein‐protein interaction studies, and is dependent on localization of RXFP1 and Gα i/o in membrane raft microdomains . This finding suggests that cAMP activity at RXFP1 is compartmentalized, which is consistent with the finding that in HEK‐RXFP1 cells, Gα s ‐induced cAMP activity affects CRE transcription, whereas Gα i3 ‐induced cAMP activity affects NFκB transcription .…”

“…Biased signaling has been shown to occur at relaxin family peptide receptors. For example, at RXFP1, H2 relaxin promotes ERK1/2 activation but ML290 does not . Relatedly, system bias has been shown, particularly for RXFP1.…”

“…Relatedly, system bias has been shown, particularly for RXFP1. For example, HEK‐RXFP1 cells signal via cAMP but not cGMP, whereas fibroblasts that natively express RXFP1 signal via cGMP but not cAMP, and human vascular cells that express RXFP1 signal through both . Measuring the temporal aspects of signaling facilitates accurate detection of biased ligands, and the use of native cells will facilitate understanding of how relaxin family peptide receptors signal in different cell backgrounds.…”

“…However, patterns of cAMP activity also depend on the cell type being stimulated. For example, fibroblasts that natively express RXFP1 show little or no increases in cAMP activity when stimulated with an RXFP1 agonist …”

Therapeutic effects of a small molecule agonist of the relaxin receptor ML290 in liver fibrosis

“…A partial list of biological processes, cellular components, and molecular functions revealed by Kyoto Encyclopedia of Genes and Genomes (KEGG; https://mww.genome.jp/kegg/kegg1.html) pathway and Reactome Pathway (https//reactome.org/) analysis is presented in Fig. 4 D . ML290 significantly affected ECM and collagen formation as well as the cellular pathways previously shown to be activated by relaxin or ML290/RXFP1 signaling, such as the MAPK, PI3K–protein kinase B, p53, cytokine, and IL signaling pathways (5, 28).…”