In search of a small molecule agonist of the relaxin receptor RXFP1 for the treatment of liver fibrosis

Andrew Mcbride,Ivan Caballero-Hernandez,Scott P Webster,Jonathan A Fallowfield,Rebecca L Aucott,Duncan S Holmes,Sapna Desai,Mark J Bamford,Theresa Pell,Jeremy Griggs,Martin P Ruediger,Danuta E Mossakowska,Kate Simpson,Anna M Hoy,John P Iredale

doi:10.1038/s41598-017-10521-9

Abstract

The peptide hormone human relaxin-2 (H2-RLX) has emerged as a potential therapy for cardiovascular and fibrotic diseases, but its short in vivo half-life is an obstacle to long-term administration. The discovery of ML290 demonstrated that it is possible to identify small molecule agonists of the cognate G-protein coupled receptor for H2-RLX (relaxin family peptide receptor-1 (RXFP1)). In our efforts to generate a new medicine for liver fibrosis, we sought to identify improved small molecule functional mimetics of H2-RLX with selective, full agonist or positive allosteric modulator activity against RXFP1. First, we confirmed expression of RXFP1 in human diseased liver. We developed a robust cellular cAMP reporter assay of RXFP1 signaling in HEK293 cells transiently expressing RXFP1. A high-throughput screen did not identify further specific agonists or positive allosteric modulators of RXFP1, affirming the low druggability of this receptor. As an alternative approach, we generated novel ML290 analogues and tested their activity in the HEK293-RXFP1 cAMP assay and the human hepatic cell line LX-2. Differences in activity of compounds on cAMP activation compared with changes in expression of fibrotic markers indicate the need to better understand cell- and tissue-specific signaling mechanisms and their disease-relevant phenotypes in order to enable drug discovery.

Highlights

Fibrotic disorders represent an increasing cause of morbidity and mortality worldwide, contributing to an estimated 45% of all-cause mortality in the United States[1]
We investigate generation of additional RXFP1 agonists through a cAMP-mediated assay; high-throughput screening afforded no further specific agonists, we describe the identification of novel, ML290-related activators of RXFP1-mediated cAMP and their use, together with H2-RLX and ML290 in understanding pathways relevant to liver fibrosis, primarily in the hHSC line LX-2
To further explore the clinical relevance of RXFP1 as an antifibrotic target, we examined the expression and distribution of RXFP1 mRNA by in situ hybridization in liver biopsies from patients diagnosed with either early or advanced liver fibrosis due to non-alcoholic steatohepatitis (NASH; n = 2) and autoimmune hepatitis (AIH, n = 2)

Summary

Introduction

Fibrotic disorders represent an increasing cause of morbidity and mortality worldwide, contributing to an estimated 45% of all-cause mortality in the United States[1]. In the United Kingdom, liver fibrosis is an extraordinary exception to the major improvements made over the past 30 years in the treatment and outcomes for chronic disorders such as heart disease, stroke and many cancers[2]. Standardized mortality rates for liver disease have increased inexorably - by 400% since 1970, and in patients younger than 65 years by almost 500%2. Despite this significant clinical burden, and major advances in our understanding of the pathogenesis of liver fibrosis, there are still no approved antifibrotic therapies. The major profibrogenic cell type is the activated hepatic stellate cell-myofibroblast (HSC-MF), which synthesizes scar tissue and contributes to portal hypertension (PHT) by increasing intra-hepatic vascular resistance through sinusoidal contraction. Mutagenesis studies have shown that H2-RLX binds with high affinity to the LRRs of the extracellular domain and with lower affinity to the extracellular loops of the transmembrane domain[6,7,8,9]

Methods

Results

Conclusion