107P London, UK
Pharmacology 2016

 

 

ML290, a small allosteric biased agonist at RXFP1

M. Kocan1, M. Sarwar1, S. Ang1, J. Xiao2, J. J. Marugan2, M. A. Hossain3, C. Wang4, D. S. Hutchinson1, C. Samuel4, A. I. Agoulnik5, R. A. Bathgate3, R. J. Summers1. 1Drug Discovery Biology, Monash University, Parkville, AUSTRALIA, 2NIH Chemical Genomics Center, NIH, Maryland, 3The Florey Institute of Neuroscience and Mental Health, Parkville, AUSTRALIA, 4Pharmacology, Monash University, Clayton, AUSTRALIA, 5Human and Molecular Genetics, Florida International University, Florida.

Introduction: Relaxin, an insulin-like peptide hormone that is the cognate ligand at relaxin family peptide receptor 1 (RXFP1) has vasodilator and anti-fibrotic properties and is in Phase III clinical trials for the treatment of acute heart failure. ML290 (2-Isopropoxy-N-(2-(3-(trifluoromethylsulfonyl)phenylcarbamoyl)phenyl)benzamide) is a small molecule allosteric agonist acting at RXFP11 but relatively little is known of its signaling profile.

Method: This study examined ML290-mediated MAPK, cAMP and cGMP signalling using Surefire or Alphascreen kits in human cells endogenously and recombinantly expressing RXFP12 and longer-term actions on markers of fibrosis including MMP2 expression and Smad2/3 phosphorylation in human cardiac fibroblasts (HCFs)2. Data points represent mean±SEM of n experiments.

Results: ML290 (1μM) did not affect the rate of association or dissociation of 125I-H2 relaxin, but did increase total binding to RXFP1 from 102.7±7.3% to 135.2±6.5% (P<0.05, n=6). In HEK-RXFP1 cells, ML290 stimulated cAMP accumulation and phosphorylation of p38MAPK but did not promote cGMP accumulation or phosphorylation of ERK1/2 and JNK1/2/3. In human primary vascular cells, ML290 increased cAMP and cGMP accumulation but not p-ERK1/2 in coronary artery (HCAEC) and umbilical vein endothelial cells (HUVEC), in umbilical artery (HUASMC) and umbilical vein smooth muscle cells (HUVSMC) but not in umbilical artery endothelial cells that do not display cell surface expression of RXFP1. In human cardiac fibroblasts (HCF), ML290 increased cGMP accumulation but had no effect on p-ERK1/2 and given chronically also activated MMP-2 expression and inhibited TGF-β1-induced Smad2 and Smad3 phosphorylation. ML290 increased p-p38MAPK only in smooth muscle but not endothelial cells. In vascular cells, ML290 was ten times more potent for cGMP accumulation and p-p38MAPK than for cAMP accumulation. ML290-mediated cAMP and cGMP accumulation was inhibited by NF449 (Gαs inhibitor) but not NF023 (Gαi inhibitor) in all vascular cells. In BRET studies ML290 caused strong coupling of RXFP1 to Gαs and GαoB but weak coupling to Gαi3.

Table 1 pEC50 values for signalling pathways activated by H2 relaxin and ML290 (n=3-7)

pERK1/2 pERK1/2 p-p38MAPK cGMP cAMP
H2-relaxin ML290 ML290 ML290 ML290
HEK-RXFP1 9.5 ± 0.3 NE 9.3 ± 0.6 NE 6.4 ± 0.1
HCAEC ND NE NE 7.2 ± 0.4 6.1 ± 0.3
HUVEC 9.2 ± 0.4# NE NE 7.2 ± 0.5 6.1 ± 0.5
HUASMC 9.1 ± 0.4# NE 8.6 ± 0.6 7.2 ± 0.5 6.1 ± 0.7
HUVSMC 9.2 ± 0.4# NE 8.5 ± 1.0 7.2 ± 0.6 6.2 ± 0.5

Conclusion: ML290 exhibited signalling and system bias at RXFP1 displaying a signalling profile indicative of vasodilator and anti-fibrotic properties. Understanding the signalling profile of drugs acting at RXFP1 is vital for drug development targeting this receptor.

References:

1. Xiao et al., (2013) Nature Comm 4: 1-7

2. Sarwar M et al. (2015) Br J Pharmacol 172: 1005-19