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 Gα_s and Gα_oB but weak coupling to Gα_i3. ML290 exhibited signalling bias at RXFP1 possessing a signalling profile indicative of vasodilator and anti-fibrotic properties.

Figure 1

ML290 and binding of ¹²⁵I-H2 relaxin to RXFP1 stably expressed in HEK293 cells. In (A) co-incubation with ML290 (90 min), increased total binding of ¹²⁵I-H2 relaxin to RXFP1 in a concentration-dependent manner (n = 6). In (B) ML290 (1 μM) had no significant effect on competition by H2 relaxin for ¹²⁵I-H2 relaxin binding (n = 8). In kinetic studies, ML290 (1 μM) had no significant effect on (C) dissociation (n = 3) or (D) association rate (n = 6–7) of ¹²⁵I-H2 relaxin from RXFP1. Data are mean ± SEM of ‘n’ experiments.

Figure 2

RXFP1 - G protein interactions and treatment with H2 relaxin or ML290. HEK-RXFP1-Rluc8 cells were transiently co-transfected with Gγ2-Venus, Gβ1 and one of Gα subunits (Gα_s, Gα_oB, Gα_i3). Interactions between RXFP1 and G proteins were detected prior to and after treatment with H2 relaxin (0.1 μM) or ML290 (0.1 μM or 10 μM) using real-time BRET assays. Both ML290 and H2 relaxin induced interactions between RXFP1-Rluc8 and Gα_s, Gα_oB, and to a lesser extent Gα_i3 (A–C). Shifts in BRET ratio between RXFP1 and Gα_s were quantitatively and qualitatively similar with ML290 and H2 relaxin. However the ML290 RXFP1- Gα_oB ligand-induced BRET ratio moved in the opposite direction to that to H2 relaxin (B) suggesting that each ligand induces a different receptor conformation. Interactions between RXFP1 and Gα_i3 with H2 relaxin and ML290 were weak but qualitatively similar. There were no interactions between RXFP1-Rluc8 and Gγ2-Venus in the absence of Gα subunits (D). Ligand-induced BRET ratios were calculated by subtracting the BRET ratio for the vehicle-treated sample from that obtained from each ligand-treated sample as described in Materials and Methods. Data are mean ± SEM of 4 independent experiments.

Figure 3

Activation of ERK1/2, p38MAPK and generation of cAMP by H2 relaxin and ML290. In HEK-RXFP1 cells, H2 relaxin activated p-ERK1/2 (A) 5 min), p-p38MAPK (B) 15 min) and cAMP accumulation (C) 30 min) in a concentration-dependent manner. ML290 did not directly activate p-ERK1/2 (A), but did activate p38MAPK (B) with lower efficacy and cAMP accumulation with similar efficacy but significantly lower potency than H2 relaxin (C). 10 min pretreatment with ML290 enhanced p-ERK1/2 activation produced by relaxin (D) 4 min). Data are mean ± SEM for 4–8 independent experiments.

Figure 4

Signal transduction pathways activated by ML290 in human primary vascular cells and in human cardiac fibroblasts (HCF). In (A) ML290 (30 min) concentration-dependently increased cAMP accumulation in HCAECs (, n = 7), HUVECs (, n = 7), HUASMCs (, n = 5), HUVSMCs (, n = 4) but not in HUAECs that do not express cell surface RXFP1 (, n = 4); in (B) ML290 (30 min) also increased cGMP accumulation in HCAECs (n = 7), HUVECs (n = 7), HUASMCs (n = 7), HUVSMCs (n = 4) but not in HUAECs (n = 4); In (C) ML290 increased p-p38MAPK (15 min) but only in HUASMC and HUVSMCs (n = 3); In (D) ML290 did not cause ERK1/2 phosphorylation in HCAECs (n = 3), HUVECs (n = 3), HUASMCs (n = 3), HUVSMCs (n = 3) or HUAECs (n = 3). In HCFs, H2 relaxin activated cGMP accumulation in a concentration-dependent manner ((E) n = 5) (40 min) and p-ERK1/2 ((F) n = 3) (5 min). ML290 did not activate p-ERK1/2 (F; n = 3) but did induce cGMP accumulation (E; n = 5) in a concentration-dependent manner albeit with lower potency than H2 relaxin. Data shown are mean ± SEM of ‘n’ independent experiments.

Figure 5

The role of G proteins, βγ subunits and PI3-kinase in ML290-mediated cAMP and cGMP accumulation in human primary vascular cells. ML290 (10 μM, 30 min) increased cAMP accumulation in HCAECs ((A) n = 5), HUVECs ((B) n = 8), HUASMCs ((C) n = 8) and HUVSMCs ((D) n = 4). Treatment with the Gαs inhibitor NF449 (10 μM, 30 min) of HCAECs ((A) n = 3) and HUVECs ((B) n = 4) abolished ML290-mediated cAMP accumulation (30 min) whereas in HUASMCs ((C) n = 4) and HUVSMCs ((D) n = 4) it reduced the maximum cAMP response. The Gαi/Gα_OB inhibitor NF023 (10 μM, 30 min) in HCAECs ((A) n = 4) and HUVECs ((B) n = 4), HUASMCs ((C) n = 4) and HUVSMCs ((D) n = 4) had no effect on ML290-mediated cAMP accumulation. Pre-treatment of HCAECs ((A) n = 3) and HUVECs ((B) n = 3) with the Gβγ inhibitors mSIRK (5 μM, 30 min) and gallein (50 μM, 45 min) and the PI3-kinase inhibitor Wortmannin (100 nM, 30 min) had no effect on ML290-mediated cAMP accumulation whereas pre-treatment of HUASMCs ((C) n = 3) or HUVSMCs ((D) n = 3) with both Gβγ inhibitors and the PI3-kinase inhibitor reduced the maximum cAMP response to ML290. ML290 (10 μM, 30 min) also increased cGMP accumulation in HCAECs ((E) n = 3), HUVECs ((F) n = 4), HUASMCs ((G) n = 3) and HUVSMCs ((H) n = 3). NF449 (10 μM, 30 min) pre-treatment of HCAECs ((E) n = 4); HUVECs ((F) n = 3), HUASMCs ((G) n = 6) and HUVSMCs ((H) n = 4) reduced the maximum ML290-mediated cGMP response. Pretreatment with NF023 (10 μM, 30 min) of HCAECs ((E) n = 3), HUVECs ((F) n = 3), HUASMCs ((G) n = 4) and HUVSMCs ((H) n = 4) had no effect on ML290-mediated cGMP accumulation. Pretreatment with the Gβγ inhibitors mSIRK (5 μM, 30 min) and gallein (50 μM, 45 min) and the PI3-kinase inhibitor Wortmannin (100 nM, 30 min) had no effect on cGMP accumulation in HCAECs ((E) n = 3) and HUVECs ((F) n = 3) whereas in HUASMCs ((G) n = 5) and HUVSMCs ((H) n = 4), it reduced the maximum response. Pre-treatment of HUASMCs ((C,G) n = 4–11) and HUVSMCs ((D,H) n = 4–5) with mSIRK control peptide L9A (5 μM, 30 min) had no significant effect on ML290-mediated cAMP or cGMP accumulation. Statistical significance was assessed using a one-way ANOVA with a Dunnet’s post-hoc test compared to ML290 alone: **p < 0.01 and *p < 0.05. Data shown are mean ± SEM of ‘n’ independent experiments.

Figure 6

ML290 effects on MMP-2 expression and TGF-β1-induced Smad2 and Smad3 phosphorylation in HCFs. ML290 (1 μM) promoted MMP-2 activity to an equivalent extent to H2 relaxin (0.1 μM) over 72 hours. In (A upper) a representative cropped zymograph (see Figure S4) of duplicate samples from two separate experiments; in (A lower) mean ± SE OD MMP-2, expressed as the ratio of that of in the untreated control group. **p < 0.01 versus untreated control group. In HCFs treated for 72 hours, ML290 (1 μM) inhibited TGF-β1-induced increases in p-Smad2 (B, n = 4) and increases in p-Smad3 (C, n = 4) to a level similar to that with H2 relaxin (0.1 μM). TGF-β1 (2 ng/ml) increased p-Smad2 (B) and p-Smad3 (C) compared to vehicle treated samples. Neither H2 relaxin (0.1 μM) nor ML290 (1 μM) had a significant effect on p-Smad2 or p-Smad3 in cells not treated with TGF-β1 (B,C). Data are mean ± SEM of 4 independent experiments performed in duplicate, **p < 0.01 and *p < 0.05.

Figure 7

Chemical structure of ML290. 2-Isopropoxy-N-(2-(3-(tri fluoromethyl sulfonyl) phenyl carbamoyl) phenyl) benzamide.

Figure 8

Comparison of signalling pathways activated by ML290 and H2 relaxin through RXFP1 expressed in human primary vascular cells and myofibroblasts. In human primary myofibroblasts both H2 relaxin and ML290 have a profile corresponding to anti-fibrotic activity yet ML290 achieves this without activating ERK. In human primary vascular cells H2 relaxin activates cAMP, cGMP and pERK with similar potency whereas ML290 is biased towards cGMP signalling compared with cAMP and does not activate pERK.