Abstract Body (Do not enter title and authors here): Background: Dysregulated smooth muscle cell (SMC) hypertrophy and proliferation contribute to vascular remodeling in pulmonary arterial hypertension (PAH). Relaxin-2 (RLN2), a peptide hormone known to exert potent vasodilator, anti-fibrotic, anti-apoptotic, and anti-inflammatory effects in diverse myogenic lineages, signals via G-protein coupled receptor (GPCR) Relaxin Family Peptide Receptor 1 (RXFP1). Therapeutic use of RLN2 has been limited by its short half-life and need for parenteral administration. ML290, an orally bioavailable small molecule allosteric agonist of human RXFP1, exhibits properties of biased GPCR signaling compared to RLN2.

Hypothesis: Biased modulation of relaxin-RXFP1 signaling via ML290 attenuates experimental PH.

Methods and Results: RXFP1 expression was upregulated in the smooth muscle layer of lung sections from patients with PAH compared to controls. In human pulmonary artery SMCs (HPASMCs), RLN2 (100 ng/mL) and ML290 (10 mM) inhibited TGFβ-SMAD2/3 signaling, and inhibited myofibrogenic differentiation of HPASMCs, blocking TGFβ-mediated expression of αSMA, Calponin, Caldesmon, fibronectin and type 1 Collagen. Both RLN2 and ML290 inhibited serum-induced and PDGF-BB-induced migration of HPASMCs. TRUPATH BRET assays performed in HEK cells revealed ML290 to be less potent in activating Gαi3, GαoB, and GαS than RLN2, and that ML290 elicted partial cAMP and cGMP responses. In contrast to RLN2, ML290 did not recruit β-arrestin 1 and had diminished activation of β-arrestin 2. Continuous infusion of RLN2 did not improve experimental PH in several rodent models. In contrast, ML290 (10 mg/kg daily oral gavage) attenuated right ventricular systolic pressure, RV hypertrophy and muscularization in humanized RXFP1 knock-in (hRXFP1^KI/KI) mice exposed to Sugen-hypoxia (SuHx). but not in WT or Rxfp1^-/- mice, demonstrating specificity for human RXFP1.

Conclusions: ML290 exhibits biased GPCR-mediated signaling of RXFP1 in comparison to the native ligand RLN2. ML290 potently inhibits TGFb-mediated plasticity of vascular SMCs, and demonstrates efficacy in experimental PH in RXFP1 KI mice. The efficacy of ML290 in contrast to RLN2 in experimental PH may be related to its biased signaling, pharmacokinetics, or pharmacodynamics.