Discovery of RXFP2 genetic association in resistant hypertensive men and RXFP2 antagonists for the treatment of resistant hypertension

Abstract

Hypertension remains a leading cause of cardiovascular and kidney diseases. Failure to control blood pressure with ≥ 3 medications or control requiring ≥ 4 medications is classified as resistant hypertension (rHTN) and new therapies are needed to reduce the resulting increased risk of morbidity and mortality. Here, we report genetic evidence that relaxin family peptide receptor 2 (RXFP2) is associated with rHTN in men, but not in women. This study shows that adrenal gland gene expression of RXFP2 is increased in men with hypertension and the RXFP2 natural ligand, INSL3, increases adrenal steroidogenesis and corticosteroid secretion in human adrenal cells. To address the hypothesis that RXFP2 activation is an important mechanism in rHTN, we discovered and characterized small molecule and monoclonal antibody (mAb) blockers of RXFP2. The novel chemical entities and mAbs show potent, selective inhibition of RXFP2 and reduce aldosterone and cortisol synthesis and release. The RXFP2 mAbs have suitable rat pharmacokinetic profiles to evaluate the role of RXFP2 in the development and maintenance of rHTN. Overall, we identified RXFP2 activity as a potential new mechanism in rHTN and discovered RXFP2 antagonists for the future interrogation of RXFP2 in cardiovascular and renal diseases.

Figure 1

RXFP2 is associated with resistant hypertension in men. (a) Manhattan plot of GWAS on resistant hypertension versus non-resistant hypertension in the 23andMe cohort. Locuszoom plots of the association upstream of RXFP2 in both men and women (b) and in women and men separately (c). The lead SNP (rs2146377) is highlighted. (d) RXFP2 allelic status is associated with RXFP2 gene expression changes in the adrenal gland (GTEx v8, www.gtexportal.org/home/snp/rs2146377, p = 1.4e-14). (e) Endogenous RXFP2 mRNA expression relative to beta-actin in adrenal gland of hypertensive men (n = 6) is increased compared to normotensive men (n = 6, p = 0.041, Mann–Whitney U test).

Figure 2

Characterization of RXFP activity assays and RXFP2 small molecule blockers in HTS and profiling assays. (a) RXFP2 ligands stimulate cAMP generation in cells with stable expression of a RXFP receptor. Treatment with INSL3 for 30 min dose-dependently increases cAMP in HEK293 cells with Bacmam expression of human RXFP2 (hRXFP2, n = 3) or rat RXFP2 (rRXFP2, n = 4). Treatment with relaxin (H2 Relaxin) for 30 min dose-dependently increases cAMP in HEK293 cells with Bacmam expression of human RXFP1 (n = 3). (b) Dose–response curves for RXFP2 antagonists in INSL3-stimulated cAMP in HEK293 cells with Bacmam expression of human RXFP2 (HEK hRXFP2) or rat RXFP2 (HEK rRXFP2). Dose–response curves for RXFP2 antagonists in relaxin-stimulated cAMP generation in HEK293 cells with Bacmam expression of human RXFP1 (HEK hRXFP1).

Figure 3

Characterization of RXFP2 mAbs in screening and profiling assays. (a) ELISA and cell binding data showing the binding distribution of all antibodies generated against RXFP2. The majority of antibodies generated bind to the LDLa-linker domain. (b) Biacore S200 ka vs kd binding data against rat LDLa-linker protein. (c) Biacore S200 ka vs kd binding data against human LDLa-linker protein. Three antibodies within the anti-RXFP2 panel show cross reactivity to the human LDLa-linker domain. KD values against human LDLa-linker protein: 2.75 nM (4B1), 11.7 nM (4F6), and 2.91 nM (4G6). (d, e) Representative dose–response curves of RXFP2 mAb treatment in INSL3 (EC₈₀)-stimulated cAMP assay in HEK cells stably expressing rat RXFP2 (d, n = 2) or human RXFP2 (e, n = 2). The INSL3 EC₈₀ of 13.96 nM and 2.92 nM were used, respectively. (f) Three-point RXFP2 mAb selectivity screen for activity against relaxin-stimulated cAMP activity in HEK cells stably expressing rat RXFP1 (n = 2). (g) Three-point RXFP2 mAb agonism screen for RXFP2 mAb activity in cAMP assay in HEK cell stably expressing rat RXFP2 (n = 2).

Figure 4

RXFP2 ligand INSL3 stimulates cAMP generation, steroidogenesis, and steroid hormone release in human adrenal cells H295R with stable expression of RXFP2. (a) Demonstration that treatment for 30 min with the RXFP2 ligand human INSL3 dose-dependently stimulates cAMP generation with EC₅₀ = 4 nM in H295R stably expressing human RXFP2 (n = 2). (b, c) INSL3 treatment for 48 h dose-dependently increases CYP11B1 (steroid 11β-hydroxylase enzyme) and CYP11B2 (aldosterone synthase) mRNA expression levels, fold change normalized to GAPDH, in H295R stably expressing human RXFP2 (n = 3). INSL3 EC₅₀ is 1.3 nM (CYP11B1) and 2.0 nM (CYP11B2). (d, e) Using ELISA detection, rat INSL3 dose-dependently increases cortisol secretion (EC₅₀ = 0.16 nM) and aldosterone secretion (EC₅₀ = 2.2 nM) in H295R cells stably expressing rat RXFP2 (n = 2).

Figure 5

RXFP2 antagonists block INSL3-stimulated steroidogenesis and corticosteroid secretion in human adrenal cortex cells H295R stably expressing RXFP2. (a) Dose–response curves of RXFP2 mAb treatment in H295R cells stably expressing rat RXFP2 stimulated with 3.5 nM rat INSL3 (EC₈₀) on CYP11B1 (steroid 11β-hydroxylase enzyme) and CYP11B2 (aldosterone synthase) mRNA expression levels (qPCR, relative to GAPDH control). IgG n = 6; all others n = 2. (b) The supernatants of cells stimulated with INSL3 and treated with the RXFP2 mAbs 1F2, 4G6, and 2D4 were assayed for cortisol (n = 2) and 2D4 was also evaluated for aldosterone (n = 2) concentration (ELISA). (c) Dose–response curve of RXFP2 small molecule antagonist GSK618069 in H295R cells stably expressing rat RXFP2 and stimulated with 3.5 nM rat INSL3 (EC₈₀) on CYP11B2 (aldosterone synthase) mRNA expression levels (qPCR, relative to GAPDH). (d) Proposed hypothesis for a role of RXFP2 in causing hypertension through adrenal steroidogenesis and secretion. Graphic was created using BioRender.com. Small molecule compound (SMC).

Figure 6

Pharmacokinetic study of RXFP2 mAbs in rat. (a) Six RXFP2 mAbs (1G1, 2D4, 4B1, 4G6, 1F2, 3F3) were discretely dosed to male Wistar Han rats (1 mg/kg, s.c., n = 3/mAb) once and the plasma concentration of RXFP2 mAbs was determined over 28 days. (b) Rat pharmacokinetic parameters calculated by non-compartmental analysis (NCA) analysis. Values are reported in mean ± standard deviation. ND: not determined due to insufficient data points or AUC extrapolated% > 20. C_max is the maximum concentration, T_max is the time to the maximum concentration, T_1/2 is the terminal half-life, Cl/F is the apparent clearance, AUC_last is the area under the concentration–time profile from time zero to the time of last quantifiable concentration.