Identification and optimization of small-molecule agonists of the human relaxin hormone receptor RXFP1

Abstract

The anti-fibrotic, vasodilatory and pro-angiogenic therapeutic properties of recombinant relaxin peptide hormone have been investigated in several diseases, and recent clinical trial data has shown benefit in treating acute heart failure. However, the remodelling capacity of these peptide hormones is difficult to study in chronic settings because of their short half-life and the need for intravenous administration. Here we present the first small-molecule series of human relaxin/insulin-like family peptide receptor 1 agonists. These molecules display similar efficacy as the natural hormone in several functional assays. Mutagenesis studies indicate that the small molecules activate relaxin receptor through an allosteric site. These compounds have excellent physical and in vivo pharmacokinetic properties to support further investigation of relaxin biology and animal efficacy studies of the therapeutic benefits of relaxin/insulin-like family peptide receptor 1 activation.

Figure 1. Chemical structures of hit molecules 1 and 2 identified in a RXFP1 cAMP primary screening assay

Both molecules share a common chemical scaffold containing a core substructure of 2-acetamido-N-phenylbenzamide (in blue).

Figure 2. Structure activity relationship optimization campaign

Ten representative compounds, 1 and 3–11, highlight the key steps in the hit-to-lead evolution. The EC₅₀ and relative activity for each compound are shown using the RXFP1 primary screening assay. 100% relative activity was normalized to 57.7 µM forskolin stimulation, and 0% relative activity was normalized to compound vehicle control (0.58% DMSO). Complete concentration-response data are also provided (Supplementary Fig. 1).

Figure 3. Activation of VEGF gene expression in THP1 cells

THP1 cells were treated with compound vehicle control (0.58% DMSO), relaxin (10 ng/mL; 1.66 nM) or seven representative compounds 5–11 at 250 nM for 2 h. The level of VEGF gene expression was measured by quantitative real-time PCR and normalized to GAPDH expression and vehicle control as 1 (n=4 for each). All analogs other than compound 5 show significant (*P<0.05; **P<0.01; ***P<0.001) up-regulation of VEGF gene expression between treatment groups and vehicle control. All bars represent the mean values ± s.e.m.; statistical analysis was performed using two-tailed, Student’s t-test.

Figure 4. Compound 8 exhibits concentration-dependent increase of cellular impedance in RXFP1 transfected HEK293 cells

(a) Effect of relaxin (10 ng/mL; 1.66 nM), compared to compound 8 at 250 nM, 500 nM, and 750 nM on cell impedance in HEK293 cells stably transfected with RXFP1 (n=4 for each). The values at each point were normalized to the values of vehicle treatment. The subset of data points was used for the graph drawing. All points represent the mean values ± s.e.m. (b) Relative cell impedance normalized to the relaxin treatment group (100) at 30 min after addition of relaxin or compound 8. Columns represent the mean values ± s.e.m.. Differences, evaluated by two-tailed, Student’s t-test, between treatment groups and vehicle control are significant (***P<0.001). (c) Relaxin (10 ng/mL; 1.66 nM) and compound 8 at 750 nM did not affect cell impedance in parental HEK293 cells.

Figure 5. Three dimensional conformation of compound 8 determined by X-ray diffraction crystallography

Two intramolecular hydrogen binding interactions (dash lines) are identified with a bond length of 2.70 Å and 2.72 Å, respectively. Full parameters are also provided (Supplementary Table S3).

Figure 6. In vivo pharmacokinetic profile of compound 8

Mean plasma and heart concentration-time profiles of compound 8 ± s.e.m. after a single intraperitoneal (IP) dose of 30 mg/kg in male C57BL/6 mice (n = 3). No abnormal clinical observation was found during the in life phase. The IP dosing formulation solution was prepared in 10% NMP + 10% Solutol HS15 + 10% PEG400 + 70% saline. Full pharmacokinetic data and parameters are also provided (Supplementary Table S5).

Figure 7. Identification of RXFP1 region responsible for activation by compound 8

Human RXFP1 (for clarity denoted hRXFP1 in black) is fully activated (100%) after treatment with relaxin (15 nM) or compound 8 (66 µM). Mouse RXFP1 (denoted mRXFP1 in red) does not respond to compound 8 (marked as 0%) at 66 µM. RXFP1 contains extracellular, transmembrane, and intracellular (ICD) domains. Using chimeric mouse-human receptors the region responsible for RXFP1 activation by compound 8 was mapped to the part containing extracellular loop 3 (ECL3) of the transmembrane domain. Alignment of hRXFP1 and mRXFP1 shows two pairs of divergent amino acids within ECL3. The N-terminal IL to VV substitution in the mouse construct (mRXFP1-M10) did not rescue mouse receptor response, whereas C-terminal GT to DS substitution in human RXFP1 (hRXFP1-M11) abolished its compound 8 dependent activation. The mouse construct with (mRXFP1-M11) mutant was partially active and the mouse receptor with humanized ECL3 (mRXFP1-M10M11) was fully active after stimulation with compound 8. The cAMP response to compound 8 (66 µM) in cells transfected with a specific construct was normalized to the response of the same cells to relaxin (15 nM). The results represent the average of 3 independent experiments ± s.e.m. repeated in quadruplicates. **P<0.01 vs mRXFP1 by Student’s t-test.