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. 2023 May 3;6(5):842-853.
doi: 10.1021/acsptsci.3c00053. eCollection 2023 May 12.

Development of Novel High-Affinity Antagonists for the Relaxin Family Peptide Receptor 1

Mohammed Akhter Hossain  1   2   3 , Praveen Praveen  1 , Nurhayati Ahmad Noorzi  4   5 , Hongkang Wu  1   3 , Ian P Harrison  4   5 , Thomas Handley  1 , Stavros Selemidis  6 , Chrishan S Samuel  4   5 , Ross A D Bathgate  1   3
Affiliations

Development of Novel High-Affinity Antagonists for the Relaxin Family Peptide Receptor 1

Mohammed Akhter Hossain et al. ACS Pharmacol Transl Sci. .

Abstract

H2 relaxin is a peptide hormone that exerts its biological actions through the G protein-coupled receptor, RXFP1. The numerous important biological functions of H2 relaxin, including potent renal, vasodilatory, cardioprotective, and anti-fibrotic actions, have resulted in considerable interest in its use as a therapeutic for various cardiovascular diseases and other fibrotic indications. Interestingly though, H2 relaxin and RXFP1 have been shown to be overexpressed in prostate cancer, allowing for the downregulation or blocking of relaxin/RXFP1 to decrease prostate tumor growth. These findings suggest the application of an RXFP1 antagonist for the treatment of prostate cancer. However, these therapeutically relevant actions are still poorly understood and have been hindered by the lack of a high-affinity antagonist. In this study, we chemically synthesized three novel H2 relaxin analogues that have complex insulin-like structures with two chains (A and B) and three disulfide bridges. We report here the structure-activity relationship studies on H2 relaxin that resulted in the development of a novel high-affinity RXFP1 antagonist, H2 B-R13HR (∼40 nM), that has only one extra methylene group in the side chain of arginine 13 in the B-chain (ArgB13) of H2 relaxin. Most notably, the synthetic peptide was shown to be active in a mouse model of prostate tumor growth in vivo where it inhibited relaxin-mediated tumor growth. Our compound H2 B-R13HR will be an important research tool to understand relaxin actions through RXFP1 and may be a potential lead compound for the treatment of prostate cancer.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Amino acid sequences, structure of H2 relaxin, and predicted structures of H2 variants. (A) Primary sequence of human relaxin 2 (H2 relaxin) and its variants, H2 B-R13HR, H2 B-R17HR, and H2 B-R13/17HR. (B) Solution NMR structure of H2 relaxin (Protein Data Bank (PDB): 2MV1) and simulated structures of HR-incorporated H2 relaxin analogues. Structural estimation of HR substitution into H2 relaxin was achieved with PyMOL v 2.0 using the SwissSideChain plugin, and arginine residues were replaced with HR before the side chains were manually aligned to the original arginine side chain. (C) Structures of arginine (R) and homoarginine (HR).
Figure 2
Figure 2
Activity of B-R13/17 modified peptides in comparison to H2 relaxin in HEK-RXFP1 cells. (A) Whole cell Eu-H2 relaxin competition binding assays. (B) cAMP activity expressed as percent maximum H2 relaxin response from CRE reporter gene assays. Data are expressed as the mean ± SEM of at least three experiments performed in triplicate.
Figure 3
Figure 3
cAMP accumulation activity of B-R13/17HR modified peptides in comparison to H2 relaxin in THP1 cells. Data are expressed as the percent maximum H2 relaxin response and represent the mean ± SEM of at least three experiments performed in triplicate.
Figure 4
Figure 4
Evaluation of the effects of H2 B-R13/17K, H2 B-R13/17HR, and H2 B-R13HR on the MMP-promoting effects of H2 relaxin and effects of H2 B-R13/17HR and H2 B-R13HR on the collagen-inhibitory effects of H2 relaxin in human cardiac myofibroblasts (HCMFs). (A) Representative gelatin zymograph of MMP-2 levels (gelatinase A; 72 kDa) that were secreted into the cell media from TGF-β1 (2 ng/mL)-stimulated HCMFs alone (lane 1) and TGF-β1-stimulated HCMFs that were co-treated with H2 relaxin (16.8 nM) alone (lane 2) or H2 relaxin and two different concentrations (1 or 0.1 μM) of H2 B-R13/17K (lanes 3 and 4, respectively), H2 B-R13/17HR (lanes 5 and 6, respectively) or H2 B-R13HR (lanes 7 and 8, respectively) or 0.1 μM H2 B-R13/17K (lanes 9), H2 B-R13/17HR (lanes 10) or H2 B-R13HR (lane 3) alone for 72 h in culture. Also shown is the relative mean ± SEM OD of MMP-2 from each of the groups evaluated, from n = 3 separate experiments conducted in duplicate. (B) Mean ± SEM collagen content from 500,000 TGF-β1-stimulated HCMFs as well as TGF-β1-stimulated HCMFs that were co-treated with H2 relaxin (16.8 nM) alone, H2 B-R13/17HR (1 μM) or H2 B-R13HR (1 μM) alone, or H2 relaxin+H2 B-R13/17HR or H2 relaxin+H2 B-R13HR for 72 h in culture, from n = 4 separate experiments conducted in duplicate. *p < 0.05, **p < 0.01 vs TGF-β1 alone; #p < 0.05, ##p < 0.01 vs (TGF-β1+)H2 relaxin-treated group.
Figure 5
Figure 5
Evaluation of the effects of H2 B-R13/17HR and H2 B-R13/HR on the prostate tumor growth-promoting effects of H2 relaxin. Shown is the prostate tumor size (mg) of RM1 cell-injected mice that were left untreated for 10 days versus RM1 cell-injected mice that were treated with H2 relaxin (0.15 mg/kg/day) alone, H2 B-R13/17HR (1.5 mg/kg/day) or H2 B-R13HR (1.5 mg/kg/day) alone, or H2 relaxin+H2 B-R13/17HR or H2 relaxin+H2 B-R13HR from days 2–10 post-RM1 cell administration; from n = 4–6 mice/group. **p < 0.01 vs untreated group; ##p < 0.01 vs H2 relaxin alone-treated group.
Figure 6
Figure 6
Evaluation of the effects of H2 B-R13/17HR and H2 B-R13HR on the MMP-promoting effects of H2 relaxin in mouse prostate tumors. (A) Representative zymograph of MMP-9 (gelatinase B; 92 kDa) and MMP-2 (gelatinase A; 72 kDa) levels from mouse prostate tumor-derived protein extracts from untreated mice (lanes 1 and 2) and mice treated with 0.15 mg/kg/day H2 relaxin alone (lanes 3 and 4); 0.15 mg/kg/day H2 relaxin+1.5 mg/kg/day H2 B-R13/17HR (lanes 5 and 6); 1.5 mg/kg/day H2 B-R13/17HR alone (lanes 7 and 8); 0.15 mg/kg/day H2 relaxin+1.5 mg/kg/day H2 B-R13HR (lanes 9 and 10); or 1.5 mg/kg/day H2 B-R13HR alone (lanes 11 and 12). Three separate zymographs analyzing 4–6 samples per group produced similar results. Relative mean ± SEM OD of (B) MMP-9 and (C) MMP-2 from each of the six groups analyzed, from n = 4–6 mice/group. **p < 0.01 vs untreated group; #p < 0.05, ##p < 0.01 vs H2 relaxin alone-treated group.
Figure 7
Figure 7
Circular dichroism spectra of H2 relaxin and HR-incorporated variants in phosphate buffer (pH 7.5) at 25 °C.
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