The Minimal Active Structure of Human Relaxin-2

Abstract

H2 relaxin is a peptide hormone associated with a number of therapeutically relevant physiological effects, including regulation of collagen metabolism and multiple vascular control pathways. It is currently in phase III clinical trials for the treatment of acute heart failure due to its ability to induce vasodilation and influence renal function. It comprises 53 amino acids and is characterized by two separate polypeptide chains (A-B) that are cross-linked by three disulfide bonds. This size and complex structure represents a considerable challenge for the chemical synthesis of H2 relaxin, a major limiting factor for the exploration of modifications and derivatizations of this peptide, to optimize effect and drug-like characteristics. To address this issue, we describe the solid phase peptide synthesis and structural and functional evaluation of 24 analogues of H2 relaxin with truncations at the termini of its peptide chains. We show that it is possible to significantly truncate both the N and C termini of the B-chain while still retaining potent biological activity. This suggests that these regions are not critical for interactions with the H2 relaxin receptor, RXFP1. In contrast, truncations do reduce the activity of H2 relaxin for the related receptor RXFP2 by improving RXFP1 selectivity. In addition to new mechanistic insights into the function of H2 relaxin, this study identifies a critical active core with 38 amino acids. This minimized core shows similar antifibrotic activity as native H2 relaxin when tested in human BJ3 cells and thus represents an attractive receptor-selective lead for the development of novel relaxin therapeutics.Human relaxin-2 or H2 relaxin is a peptide hormone with multiple pleiotropic actions (1). Initially thought to be only a reproductive hormone involved in facilitating delivery of the young, more recent studies have demonstrated that H2 relaxin plays a key role in inflammatory and matrix remodeling processes and possesses potent vasodilatory, angiogenic, and other cardioprotective actions (2). The vasodilatory effects of H2 relaxin are thought to involve promotion of nitric oxide and the gelatinases, matrix metalloproteinase-2 and matrix metalloproteinase-9, in addition to antagonism of the vasoconstricting actions of endothelin-1 and angiotensin II (3). This causes systemic and renal vasodilation, increased arterial compliance, and other vascular changes. These findings have led to current clinical trial evaluation of relaxin as drug for the treatment of patients with acute heart failure (AHF) 6 (4, 5). Furthermore, the matrix remodeling actions of H2 relaxin have enhanced its reputation as a rapidly acting but safe antifibrotic agent, which has been further supported by its ability to successfully inhibit and/or reverse fibrosis in every preclinical model of experimental disease evaluated to date (6, 7).All of the above actions of H2 relaxin are thought to be mediated through its native receptor RXFP1 (originally named LGR7), which is a leucine-rich repeat containing G-prote...

Identification of Key Residues Essential for the Structural Fold and Receptor Selectivity within the A-chain of Human Gene-2 (H2) Relaxin

“…Because of the numerous truncations, it was unclear which specific residue(s) contributed to the complete loss of RXFP2 activity, although Trp-28 is believed to be one of them (14,35). The alanine scanning results described above now suggest that Tyr-3 in the A-chain is another important residue for RXFP2 activity.…”

“…We have recently designed and created a truncated relaxin analog, H2:(A4 -24)(B7-24), having an active core of 38 amino acids that displayed high RXFP1 selectivity over RXFP2 (35). Because of the numerous truncations, it was unclear which specific residue(s) contributed to the complete loss of RXFP2 activity, although Trp-28 is believed to be one of them (14,35).…”

“…These treatments were carried out three or four separate times in duplicate or triplicate. The amount of collagen deposited by BJ3 cells into the matrix was then determined, as described before (35).…”

“…Hydroxyproline values were then converted to collagen content as detailed previously (35,39) and, in turn, divided by the dry weight of each corresponding left ventricular tissue assessed to yield collagen concentration (a measure of fibrosis).…”

“…BJ3 cells were maintained and plated for experimentation as detailed previously (35). TGF-␤1 (2 ng/ml)-stimulated cells were treated for 72 h with either native H2 relaxin (100 ng/ml; 16.8 nM) or an equivalent concentration of the H2:A(4 -24)(F23A) analog (16.8 nM), whereas TGF-␤1-unstimulated cells treated with or without the H2:A(4 -24)(F23A) analog were used as appropriate controls (from which basal collagen deposition could be measured).…”