Development of a synthetic relaxin-3/INSL5 chimeric peptide ligand for NanoBiT complementation binding assays.

INSL5 and relaxin-3 are relaxin family peptides with important roles in gut and brain function, respectively. They mediate their actions through the class A GPCRs RXFP4 and RXFP3. RXFP4 has been proposed to be a therapeutic target for colon motility disorders whereas RXFP3 targeting could be effective for neurological conditions such as anxiety. Validation of these targets has been limited by the lack of specific ligands and the availability of robust ligand-binding assays for their development. In this study, we have utilized NanoBiT complementation to develop a SmBiT-conjugated tracer for use with LgBiT-fused RXFP3 and RXFP4. The low affinity between LgBiT:SmBiT should result in a low non-specific luminescence signal and enable the quantification of binding without the tedious separation of non-bound ligands. We used solid-phase peptide synthesis to produce a SmBiT-labelled RXFP3/4 agonist, R3/I5, where SmBiT was conjugated to the B-chain N-terminus via a PEG12 linker. Both SmBiT-R3/I5 and R3/I5 were synthesized and purified in high purity and yield. Stable HEK293T cell lines expressing LgBiT-RXFP3 and LgBiT-RXFP4 were produced and demonstrated normal signaling in response to the synthetic R3/I5 peptide. Binding was first characterized in whole-cell binding kinetic assays validating that the SmBiT-R3/I5 bound to both cell lines with nanomolar affinity with minimal non-specific binding without bound and free SmBiT-R3/I5 separation. We then optimized membrane binding assays, demonstrating easy and robust analysis of both saturation and competition binding from frozen membranes. These assays therefore provide an appropriate rigorous binding assay for the high-throughput analysis of RXFP3 and RXFP4 ligands.

Hepatic macrophages act as a central hub for relaxin-mediated alleviation of liver fibrosis.

Relaxin is an antifibrotic peptide hormone previously assumed to directly reverse the activation of hepatic stellate cells for liver fibrosis resolution. Using nanoparticle-mediated delivery, here we show that, although relaxin gene therapy reduces liver fibrosis in vivo, in vitro treatment fails to induce quiescence of the activated hepatic stellate cells. We show that hepatic macrophages express the primary relaxin receptor, and that, on relaxin binding, they switch from the profibrogenic to the pro-resolution phenotype. The latter releases exosomes that promote the relaxin-mediated quiescence of activated hepatic stellate cells through miR-30a-5p. Building on these results, we developed lipid nanoparticles that preferentially target activated hepatic stellate cells in the fibrotic liver and encapsulate the relaxin gene and miR-30a-5p mimic. The combinatorial gene therapy achieves synergistic antifibrosis effects in models of mouse liver fibrosis. Collectively, our findings highlight the key role that macrophages play in the relaxin-primed alleviation of liver fibrosis and demonstrate a proof-of-concept approach to devise antifibrotic strategies through the complementary application of nanotechnology and basic science.

Effect of chimeric relaxin-like gonad-stimulating peptides on oocyte maturation and ovulation in the starfish Asterias rubens and Aphelasterias japonica.

A relaxin-like gonad-stimulating peptide (RGP), comprising two peptide chains (A- and B-chains) linked by two interchain bonds and one intrachain disulfide bond, acts as a gonadotropin in starfish. RGP orthologs have been identified in several starfish species, including Patiria pectinifera (PpeRGP), Asterias rubens (AruRGP) and Aphelasterias japonica (AjaRGP). To analyze species-specificity, this study examined the effects on oocyte maturation and ovulation in ovaries of A. rubens and A. japonica of nine RGP derivatives comprising different combinations of A- and B-chains from the three species. All nine RGP derivatives induced spawning in A. rubens and A. japonica ovaries. However, AruRGP, AjaRGP and their chimeric derivatives were more potent than peptides containing the A- or B-chain of PpeRGP. Three-dimensional models of the structures of the RGP derivatives revealed that residues in the B-chains, such as AspB6, MetB10 and PheB13 in PpeRGP and GluB7, MetB11, and TyrB14 in AruRGP and AjaRGP, respectively, are likely to be involved in receptor binding. Conversely, it is likely that ArgA18 in the A-chain of AruRGP and AjaRGP impairs binding of these peptides to the PpeRGP receptor in P. pectinifera. In conclusion, this study provides new insights into the structural basis of RGP bioactivity and RGP receptor activation in starfish.

Coatings Releasing the Relaxin Peptide Analogue B7-33 Reduce Fibrotic Encapsulation.

The development of antifibrotic materials and coatings that can resist the foreign body response (FBR) continues to present a major hurdle in the advancement of current and next-generation implantable medical devices, biosensors, and cell therapies. From an implant perspective, the most important issue associated with the FBR is the prolonged inflammatory response leading to a collagenous capsule that ultimately blocks mass transport and communication between the implant and the surrounding tissue. Up to now, most attempts to reduce the capsule thickness have focused on providing surface coatings that reduce protein fouling and cell attachment. Here, we present an approach that is based on the sustained release of a peptide drug interfering with the FBR. In this study, the biodegradable polymer poly(lactic-co-glycolic) acid (PLGA) was used as a coating releasing the relaxin peptide analogue B7-33, which has been demonstrated to reduce organ fibrosis in animal models. While in vitro protein quantification was used to demonstrate controlled release of the antifibrotic peptide B7-33 from PLGA coatings, an in vitro reporter cell assay was used to demonstrate that B7-33 retains activity against the relaxin family peptide receptor 1 (RXFP1). Subcutaneous implantation of PLGA-coated polypropylene samples in mice with and without the peptide demonstrated a marked reduction in capsule thickness (49.2%) over a 6 week period. It is expected that this novel approach will open the door to a range of new and improved implantable medical devices.

Engineering of chimeric peptides as antagonists for the G protein-coupled receptor, RXFP4.

Insulin-like peptide 5 (INSL5) is a very important pharma target for treating human conditions such as anorexia and diabetes. However, INSL5 with two chains and three disulfide bridges is an extremely difficult peptide to assemble by chemical or recombinant means. In a recent study, we were able to engineer a simplified INSL5 analogue 13 which is a relaxin family peptide receptor 4 (RXFP4)-specific agonist. To date, however, no RXFP4-specific antagonist (peptide or small molecule) has been reported in the literature. The focus of this study was to utilize the non-specific RXFP3/RXFP4 antagonist ΔR3/I5 as a template to rationally design an RXFP4 specific antagonist. Unexpectedly, we demonstrated that ΔR3/I5 exhibited partial agonism at RXFP4 when expressed in CHO cells which is associated with only partial antagonism of INSL5 analogue activation. In an attempt to improve RXFP4 specificity and antagonist activity we designed and chemically synthesized a series of analogues of ΔR3/I5. While all the chimeric analogues still demonstrated partial agonism at RXFP4, one peptide (Analogue 17) exhibited significantly improved RXFP4 specificity. Importantly, analogue 17 has a simplified structure which is more amenable to chemical synthesis. Therefore, analogue 17 is an ideal template for further development into a specific high affinity RXFP4 antagonist which will be an important tool to probe the physiological role of RXFP4/INSL5 axis.

A novel approach for preparing disulfide-rich peptide-KLH conjugate applicable to the antibody production.

To produce the antiserum against a small peptide, the target peptide-keyhole limpet hemocyanine (KLH) conjugate is generally used as an antigen, although the disulfide-rich peptide-KLH conjugate is still difficult to prepare. In our previous study, we have developed a preparation method of the disulfide-rich peptide-KLH conjugate, and this method was applied to produce the antiserum against a relaxin-like peptide. However, this method is limited to the synthetic peptide antigen, and is not applicable to a native or a recombinant peptide. In this study, to expand the applicability of this method to wide variety of peptides, we newly designed a novel thiol probe enabling the conjugation between various peptides and KLH, and applied it to produce the antiserum against relaxin-like peptide of a starfish Asterias amurensis. The antiserum obtained here showed high antibody-titer and good specificity, strongly suggesting that the method developed in this study is applicable to various peptides.

Single chain peptide agonists of relaxin receptors.

There are seven human relaxin family peptides that have two chains (A and B) and three disulfide bonds. The target receptors for four of these peptides are known as relaxin family peptide receptors, RXFP1-RXFP4. Detailed structure-activity relationship (SAR) studies of relaxin family peptides have been reported over the years and have led to the design of new analogs with agonistic and antagonistic properties. This review briefly summarizes the SAR of human relaxin 2 (H2 relaxin) and human relaxin 3 (H3 relaxin) leading to the design and development of single-B-chain only agonists, B7-33 and peptide 5. The physiological functions of these new peptides agonists in cellular and animal models are also described.

Functionality of an absolutely conserved glycine residue in the chimeric relaxin family peptide R3/I5.

The insulin superfamily is a group of homologous proteins that are further divided into the insulin family and relaxin family according to their distinct receptors. All insulin superfamily members contain three absolutely conserved disulfide linkages and a nonchiral Gly residue immediately following the first B-chain cysteine. The functionality of this conserved Gly residue in the insulin family has been studied by replacing it with natural L-amino acids or the corresponding unnatural D-amino acids. However, such analysis has not been conducted on relaxin family members. In the present study, we conducted chiral mutagenesis on the conserved B11Gly of the chimeric relaxin family peptide R3/I5, which is an efficient agonist for receptor RXFP3 and RXFP4. Similar to the effects on insulin family foldability, L-Ala or L-Ser substitution completely abolished the in vitro refolding of a recombinant R3/I5 precursor; whereas, D-Ala or D-Ser substitution had no detrimental effect on refolding of a semi-synthetic R3/I5 precursor, suggesting that the conserved Gly residue controls the foldability of relaxin family members. In contrast to the effect on insulin family activity, D-Ala or D-Ser replacement had no detrimental effect on the binding and activation potencies of the mature R3/I5 towards both RXFP3 and RXFP4, suggesting that the conserved Gly residue is irrelevant to the relaxin family's activity. The present study revealed functionality of the conserved B-chain Gly residue for a relaxin family peptide for the first time, providing an overview of its contribution to foldability and activity of the insulin superfamily.

Design and Synthesis of Potent, Long-Acting Lipidated Relaxin-2 Analogs.

Peptide hormone relaxin-2, a member of the insulin family of peptides, plays a key role in hemodynamics and renal function and has shown preclinical efficacy in multiple disease models, including acute heart failure, fibrosis, preeclampsia, and corneal wound healing. Recently, serelaxin, a recombinant version of relaxin-2, has been studied in a large phase 3 clinical trial (RELAX-AHF-2) for acute decompensated heart failure patients with disappointing outcome. The poor in vivo half-life of relaxin-2 may have limited its therapeutic efficacy and long-term cardiovascular benefit. Herein, we have developed a semisynthetic methodology and generated potent, fatty acid-conjugated relaxin analogs with long-acting pharmacokinetic (PK) profile in rodents. The enhanced PK properties translated into improved and long-lasting pharmacodynamic effect in pubic ligament elongation (PLE) studies. The resultant novel relaxin analog, R9-13, represents the first long-acting relaxin-2 analog and could potentially improve the clinical efficacy and outcome for this important peptide hormone. This semisynthetic methodology could also be applied to other cysteine-rich peptides and proteins for half-life extension.

Distinct but overlapping binding sites of agonist and antagonist at the relaxin family peptide 3 (RXFP3) receptor.

The relaxin-3 neuropeptide activates the relaxin family peptide 3 (RXFP3) receptor to modulate stress, appetite, and cognition. RXFP3 shows promise as a target for treating neurological disorders, but realization of its clinical potential requires development of smaller RXFP3-specific drugs that can penetrate the blood-brain barrier. Designing such drugs is challenging and requires structural knowledge of agonist- and antagonist-binding modes. Here, we used structure-activity data for relaxin-3 and a peptide RXFP3 antagonist termed R3 B1-22R to guide receptor mutagenesis and develop models of their binding modes. RXFP3 residues were alanine-substituted individually and in combination and tested in cell-based binding and functional assays to refine models of agonist and antagonist binding to active- and inactive-state homology models of RXFP3, respectively. These models suggested that both agonists and antagonists interact with RXFP3 via similar residues in their B-chain central helix. The models further suggested that the B-chain Trp27 inserts into the binding pocket of RXFP3 and interacts with Trp138 and Lys271, the latter through a salt bridge with the C-terminal carboxyl group of Trp27 in relaxin-3. R3 B1-22R, which does not contain Trp27, used a non-native Arg23 residue to form cation-π and salt-bridge interactions with Trp138 and Glu141 in RXFP3, explaining a key contribution of Arg23 to affinity. Overall, relaxin-3 and R3 B1-22R appear to share similar binding residues but may differ in binding modes, leading to active and inactive RXFP3 conformational states, respectively. These mechanistic insights may assist structure-based drug design of smaller relaxin-3 mimetics to manage neurological disorders.

Binding conformation and determinants of a single-chain peptide antagonist at the relaxin-3 receptor RXFP3.

The neuropeptide relaxin-3 and its receptor relaxin family peptide receptor-3 (RXFP3) play key roles in modulating behavior such as memory and learning, food intake, and reward seeking. A linear relaxin-3 antagonist (R3 B1-22R) based on a modified and truncated relaxin-3 B-chain was recently developed. R3 B1-22R is unstructured in solution; thus, the binding conformation and determinants of receptor binding are unclear. Here, we have designed, chemically synthesized, and pharmacologically characterized more than 60 analogues of R3 B1-22R to develop an extensive understanding of its structure-activity relationships. We show that the key driver for affinity is the nonnative C-terminal Arg23 Additional contributors to binding include amino acid residues that are important also for relaxin-3 binding, including Arg12, Ile15, and Ile19 Intriguingly, amino acid residues that are not exposed in native relaxin-3, including Phe14 and Ala17, also interact with RXFP3. We show that R3 B1-22R has a propensity to form a helical structure, and modifications that support a helical conformation are functionally well-tolerated, whereas helix breakers such as proline residues disrupt binding. These data suggest that the peptide adopts a helical conformation, like relaxin-3, upon binding to RXFP3, but that its smaller size allows it to penetrate deeper into the orthosteric binding site, creating more extensive contacts with the receptor.

Exploring receptor selectivity of the chimeric relaxin family peptide R3/I5 by incorporating unnatural amino acids.

Relaxin family peptides perform a variety of biological functions by activating four G protein-coupled receptors, namely RXFP1-4. Our recent study demonstrated that selectivity of the chimeric relaxin family peptide R3/I5 towards the homologous RXFP3 and RXFP4 can be modulated by replacement of the highly conserved nonchiral B23Gly or B24Gly with some natural l-amino acids. To investigate the mechanism of this modulating effect, in the present study we incorporated unnatural amino acids into the B23 or B24 position of a semi-synthetic R3/I5 that was prepared by a novel sortase-catalysed ligation approach using synthetic relaxin-3 B-chain and recombinant INSL5 A-chain. R3/I5 was a weak agonist for RXFP3 after B23Gly was replaced by D-Ala or D-Ser, but a strong antagonist for this receptor after B23Gly was replaced by corresponding l-amino acids. However, these replacements always resulted in a weak agonist for RXFP4. Thus, configuration of the B23 residue of R3/I5 affected activation of RXFP3 but not RXFP4. For the B24 residue, both size and configuration affected receptor selectivity of R3/I5. l-amino acids with an appropriate size, such as L-Ser and L-Abu, had the greatest effect on increasing the selectivity of R3/I5 towards RXFP3 over the homologous RXFP4. Our present results provided new insights into receptor selectivity of R3/I5, and would facilitate design of novel agonists or antagonists for RXFP3 and RXFP4 in future studies.

The relaxin receptor as a therapeutic target - perspectives from evolution and drug targeting.

The peptide relaxin was first identified as an important circulating hormone during pregnancy over 90 years ago. Research over many years defined the numerous biological roles that relaxin plays throughout pregnancy in many mammalian species. These important biological actions have led to the testing of relaxin as a therapeutic agent for a number of indications. The discovery of the relaxin receptor, RXFP1, in 2002 facilitated the better understanding of the cellular targets of relaxin, its mechanism of action and enabled the development of relaxin mimetics and screening for small molecule agonists. Additionally, the rapid expansion of the genome databases and bioinformatics tools has significantly advanced our understanding of the evolution of the relaxin/RXFP1 signaling system. It is now clear that the relaxin-RXFP1 signaling axis is far more ancient than previously appreciated with important roles for invertebrate relaxin-like peptides in reproductive and non-reproductive functions. This review summarizes these advances as well as developments in drug targeting of RXFP1. Hence the complex mode of activation of RXFP1 is discussed as is the discovery and development of a peptide mimetic and small molecule agonist. Detailed signaling studies are summarized which highlight the cell specific signaling of a peptide mimetic and biased signaling of a small molecule agonist. These studies highlight the complexities of targeting peptide GPCRs such as RXFP1.

ML290 is a biased allosteric agonist at the relaxin receptor RXFP1.

Activation of the relaxin receptor RXFP1 has been associated with improved survival in acute heart failure. ML290 is a small molecule RXFP1 agonist with simple structure, long half-life and high stability. Here we demonstrate that ML290 is a biased agonist in human cells expressing RXFP1 with long-term beneficial actions on markers of fibrosis in human cardiac fibroblasts (HCFs). ML290 did not directly compete with orthosteric relaxin binding and did not affect binding kinetics, but did increase binding to RXFP1. In HEK-RXFP1 cells, ML290 stimulated cAMP accumulation and p38MAPK phosphorylation but not cGMP accumulation or ERK1/2 phosphorylation although prior addition of ML290 increased p-ERK1/2 responses to relaxin. In human primary vascular endothelial and smooth muscle cells that endogenously express RXFP1, ML290 increased both cAMP and cGMP accumulation but not p-ERK1/2. In HCFs, ML290 increased cGMP accumulation but did not affect p-ERK1/2 and given chronically activated MMP-2 expression and inhibited TGF-ß1-induced Smad2 and Smad3 phosphorylation. In vascular cells, ML290 was 10x more potent for cGMP accumulation and p-p38MAPK than for cAMP accumulation. ML290 caused strong coupling of RXFP1 to Gαs and GαoB but weak coupling to Gαi3. ML290 exhibited signalling bias at RXFP1 possessing a signalling profile indicative of vasodilator and anti-fibrotic properties.

Relaxin-like peptides in male reproduction - a human perspective.

The relaxin family of peptide hormones and their cognate GPCRs are becoming physiologically well-characterized in the cardiovascular system and particularly in female reproductive processes. Much less is known about the physiology and pharmacology of these peptides in male reproduction, particularly as regards humans. H2-relaxin is involved in prostate function and growth, while insulin-like peptide 3 (INSL3) is a major product of the testicular Leydig cells and, in the adult, appears to modulate steroidogenesis and germ cell survival. In the fetus, INSL3 is a key hormone expressed shortly after sex determination and is responsible for the first transabdominal phase of testicular descent. Importantly, INSL3 is becoming a very useful constitutive biomarker reflecting both fetal and post-natal development. Nothing is known about roles for INSL4 in male reproduction and only very little about relaxin-3, which is mostly considered as a brain peptide, or INSL5. The former is expressed at very low levels in the testes, but has no known physiology there, whereas the INSL5 knockout mouse does exhibit a testicular phenotype with mild effects on spermatogenesis, probably due to a disruption of glucose homeostasis. INSL6 is a major product of male germ cells, although it is relatively unexplored with regard to its physiology or pharmacology, except that in mice disruption of the INSL6 gene leads to a disruption of spermatogenesis. Clinically, relaxin analogues may be useful in the control of prostate cancer, and both relaxin and INSL3 have been considered as sperm adjuvants for in vitro fertilization. LINKED ARTICLES: This article is part of a themed section on Recent Progress in the Understanding of Relaxin Family Peptides and their Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.10/issuetoc.

Relaxin family peptides: structure-activity relationship studies.

The human relaxin peptide family consists of seven cystine-rich peptides, four of which are known to signal through relaxin family peptide receptors, RXFP1-4. As these peptides play a vital role physiologically and in various diseases, they are of considerable importance for drug discovery and development. Detailed structure-activity relationship (SAR) studies towards understanding the role of important residues in each of these peptides have been reported over the years and utilized for the design of antagonists and minimized agonist variants. This review summarizes the current knowledge of the SAR of human relaxin 2 (H2 relaxin), human relaxin 3 (H3 relaxin), human insulin-like peptide 3 (INSL3) and human insulin-like peptide 5 (INSL5). LINKED ARTICLES: This article is part of a themed section on Recent Progress in the Understanding of Relaxin Family Peptides and their Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.10/issuetoc.

Synthetic Route to Human Relaxin-2 via Iodine-Free Sequential Disulfide Bond Formation.

A new synthetic route to human relaxin-2 has been established through a sequential disulfide bond formation process in the absence of iodine. It is enabled by a combination of cysteine protection with penicillin G acylase-labile Phacm and a newly identified thiol activator bis(5-(2-methoxyethoxy)-2-pyrimidinyl disulfide. The long-standing challenges in relaxin B-chain assembly and its poor solubility have been solved by the insertion of two isoacyl dipeptide segments. The overall yield was 25% from the B chain and 5.8% from the B-chain starting resin.

Hydrocarbon stapled B chain analogues of relaxin-3 retain biological activity.

Relaxin-3 or insulin-like peptide 7 (INSL7) is the most recently discovered relaxin/insulin-like family peptide. Mature relaxin-3 consists of an A chain and a B chain held by disulphide bonds. According to structure activity relationship studies, the relaxin-3 B chain is more important in binding and activating the receptor. RXFP3 (also known as Relaxin-3 receptor 1, GPCR 135, somatostatin- and angiotensin- like peptide receptor or SALPR) was identified as the cognate receptor for relaxin-3 by expression profiles and binding studies. Recent studies imply roles of this system in mediating stress and anxiety, feeding, metabolism and cognition. Stapling of peptides is a technique used to develop peptide drugs for otherwise undruggable targets. The main advantages of stapling include, increased activity due to reduced proteolysis, increased affinity to receptors and increased cell permeability. Stable agonists and antagonists of RXFP3 are crucial for understanding the physiological significance of this system. So far, agonists and antagonists of RXFP3 are peptides. In this study, for the first time, we have introduced stapling of the relaxin-3 B chain at 14th and 18th positions (14s18) and 18th and 22nd position (18s22). These stapled peptides showed greater helicity than the unstapled relaxin-3 B chain in circular dichroism analysis. Both stapled peptides bound RXFP3 and activated RXFP3 as observed in an inhibition of forskolin-induced cAMP assay and a ERK1/2 activation assay, although with different potencies. Therefore, we conclude that stapling of the relaxin3 B chain does not compromise its ability to activate RXFP3 and is a promising method for developing stable peptide agonists and antagonists of RXFP3 to aid relaxin-3/RXFP3 research.

A perfluoroaromatic abiotic analog of H2 relaxin enabled by rapid flow-based peptide synthesis.

H2 relaxin is a pleiotropic peptide hormone with clinical potential. Here we report on the reaction and use of hexafluorobenzene as an intramolecular disulfide replacement between Cys10 and Cys15 in the A-chain of H2 relaxin. Using flow-based Fmoc solid-phase peptide synthesis methodology we were able to obtain high-quality H2 relaxin fragments that were previously reported as challenging to synthesize. Subsequent native chemical ligation and oxidative folding enabled total synthesis of both wild type H2 relaxin and a C6F4 linked analog. Cell-based activity assays revealed modest activity for the C6F4 linked H2 relaxin analog, albeit 100-fold reduced relative to wild type. This work demonstrates how perfluoroarylation-cysteine SNAr chemistry may be a useful tool for the selective replacement of native disulfide bonds in proteins.

Chemical synthesis of peptides within the insulin superfamily.

The synthesis of insulin has inspired fundamental advances in the art of peptide science while simultaneously revealing the structure-function relationship of this centrally important metabolic hormone. This review highlights milestones in the chemical synthesis of insulin that can be divided into two separate approaches: (i) disulfide bond formation driven by protein folding and (ii) chemical reactivity-directed sequential disulfide bond formation. Common to the two approaches are the persistent challenges presented by the hydrophobic nature of the individual A-chain and B-chain and the need for selective disulfide formation under mildly oxidative conditions. The extension and elaboration of these synthetic approaches have been ongoing within the broader insulin superfamily. These structurally similar peptides include the insulin-like growth factors and also the related peptides such as relaxin that signal through G-protein-coupled receptors. After a half-century of advances in insulin chemistry, we have reached a point where synthesis is no longer limiting structural and biological investigation within this family of peptide hormones. The future will increasingly focus on the refinement of structure to meet medicinal purposes that have long been pursued, such as the development of a glucose-sensitive insulin. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.