Targeting the relaxin-3/RXFP3 system: a patent review for the last two decades.

INTRODUCTION: The neuropeptide relaxin-3/RXFP3 system belongs to the relaxin/insulin superfamily and is involved in many important physiological processes, such as stress responses, appetite control, and motivation for reward. Although relaxin-3 is the endogenous agonist for RXFP3, it can also bind to and activate RXFP1 and RXFP4. Consequently, research has been focused on the development of RXFP3-specific peptides and small-molecule ligands to validate the relaxin-3/RXFP3 system as a novel drug target. AREAS COVERED: This review provides an overview of patents on the relaxin-3/RXFP3 system covering ligand development and pharmacological studies since 2003. Related patents and literature reports were obtained from established sources including SciFinder, Google Patents, and Espacenet for patents and SciFinder, PubMed, and Google Scholar for literature reports. EXPERT OPINION: There has been an increasing amount of patent activities around relaxin-3/RXFP3, highlighting the importance of this novel neuropeptide system for drug discovery. The development of relaxin-3 derived peptides and small-molecule modulators, as well as behavioral studies in rodents, have shown that the relaxin-3/RXFP3 system is a promising drug target for treating various metabolic and neuropsychiatric diseases including obesity, anxiety, and alcohol addiction.

Human recombinant relaxin-2 (serelaxin) regulates the proteome, lipidome, lipid metabolism and inflammatory profile of rat visceral adipose tissue.

Recombinant human relaxin-2 (serelaxin) has been widely proven as a novel drug with myriad effects at different cardiovascular levels, which support its potential therapeutic efficacy in several cardiovascular diseases (CVD). Considering these effects, together with the influence of relaxin-2 on adipocyte physiology and adipokine secretion, and the connection between visceral adipose tissue (VAT) dysfunction and the development of CVD, we could hypothesize that relaxin-2 may regulate VAT metabolism. Our objective was to evaluate the impact of a 2-week serelaxin treatment on the proteome and lipidome of VAT from Sprague-Dawley rats. We found that serelaxin increased 1 polyunsaturated fatty acid and 6 lysophosphatidylcholines and decreased 4 triglycerides in VAT employing ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) based platforms, and that regulates 47 phosphoproteins using SWATH/MS analysis. Through RT-PCR, we found that serelaxin treatment also caused an effect on VAT lipolysis through an increase in the mRNA expression of hormone-sensitive lipase (HSL) and a decrease in the expression of adipose triglyceride lipase (ATGL), together with a reduction in the VAT expression of the fatty acid transporter cluster of differentiation 36 (Cd36). Serelaxin also caused an anti-inflammatory effect in VAT by the decrease in the mRNA expression of tumor necrosis factor α (TNFα), interleukin-1ß (IL-1ß), chemerin, and its receptor. In conclusion, our results highlight the regulatory role of serelaxin in the VAT proteome and lipidome, lipolytic function, and inflammatory profile, suggesting the implication of several mechanisms supporting the potential benefit of serelaxin for the prevention of obesity and metabolic disorders.

Periodic injections of Relaxin 2, its pharmacokinetics and remodeling of rat hearts.

Relaxin-2 (RLX), a critical hormone in pregnancy, has been investigated as a therapy for heart failure. In most studies, the peptide was delivered continuously, subcutaneously for 2 weeks in animals or intravenously for 2-days in human subjects, for stable circulating [RLX]. However, pulsatile hormone levels may better uncover the normal physiology. This premise was tested by subcutaneously injecting Sprague Dawley rats (250 g, N = 2 males, 2 females/group) with human RLX (0, 30, 100, or 500 µg/kg), every 12 h for 1 day, then measuring changes in Nav1.5, connexin43, and ß-catenin, 24 h later. Pulsatile RLX was measured by taking serial blood draws, post-injection. After an injection, RLX reached a peak in âˆ¼ 60 min, fell to 50 % in 5-6 h; injections of 0, 30, 100 or 500 µg/kg yielded peak levels of 0, 11.26 ± 3.52, 58.33 ± 16.10, and 209.42 ± 29.04 ng/ml and residual levels after 24-hrs of 0, 4.9, 45.1 and 156 pg/ml, respectively. The 30 µg/kg injections had no effect and 100 µg/kg injections increased Nav1.5 (25 %), Cx43 (30 %) and ß-catenin (90 %). The 500 µg/kg injections also increased Nav1.5 and Cx43 but were less effective at upregulating ß-catenin (up by 25 % vs. 90 %). Periodic injections of 100 µg/kg were highly effective at increasing the expression of Nav1.5 and Cx43 which are key determinants of conduction velocity in the heart and the suppression of arrhythmias. Periodic RLX is effective at eliciting changes in cardiac protein expression and may be a better strategy for its longer-term delivery in the clinical setting.

Silica-induced macrophage pyroptosis propels pulmonary fibrosis through coordinated activation of relaxin and osteoclast differentiation signaling to reprogram fibroblasts.

Silica nanoparticle (SiNP) exposure induces severe pulmonary inflammation and fibrosis, but the pathogenesis remains unclear, and effective therapies are currently lacking. To explore the mechanism underlying SiNPs-induced pulmonary fibrosis, we constructed in vivo silica exposure animal models and in vitro models of silica-induced macrophage pyroptosis and fibroblast transdifferentiation. We found that SiNP exposure elicits upregulation of pulmonary proteins associated with pyroptosis, including NLRP3, ASC, IL-1ß, and GSDMD, while the immunofluorescence staining co-localized NLRP3 and GSDMD with macrophage-specific biomarker F4/80 in silica-exposed lung tissues. However, the NLRP3 inhibitor MCC950 and classical anti-fibrosis drug pirfenidone (PFD) were found to be able to alleviate silica-induced collagen deposition in the lungs. In in vitro studies, we exposed the fibroblast to a conditioned medium from silica-induced pyroptotic macrophages and found enhanced expression of α-SMA, suggesting increased transdifferentiation of fibroblast to myofibroblast. In line with in vivo studies, the combined treatment of MCC950 and PFD was demonstrated to inhibit the expression of α-SMA and attenuate fibroblast transdifferentiation. Mechanistically, we adopted high throughput RNA sequencing on fibroblast with different treatments and found activated signaling of relaxin and osteoclast differentiation pathways, where the expression of the dysregulated genes in these two pathways was examined and found to be consistently altered both in vitro and in vivo. Collectively, our study demonstrates that SiNP exposure induces macrophage pyroptosis, which subsequently causes fibroblast transdifferentiation to myofibroblasts, in which the relaxin and osteoclast differentiation signaling pathways play crucial roles. These findings may provide valuable references for developing new therapies for pulmonary fibrosis.

Mechanisms of biased agonism by Gαi/o-biased stapled peptide agonists of the relaxin-3 receptor.

The neuropeptide relaxin-3 is composed of an A chain and a B chain held together by disulfide bonds, and it modulates functions such as anxiety and food intake by binding to and activating its cognate receptor RXFP3, mainly through the B chain. Biased ligands of RXFP3 would help to determine the molecular mechanisms underlying the activation of G proteins and ß-arrestins downstream of RXFP3 that lead to such diverse functions. We showed that the i, i+4 stapled relaxin-3 B chains, 14s18 and d(1-7)14s18, were Gαi/o-biased agonists of RXFP3. These peptides did not induce recruitment of ß-arrestin1/2 to RXFP3 by GPCR kinases (GRKs), in contrast to relaxin-3, which enabled the GRK2/3-mediated recruitment of ß-arrestin1/2 to RXFP3. Relaxin-3 and the previously reported peptide 4 (an i, i+4 stapled relaxin-3 B chain) did not exhibit biased signaling. The staple linker of peptide 4 and parts of both the A chain and B chain of relaxin-3 interacted with extracellular loop 3 (ECL3) of RXFP3, moving it away from the binding pocket, suggesting that unbiased ligands promote a more open conformation of RXFP3. These findings highlight roles for the A chain and the N-terminal residues of the B chain of relaxin-3 in inducing conformational changes in RXFP3, which will help in designing selective biased ligands with improved therapeutic efficacy.

Specific expression and blood kinetics for relaxin 2, lipocalin 2, and tissue factor pathway inhibitor 2 at the canine placenta and pregnant bloods.

In general, humoral factors released from the placenta influence pregnancy progression, but the involvement of the canine placenta is often unidentified. We investigated specific genes in canine placentas and analyzed the blood dynamics of the translated proteins. Furthermore, RNAs are known to be released from placentas embedding in exosomes, a type of extracellular vesicles. Here, the presence of cell-free RNAs in pregnant serums was also confirmed. RNA specimens were purified from the normal healthy dog placentas and applied to RNA-Seq analysis. Expressions of frequent genes were confirmed by RT-PCR using placentas from other individuals and breeds. Relaxin (RLN) 2, lipocalin (LCN) 2, and tissue factor pathway inhibitor (TFPI) 2 were selected as high-expressed and placenta-specific genes. By western blot, the three factors were clearly detected in the pregnant serums. Quantitative analysis revealed that the amount of RLN2 increased significantly from non-pregnancy to day 41 of pregnancy. Regarding LCN2 and TFPI2, the protein serum levels elevated during pregnancy, but the statistical differences were not detected. Exosomes were found in all pregnant serums; however, the percentage was less than 6% in total extracellular vesicles. The cell-free RNA related to RLN2 was detected, but no elevation was confirmed during pregnancy. We found specific genes in the canine placenta and the transition of their translated protein into the blood. These factors may become useful tools for research on canine pregnancy and monitoring of reproductive management. Exosomes and cell-free RNA could not be found to be valid in canine reproduction.

Specific knockout of notch-1 attenuates non-alcoholic fatty liver disease by promoting SHP2 phosphorylation.

OBJECTIVE: To investigate the effect of Notch-1 signaling on NAFLD and its molecular mechanism. METHODS: The lipid deposition in liver tissues was detected by oil red O staining. Western blotting was performed to detect the expressions of SREBP1C, SREBP2, LXR, IL-1ß, IL-18, NLRP3, Notch-1, NOX2, NOX4, p-PI3K and p-SHP2 in macrophages, and the expressions of ALIX, CD9, IL-1ß and SREBP1C in exosomes. Macrophages in the Notch-1MAC-KO group and Notch-1WT group were treated with FFA, and those in the Notch-1WT+FFA group and Notch-1MAC-KO+FFA group were treated with SHP2 inhibitors PHPS1 and Relaxin. RESULTS: It was observed by oil red O staining that lipid deposition in mice with NAFLD was reduced in the Notch-1MAC-KO group. The results of Western blotting showed that the expressions of ALIX, CD9, IL-1ß and SREBP1C in macrophage exosomes were significantly lower in the Notch-1MAC-KO group than in the Notch-1WT group. In macrophages, the expressions of SREBP1C, SREBP2, LXR, IL-1ß, IL-18, Notch-1, NOX2, NOX4 and p-PI3K significantly decreased, while the expression of p-SHP2 significantly increased in the Notch-1MAC-KO group compared with the Notch-1WT group. The Notch-1MAC-KO+FFA group had significantly decreased expressions of SREBP1C, NLRP3, IL-1ß, IL-18, SREBP2, NOX2, NOX4 and p-PI3K and a significantly increased expression of p-SHP2 compared with the Notch-1WT+FFA group. However, the differences in the above proteins were all eliminated after PHPS1 and Relaxin were added. CONCLUSION: Specific knockout of Notch-1 attenuates NAFLD, and reduces inflammation and lipid deposition in the liver by promoting SHP2 phosphorylation.

Investigating the role of the relaxin-3/RXFP3 system in neuropsychiatric disorders and metabolic phenotypes: A candidate gene approach.

The relaxin-3/RXFP3 system has been implicated in the modulation of depressive- and anxiety-like behaviour in the animal literature; however, there is a lack of human studies investigating this signalling system. We seek to bridge this gap by leveraging the large UK Biobank study to retrospectively assess genetic risk variants linked with this neuropeptidergic system. Specifically, we conducted a candidate gene study in the UK Biobank to test for potential associations between a set of functional, candidate single nucleotide polymorphisms (SNPs) pertinent to relaxin-3 signalling, determined using in silico tools, and several outcomes, including depression, atypical depression, anxiety and metabolic syndrome. For each outcome, we used several rigorously defined phenotypes, culminating in subsample sizes ranging from 85,881 to 386,769 participants. Across all outcomes, there were no associations between any candidate SNP and any outcome phenotype, following corrections for multiple testing burden. Regression models comprising several SNPs per relevant candidate gene as exploratory variables further exhibited no prediction of outcome. Our findings corroborate conclusions from previous literature about the limitations of candidate gene approaches, even when based on firm biological hypotheses, in the domain of genetic research for neuropsychiatric disorders.

The dual and multifaceted role of relaxin-2 in cancer

The continuous increase in cancer-associated deaths despite the substantial improvement in diagnosis and treatment has sparked discussions on the need for novel biomarkers and therapeutic strategies for cancer. Although increasing evidence has demonstrated the pivotal role of relaxin-2 in multiple cancers, their role is a double-edged sword with both protumor and antitumor having been reported in various malignant tumors. Considering this dual role, it appears the biological mechanism underpinning the action of relaxin-2 in cancer is not clear and further studies to elucidate their potential as a preventive factor for cancers are of prime importance. Herein, a summarized up-to-date report on the role of relaxin-2 in human cancer including detailed clinical and experimental evidence supporting their tumor-promoting and inhibitory functions in cancer development and progression has been elucidated. Also, signaling pathways and other factors orchestrating the activities of relaxin-2 in the tumor microenvironment has been discussed. Collectively, the evidence from this review has demonstrated the need for further evaluation of the role of relaxin-2 as a diagnostic and or prognostic biomarker for cancer (AU)

Novel RXFP3 negative allosteric modulator RLX-33 reduces alcohol self-administration in rats.

There is much interest in identifying novel pharmacotherapeutic targets that improve clinical outcomes for the treatment of alcohol use disorder (AUD). One promising target for therapeutic intervention is the relaxin family peptide 3 (RXFP3) receptor, a cognate receptor for neuropeptide relaxin-3, which has previously been implicated in regulating alcohol drinking behavior. Recently, we developed the first small-molecule RXFP3-selective negative allosteric modulator (NAM) RLX-33. Therefore, the goal of the present work was to characterize the impact of this novel NAM on affective-related behaviors and alcohol self-administration in rats. First, the effects of RLX-33 were tested on alcohol and sucrose self-administration in Wistar and alcohol-preferring P rats to determine the dose-response profile and specificity for alcohol. Then, we assessed the effects of systemic RLX-33 injection in Wistar rats in a battery of behavioral assays (open-field test, elevated zero maze, acoustic startle response test, and prepulse inhibition) and tested for alcohol clearance. We found that the lowest effective dose (5 mg/kg) reduced alcohol self-administration in both male and female Wistar rats, while in alcohol-preferring P rats, this effect was restricted to males, and there were no effects on sucrose self-administration or general locomotor activity. The characterization of affective and metabolic effects in Wistar rats generally found few locomotor, affective, or alcohol clearance changes, particularly at the 5 mg/kg dose. Overall, these findings are promising and suggest that RXFP3 NAM has potential as a pharmacological target for treating AUD.

Noncovalent Peptide Stapling Using Alpha-Methyl-l-Phenylalanine for α-Helical Peptidomimetics.

Peptides and peptidomimetics are attractive drug candidates because of their high target specificity and low-toxicity profiles. Developing peptidomimetics using hydrocarbon (HC)-stapling or other stapling strategies has gained momentum because of their high stability and resistance to proteases; however, they have limitations. Here, we take advantage of the α-methyl group and an aromatic phenyl ring in a unique unnatural amino acid, α-methyl-l-phenylalanine (αF), and propose a novel, noncovalent stapling strategy to stabilize peptides. We utilized this strategy to create an α-helical B-chain mimetic of a complex insulin-like peptide, human relaxin-3 (H3 relaxin). Our comprehensive data set (in vitro, ex vivo, and in vivo) confirmed that the new high-yielding B-chain mimetic, H3B10-27(13/17αF), is remarkably stable in serum and fully mimics the biological function of H3 relaxin. H3B10-27(13/17αF) is an excellent scaffold for further development as a drug lead and an important tool to decipher the physiological functions of the neuropeptide G protein-coupled receptor, RXFP3.

The role of relaxin in anterior cruciate ligament injuries: a systematic review.

OBJECTIVE: Anterior cruciate ligament (ACL) tears are exceedingly common among the athletic population and are seen with higher incidence in females. Observational studies have noted peak ACL tear rates in the luteal phase of the menstrual cycle, a time in which the hormone relaxin peaks in serum concentration. METHODS: A systematic review of the literature was performed. Inclusion criteria specified all prospective and retrospective studies which included the role of relaxin in the pathogenesis of ACL tears. RESULTS: Six studies met inclusion criteria yielding 189 subjects from clinical studies and 51 in vitro samples. Included studies found that ACL samples exhibit selective relaxin binding. When pre-treated with estrogen prior to relaxin exposure, female ACL tissue samples exhibit increased expression of collagen degrading receptors. CONCLUSION: Relaxin displays binding specificity to the female ACL and increased serum concentrations are correlated with increased ACL tear rates in female athletes. Further research is needed in this area. LEVEL OF EVIDENCE: V.

Localization of relaxin-like gonad-stimulating peptide expression in starfish reveals the gonoducts as a source for its role as a regulator of spawning.

Oocyte maturation and gamete release (spawning) in starfish are triggered by relaxin-like gonad-stimulating peptide (RGP), a neuropeptide that was first isolated from the radial nerve cords of these animals. Hitherto, it has generally been assumed that the radial nerve cords are the source of RGP that triggers spawning physiologically. To investigate other sources of RGP, here we report the first comprehensive anatomical analysis of its expression, using both in situ hybridization and immunohistochemistry to map RGP precursor transcripts and RGP, respectively, in the starfish Asterias rubens. Cells expressing RGP precursor transcripts were revealed in the ectoneural epithelium of the radial nerve cords and circumoral nerve ring, arm tips, tube feet, cardiac stomach, pyloric stomach, and, most notably, gonoducts. Using specific antibodies to A. rubens RGP, immunostaining was revealed in cells and/or fibers in the ectoneural region of the radial nerve cords and circumoral nerve ring, tube feet, terminal tentacle and other arm tip-associated structures, body wall, peristomial membrane, esophagus, cardiac stomach, pyloric stomach, pyloric caeca, and gonoducts. Our discovery that RGP is expressed in the gonoducts of A. rubens proximal to its gonadotropic site of action in the gonads is important because it provides a new perspective on how RGP may act as a gonadotropin in starfish. Thus, we hypothesize that it is the release of RGP from the gonoducts that triggers gamete maturation and spawning in starfish, while RGP produced in other parts of the body may regulate other physiological/behavioral processes.

The relaxin receptor RXFP1 signals through a mechanism of autoinhibition.

The relaxin family peptide receptor 1 (RXFP1) is the receptor for relaxin-2, an important regulator of reproductive and cardiovascular physiology. RXFP1 is a multi-domain G protein-coupled receptor (GPCR) with an ectodomain consisting of a low-density lipoprotein receptor class A (LDLa) module and leucine-rich repeats. The mechanism of RXFP1 signal transduction is clearly distinct from that of other GPCRs, but remains very poorly understood. In the present study, we determine the cryo-electron microscopy structure of active-state human RXFP1, bound to a single-chain version of the endogenous agonist relaxin-2 and the heterotrimeric Gs protein. Evolutionary coupling analysis and structure-guided functional experiments reveal that RXFP1 signals through a mechanism of autoinhibition. Our results explain how an unusual GPCR family functions, providing a path to rational drug development targeting the relaxin receptors.

C1q/TNF-Related Proteins 1, 6 and 8 Are Involved in Corneal Epithelial Wound Closure by Targeting Relaxin Receptor RXFP1 In Vitro.

Inadequate wound healing of ocular surface injuries can lead to permanent visual impairment. The relaxin ligand-receptor system has been demonstrated to promote corneal wound healing through increased cell migration and modulation of extracellular matrix formation. Recently, C1q/tumor necrosis factor-related protein (CTRP) 8 was identified as a novel interaction partner of relaxin receptor RXFP1. Additional data also suggest a role for CTRP1 and CTRP6 in RXFP1-mediated cAMP signaling. However, the role of CTRP1, CTRP6 and CTRP8 at the ocular surface remains unclear. In this study, we investigated the effects of CTRP1, CTRP6, and CTRP8 on epithelial ocular surface wound closure and their dependence on the RXFP1 receptor pathway. CTRP1, CTRP6, and CTRP8 expression was analyzed by RT-PCR and immunohistochemistry in human tissues and cell lines derived from the ocular surface and lacrimal apparatus. In vitro ocular surface wound modeling was performed using scratch assays. We analyzed the effects of recombinant CTRP1, CTRP6, and CTRP8 on cell proliferation and migration in human corneal and conjunctival epithelial cell lines. Dependence on RXFP1 signaling was established by inhibiting ligand binding to RXFP1 using a specific anti-RXFP1 antibody. We detected the expression of CTRP1, CTRP6, and CTRP8 in human tissue samples of the cornea, conjunctiva, meibomian gland, efferent tear ducts, and lacrimal gland, as well as in human corneal, conjunctival, and meibomian gland epithelial cell lines. Scratch assays revealed a dose-dependent increase in the closure rate of surface defects in human corneal epithelial cells after treatment with CTRP1, CTRP6, and CTRP8, but not in conjunctival epithelial cells. Inhibition of RXFP1 fully attenuated the effect of CTRP8 on the closure rate of surface defects in human corneal epithelial cells, whereas the CTRP1 and CTRP6 effects were not completely suppressed. Conclusions: Our findings demonstrate a novel role for CTRP1, CTRP6, and CTRP8 in corneal epithelial wound closure and suggest an involvement of the relaxin receptor RXFP1 signaling pathway. This could be a first step toward new approaches for pharmacological and therapeutic intervention.

The dual and multifaceted role of relaxin-2 in cancer.

The continuous increase in cancer-associated deaths despite the substantial improvement in diagnosis and treatment has sparked discussions on the need for novel biomarkers and therapeutic strategies for cancer. Although increasing evidence has demonstrated the pivotal role of relaxin-2 in multiple cancers, their role is a double-edged sword with both protumor and antitumor having been reported in various malignant tumors. Considering this dual role, it appears the biological mechanism underpinning the action of relaxin-2 in cancer is not clear and further studies to elucidate their potential as a preventive factor for cancers are of prime importance. Herein, a summarized up-to-date report on the role of relaxin-2 in human cancer including detailed clinical and experimental evidence supporting their tumor-promoting and inhibitory functions in cancer development and progression has been elucidated. Also, signaling pathways and other factors orchestrating the activities of relaxin-2 in the tumor microenvironment has been discussed. Collectively, the evidence from this review has demonstrated the need for further evaluation of the role of relaxin-2 as a diagnostic and or prognostic biomarker for cancer.

A Relaxin-Like Gonad-Stimulating Peptide Appears in the Early Development of the Starfish Patiria pectinifera.

Relaxin-like gonad-stimulating peptide (RGP) is a hormone with gonadotropin-like activity in starfish. This study revealed that spawning inducing activity was detected in an extract of brachiolaria larvae of Patiria pectinifera. Spawning inducing activity in the extract was due to P. pectinifera RGP (PpeRGP), not 1-methyladenine. The expression of PpeRGP mRNA was also found in brachiolaria. Immunohistochemical observation with specific antibodies for PpeRGP showed that PpeRGP was distributed in the peripheral adhesive papilla of the brachiolaria arms. In contrast, PpeRGP was not detected in the adult rudiment or ciliary band regions, which are present in the neural system. These findings strongly suggest that RGP exists in the larvae before metamorphosis. Because gonads are not developed in starfish larvae, it seems likely that RGP plays another role other than gonadotropic action in the early development of starfish.

Development of a long-acting relaxin analogue, LY3540378, for treatment of chronic heart failure.

BACKGROUND AND PURPOSE: Chronic heart failure, a progressive disease with limited treatment options currently available, especially in heart failure with preserved ejection fraction (HFpEF), represents an unmet medical need as well as an economic burden. The development of a novel therapeutic to slow or reverse disease progression would be highly impactful to patients and society. Relaxin-2 (relaxin) is a human hormone regulating cardiovascular, renal, and pulmonary adaptations during pregnancy. A short-acting recombinant relaxin, Serelaxin, demonstrated short-term heart failure symptom relief and biomarker improvement in acute heart failure trials. Here, we present the development of a long-acting relaxin analogue to be tested in the treatment of chronic heart failure. EXPERIMENTAL APPROACH: LY3540378 is a long-acting protein therapeutic composed of a human relaxin analogue and a serum albumin-binding VHH domain. KEY RESULTS: LY3540378 is a potent agonist of the relaxin family peptide receptor 1 (RXFP1) and maintains selectivity against RXFP2/3/4 comparable to native relaxin. The half-life of LY3540378 in preclinical species is extended through high affinity binding of the albumin-binding VHH domain to serum albumin. When tested in a single dose administration, LY3540378 elicited relaxin-mediated pharmacodynamic responses, such as reduced serum osmolality and increased renal blood flow in rats. In an isoproterenol-induced cardiac hypertrophy mouse model, treatment with LY3540378 significantly reduced cardiac hypertrophy and improved isovolumetric relaxation time. In a monkey cardiovascular safety study, there were no adverse observations from administration of LY3540378. CONCLUSION AND IMPLICATIONS: LY3540378 demonstrated to be a suitable clinical development candidate, and is progressing in clinical trials.

Ligand recognition mechanism of the human relaxin family peptide receptor 4 (RXFP4).

Members of the insulin superfamily regulate pleiotropic biological processes through two types of target-specific but structurally conserved peptides, insulin/insulin-like growth factors and relaxin/insulin-like peptides. The latter bind to the human relaxin family peptide receptors (RXFPs). Here, we report three cryo-electron microscopy structures of RXFP4-Gi protein complexes in the presence of the endogenous ligand insulin-like peptide 5 (INSL5) or one of the two small molecule agonists, compound 4 and DC591053. The B chain of INSL5 adopts a single α-helix that penetrates into the orthosteric pocket, while the A chain sits above the orthosteric pocket, revealing a peptide-binding mode previously unknown. Together with mutagenesis and functional analyses, the key determinants responsible for the peptidomimetic agonism and subtype selectivity were identified. Our findings not only provide insights into ligand recognition and subtype selectivity among class A G protein-coupled receptors, but also expand the knowledge of signaling mechanisms in the insulin superfamily.

Single-Shot Solid-Phase Synthesis of Full-Length H2 Relaxin Disulfide Surrogates.

Chemical synthesis of insulin superfamily proteins (ISPs) has recently been widely studied to develop next-generation drugs. Separate synthesis of multiple peptide fragments and tedious chain-to-chain folding are usually encountered in these studies, limiting accessibility to ISP derivatives. Here we report the finding that insulin superfamily proteins (e.g. H2 relaxin, insulin itself, and H3 relaxin) incorporating a pre-made diaminodiacid bridge at A-B chain terminal disulfide can be easily and rapidly synthesized by a single-shot automated solid-phase synthesis and expedient one-step folding. Our new H2 relaxin analogues exhibit almost identical structures and activities when compared to their natural counterparts. This new synthetic strategy will expediate production of new ISP analogues for pharmaceutical studies.