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Insulin-like peptide 5 (INSL5) targets the G protein-coupled receptor, relaxin family peptide receptor 4 (RXFP4), predominantly coexpressed in the colorectum. While INSL5 also binds to the related receptor RXFP3, it does not activate it. The INSL5/RXFP4 axis is a promising target for treating gastrointestinal disorders such as constipation. Despite its therapeutic potential, the clinical application of INSL5 has been hindered by synthesis complexities, necessitating the need for more accessible yet potent mimetics. In this study, we engineered an INSL5 analogue A13:B7-24-GG, featuring a simplified two-chain, two-disulfide scaffold with 32 amino acids, as opposed to the 45 amino acids found in native INSL5 (two-chain, three-disulfide), improving the synthesis yield by 19.5-fold. Additionally, A13:B7-24-GG demonstrates ∼4-fold higher potency (EC 50 = 1.17 nM vs 4.57 nM) and ∼11 times greater selectivity than native INSL5, with significantly reduced RXFP3 binding affinity, positioning it as a promising new therapeutic candidate for the treatment of constipation.

Study Question: Does RXFP2 disruption impair male fertility?, Summary Answer: We identified biallelic variants in RXFP2 in patients with male infertility due to spermatogenic arrest at the spermatid stage, supporting a role of RXFP2 in human spermatogenesis, specifically in germ cell maturation., What Is Known Already: Since RXFP2, the receptor for INSL3, plays a crucial role in testicular descent during prenatal development, biallelic variants lead to bilateral cryptorchidism, as described in four families to date. While animal models have also suggested a function in spermatogenesis, the postnatal functions of RXFP2 and its ligand INSL3, produced in large amounts by the testes from puberty throughout adulthood, are largely unknown., Study Design, Size, Duration: A family with two male members affected by impaired fertility due to spermatogenic maturation arrest and a history of bilateral cryptorchidism underwent clinical, endocrinological, histological, genomic, in vitro cellular, and in silico investigations., Participants/materials, Setting, Methods: The endocrinological and histological findings were correlated with publicly available single-cell RNA sequencing (scRNA-seq) data. The genomic defects have been characterized using long-read sequencing and validated with in silico modeling and an in vitro cyclic AMP reporter gene assay., Main Results and the Role of Chance: An intragenic deletion of exon 1-5 of RXFP2 (NM_130806.5) was detected in trans with a hemizygous missense variant c.229G>A, p.(Glu77Lys). The p.(Glu77Lys) variant caused no clear change in cell surface expression or ability to bind INSL3, but displayed absence of a cAMP signal in response to INSL3, indicating a loss-of-function. Testicular biopsy in the proband showed a maturation arrest at the spermatid stage, corresponding to the highest level of RXFP2 expression in scRNA-seq data, thereby providing a potential explanation for the impaired fertility., Limitations, Reasons for Caution: Although this is so far the only study of human cases that supports the role of RXFP2 in spermatogenic maturation, this is corroborated by several animal studies that have already demonstrated a postnatal function of INSL3 and RXFP2 in spermatogenesis., Wider Implications of the Findings: This study corroborates RXFP2 as gene implicated in autosomal recessive congenital bilateral cryptorchidism due to biallelic variants, rather than autosomal-dominant cryptorchidism due to monoallelic RXFP2 variants. Our findings also support that RXFP2 is essential in human spermatogenesis, specifically in germ cell maturation, and that biallelic disruption can cause male infertility through spermatogenic arrest at the spermatid stage., Study Funding/competing Interest(s): Funding was provided by the Bellux Society for Pediatric Endocrinology and Diabetology (BELSPEED) and supported by a Research Foundation Flanders (FWO) senior clinical investigator grant (E.D.B., 1802220N) and a Ghent University Hospital Special Research Fund grant (M.C., FIKO-IV institutional fund). The authors declare no conflict of interest., Trial Registration Number: N/A., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The peptide hormone relaxin plays a critical role in tissue remodeling in a variety of tissues through activation of its cognate receptor, RXFP1. Relaxin's ability to modify extracellular matrices has provided a strong rationale for treating fibrosis in a variety of tissues. Treatment with recombinant relaxin peptides in clinical studies of heart failure has not yet proven useful, likely due to the short half-life of infused peptide. To circumvent this particular pharmacokinetic pitfall we have used a Protein-in-Protein (PiP) antibody technology described previously, to insert a single-chain human relaxin construct into the complementarity-determining region (CDR) of an immunoglobulin G (IgG) backbone, creating a relaxin molecule with a half-life of ∼4-5 days in mice. Relaxin-PiP biologics displaced Europium-labeled human relaxin in RXFP1-expressing cells and demonstrated full agonist activity on both human and mouse RXFP1 receptors. Relaxin-PiPs did not show signal transduction bias, as they activated cAMP in THP-1 cells, and cGMP and pERK signaling in primary human cardiac fibroblasts. In an induced carbon tetrachloride mouse model of liver fibrosis one relaxin-PiP, R2-PiP, caused reduction of liver lesions, ameliorated collagen accumulation in the liver with the corresponding reduction of Collagen1a1 gene expression, and increased cell proliferation in hepatic parenchyma. These relaxin biologics represent a novel approach to the design of a long-acting RXFP1 agonist to probe the clinical utility of relaxin/RXFP1 signaling to treat a variety of human fibrotic diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The retrosplenial cortex (RSC) plays a central role in processing contextual fear conditioning. In addition to corticocortical and thalamocortical projections, the RSC receives subcortical inputs, including a substantial projection from the nucleus incertus in the pontine tegmentum. This GABAergic projection contains the neuropeptide, relaxin-3 (RLN3), which inhibits target neurons via its Gi/o-protein-coupled receptor, RXFP3. To assess this peptidergic system role in contextual fear conditioning, we bilaterally injected the RSC of adult rats with an adeno-associated-virus (AAV), expressing the chimeric RXFP3 agonist R3/I5 or a control AAV, and subjected them to contextual fear conditioning. The R3/I5 injected rats did not display any major differences to control-injected and naïve rats but displayed a significantly delayed extinction. Subsequently, we employed acute bilateral injections of the specific RXFP3 agonist peptide, RXFP3-Analogue 2 (A2), into RSC. While the administration of A2 before each extinction trial had no impact on the extinction process, treatment with A2 before each acquisition trial resulted in delayed extinction. In related anatomical studies, we detected an enrichment of RLN3-immunoreactive nerve fibers in deep layers of the RSC, and a higher level of co-localization of RXFP3 mRNA with vesicular GABA transporter (vGAT) mRNA than with vesicular glutamate transporter-1 (vGLUT1) mRNA across the RSC, consistent with an effect of RLN3/RXFP3 signalling on the intrinsic, inhibitory circuits within the RSC. These findings suggest that contextual conditioning processes in the RSC involve, in part, RLN3 afferent modulation of local inhibitory neurons that provides a stronger memory acquisition which, in turn, retards the extinction process., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2024. Published by Elsevier Inc. All rights reserved.)

A relaxin-like gonad-stimulating peptide (RGP), Aso-RGP, featuring six cysteine residues, was identified in the Crown-of-Thorns Starfish (COTS, Acanthaster cf. solaris) and initially produced through recombinant yeast expression. This method yielded a single-chain peptide with an uncleaved C-peptide (His Tag) and suboptimal purity. Our objective was to chemically synthesize Aso-RGP in its mature form, comprising two chains (A and B) and three disulfide bridges, omitting the C-peptide. Furthermore, we aimed to synthesize a newly identified relaxin-like peptide, Aso-RLP2, from COTS, which had not been previously synthesized. This paper reports the first total chemical synthesis of Aso-RGP and Aso-RLP2. Aso-RGP synthesis proceeded without major issues, whereas the A-chain of Aso-RLP2, in its reduced and unfolded state with two free thiols, presented considerable challenges. These were initially marked by "messy" RP-HPLC profiles, typically indicative of synthesis failure. Surprisingly, oxidizing the A-chain significantly improved the RP-HPLC profile, revealing the main issue was not synthesis failure but the peptide's aggregation tendency, which initially obscured analysis. This discovery highlights the critical need to account for aggregation in peptide synthesis and analysis. Ultimately, our efforts led to the successful synthesis of both peptides with purities exceeding 95 %., (© 2024 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Human insulin-like peptide 5 (INSL5) is a gut hormone produced by colonic L-cells, and its biological functions are mediated by Relaxin Family Peptide Receptor 4 (RXFP4). Our preliminary data indicated that RXFP4 agonists are potential drug leads for the treatment of constipation. More recently, we designed and developed a novel RXFP4 antagonist, A13-nR that was shown to block agonist-induced activity in cells and animal models. We showed that A13-nR was able to block agonist-induced increases in colon motility in mice of both genders that express the receptor, RXFP4. Our data also showed that colorectal propulsion induced by intracolonic administration of short-chain fatty acids was antagonized by A13-nR. Therefore, A13-nR is an important research tool and potential drug lead for the treatment of colon motility disorders, such as bacterial diarrhea. However, A13-nR acted as a partial agonist at high concentrations in vitro and demonstrated modest antagonist potency (∼35 nM). Consequently, the primary objective of this study is to pinpoint novel modifications to A13-nR that eliminate partial agonist effects while preserving or augmenting antagonist potency. In this work, we detail the creation of a series of A13-nR-modified analogues, among which analogues 3, 4, and 6 demonstrated significantly improved RXFP4 affinity (∼3 nM) with reduced partial agonist activity, enhanced antagonist potency (∼10 nM) and maximum agonist inhibition (∼80 %) when compared with A13-nR. These compounds have potential as candidates for further preclinical evaluations, marking a significant stride toward innovative therapeutics for colon motility disorders., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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 PEG 12 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Hepatorenal syndrome (HRS) is a life-threatening complication of end-stage liver disease first reported over a century ago, but its management still poses an unmet challenge. A therapeutic agent found to stabilize the condition is a short cyclic peptide, vasopressin analogue, terlipressin (TP). While TP is commonly prescribed for HRS patients in most parts of the world, it was only recently approved for use in the United States. TP exhibits short circulation half-lives and adverse side effects associated with the dose required. Herein, we present a 1,18-octadecanedioic acid (ODDA) conjugate of the cyclic peptide (ODDA-TP), which enables noncovalent binding to serum albumin via native fatty acid binding modes. ODDA-TP is demonstrated to outperform TP alone in studies including in vitro cellular receptor activation, stability in plasma, pharmacokinetics, and performance in vivo in rats. Specifically, ODDA-TP had an elimination half-life 20 times that of TP alone while exhibiting a superior safety profile., Competing Interests: The authors declare no competing financial interest., (© 2024 American Chemical Society.)

α 1A -, α 1B -, and α 1D -adrenoceptors (α 1 -ARs) are members of the adrenoceptor G protein-coupled receptor family that are activated by adrenaline (epinephrine) and noradrenaline. α 1 -ARs are clinically targeted using antagonists that have minimal subtype selectivity, such as prazosin and tamsulosin, to treat hypertension and benign prostatic hyperplasia, respectively. Abundant expression of α 1 -ARs in the heart and central nervous system (CNS) makes these receptors potential targets for the treatment of cardiovascular and CNS disorders, such as heart failure, epilepsy, and Alzheimer's disease. Our understanding of the precise physiological roles of α 1 -ARs, however, and their involvement in disease has been hindered by the lack of sufficiently subtype-selective tool compounds, especially for α 1B -AR. Here, we report the discovery of 4-[(2-hydroxyethyl)amino]-6-methyl-2H-chromen-2-one (Cpd1), as an α 1B -AR antagonist that has 10-15-fold selectivity over α 1A -AR and α 1D -AR. Through computational and site-directed mutagenesis studies, we have identified the binding site of Cpd1 in α 1B -AR and propose the molecular basis of α 1B -AR selectivity, where the nonconserved V197 45.52 residue plays a major role, with contributions from L314 6.55 within the α 1B -AR pocket. By exploring the structure-activity relationships of Cpd1 at α 1B -AR, we have also identified 3-[(cyclohexylamino)methyl]-6-methylquinolin-2(1H)-one (Cpd24), which has a stronger binding affinity than Cpd1, albeit with reduced selectivity for α 1B -AR. Cpd1 and Cpd24 represent potential leads for α 1B -AR-selective drug discovery and novel tool molecules to further study the physiology of α 1 -ARs.

The conformational ensembles of G protein-coupled receptors (GPCRs) include inactive and active states. Spectroscopy techniques, including NMR, show that agonists, antagonists and other ligands shift the ensemble toward specific states depending on the pharmacological efficacy of the ligand. How receptors recognize ligands and the kinetic mechanism underlying this population shift is poorly understood. Here, we investigate the kinetic mechanism of neurotensin recognition by neurotensin receptor 1 (NTS 1 ) using 19 F-NMR, hydrogen-deuterium exchange mass spectrometry and stopped-flow fluorescence spectroscopy. Our results indicate slow-exchanging conformational heterogeneity on the extracellular surface of ligand-bound NTS 1 . Numerical analysis of the kinetic data of neurotensin binding to NTS 1 shows that ligand recognition follows an induced-fit mechanism, in which conformational changes occur after neurotensin binding. This approach is applicable to other GPCRs to provide insight into the kinetic regulation of ligand recognition by GPCRs., (© 2023. The Author(s).)

The pre-Bötzinger complex (preBötC), a key primary generator of the inspiratory breathing rhythm, contains neurons that project directly to facial nucleus (7n) motoneurons to coordinate orofacial and nasofacial activity. To further understand the identity of 7n-projecting preBötC neurons, we used a combination of optogenetic viral transgenic approaches to demonstrate that selective photoinhibition of these neurons affects mystacial pad activity, with minimal effects on breathing. These effects are altered by the type of anesthetic employed and also between anesthetized and conscious states. The population of 7n-projecting preBötC neurons we transduced consisted of both excitatory and inhibitory neurons that also send collaterals to multiple brainstem nuclei involved with the regulation of autonomic activity. We show that modulation of subgroups of preBötC neurons, based on their axonal projections, is a useful strategy to improve our understanding of the mechanisms that coordinate and integrate breathing with different motor and physiological behaviors. This is of fundamental importance, given that abnormal respiratory modulation of autonomic activity and orofacial behaviors have been associated with the development and progression of diseases., Competing Interests: MM, AW, AC, JB, SC, SM, CM, RB, AA No competing interests declared, (© 2023, Melo et al.)

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.

Human relaxin-2 (H2 relaxin) is a peptide hormone with potent vasodilatory and anti-fibrotic effects, which is of interest for the treatment of heart failure and fibrosis. H2 relaxin binds to the Relaxin Family Peptide Receptor 1 (RXFP1). Native H2 relaxin is a two-chain, three-disulfide-bond-containing peptide, which is unstable in human serum and difficult to synthesize efficiently. In 2016, our group developed B7-33, a single-chain peptide derived from the B-chain of H2 relaxin. B7-33 demonstrated poor affinity and potency in HEK cells overexpressing RXFP1; however, it displayed equivalent potency to H2 relaxin in fibroblasts natively expressing RXFP1, where it also demonstrated the anti-fibrotic effects of the native hormone. B7-33 reversed organ fibrosis in numerous pre-clinical animal studies. Here, we detail our efforts towards a minimal H2 relaxin scaffold and attempts to improve scaffold activity through Aib substitution and hydrocarbon stapling to re-create the peptide helicity present in the native H2 relaxin.

The nucleus accumbens shell is a critical node in reward circuitry, encoding environments associated with reward. Long-range inputs from the ventral hippocampus (ventral subiculum) to the nucleus accumbens shell have been identified, yet their precise molecular phenotype remains to be determined. Here we used retrograde tracing to identify the ventral subiculum as the brain region with the densest glutamatergic (VGluT1-Slc17a7) input to the shell. We then used circuit-directed translating ribosome affinity purification to examine the molecular characteristics of distinct glutamatergic (VGluT1, VGluT2-Slc17a6) ventral subiculum to nucleus accumbens shell projections. We immunoprecipitated translating ribosomes from this population of projection neurons and analysed molecular connectomic information using RNA sequencing. We found differential gene enrichment across both glutamatergic projection neuron subtypes. In VGluT1 projections, we found enrichment of Pfkl, a gene involved in glucose metabolism. In VGluT2 projections, we found a depletion of Sparcl1 and Dlg1, genes known to play a role in depression- and addiction-related behaviours. These findings highlight potential glutamatergic neuronal-projection-specific differences in ventral subiculum to nucleus accumbens shell projections. Together these data advance our understanding of the phenotype of a defined brain circuit., (© 2023. Crown.)

Study Question: What is the impact of variants in the genes INSL3 (Insulin Like 3) and RXFP2 (Relaxin Family Peptide Receptor 2), respectively, on cryptorchidism and male infertility?, Summary Answer: Bi-allelic loss-of-function (LoF) variants in INSL3 and RXFP2 result in bilateral cryptorchidism and male infertility, whereas heterozygous variant carriers are phenotypically unaffected., What Is Known Already: The small heterodimeric peptide INSL3 and its G protein-coupled receptor RXFP2 play a major role in the first step of the biphasic descent of the testes, and variants in the INSL3 and RXFP2 genes have long been implicated in inherited cryptorchidism. However, only one single homozygous missense variant in RXFP2 has clearly been linked to familial bilateral cryptorchidism, so the effects of bi-allelic variants in INSL3 and heterozygous variants in both genes on cryptorchidism and male infertility remain unclear., Study Design, Size, Duration: Exome data of 2412 men from the MERGE (Male Reproductive Genomics) study cohort including 1902 infertile men with crypto-/azoospermia, of whom 450 men had a history of cryptorchidism, were screened for high-impact variants in INSL3 and RXFP2., Participants/materials, Setting, Methods: For patients with rare, high-impact variants in INSL3 and RXFP2, detailed clinical data were collected and the testicular phenotype was determined. Genotyping of family members was performed to analyse the co-segregation of candidate variants with the condition. Immunohistochemical staining for INSL3 in patient testicular tissue and measuring serum INSL3 concentration was performed to analyse the functional impact of a homozygous loss-of-function variant in INSL3. For a homozygous missense variant in RXFP2, its impact on the protein's cell surface expression and ability to respond to INSL3 in CRE reporter gene assay was determined., Main Results and the Role of Chance: This study presents homozygous high-impact variants in INSL3 and RXFP2 and clearly correlates these to bilateral cryptorchidism. Functional impact of the identified INSL3 variant was demonstrated by absence of INSL3-specific staining in patients' testicular Leydig cells as well as undetectable blood serum levels. The identified missense variant in RXFP2 was demonstrated to lead to reduced RXFP2 surface expression and INSL3 mediated receptor activation., Limitations, Reasons for Caution: Further investigations are needed to explore a potential direct impact of bi-allelic INSL3 and RXFP2 variants on spermatogenesis. With our data, we cannot determine whether the infertility observed in our patients is a direct consequence of the disruption of a possible function of these genes on spermatogenesis or whether it occurs secondarily due to cryptorchidism., Wider Implications of the Findings: In contrast to previous assumptions, this study supports an autosomal recessive inheritance of INSL3- and RXFP2-related bilateral cryptorchidism while heterozygous LoF variants in either gene can at most be regarded as a risk factor for developing cryptorchidism. Our findings have diagnostic value for patients with familial/bilateral cryptorchidism and additionally shed light on the importance of INSL3 and RXFP2 in testicular descent and fertility., Study Funding/competing Interest(s): This study was carried out within the frame of the German Research Foundation (DFG) funded by Clinical Research Unit 'Male Germ Cells: from Genes to Function' (DFG, CRU326). Research at the Florey was supported by an NHMRC grant (2001027) and the Victorian Government Operational Infrastructure Support Program. A.S.B. is funded by the DFG ('Emmy Noether Programme' project number 464240267). The authors declare no conflict of interest., Trial Registration Number: N/A., (© The Author(s) 2023. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The neurotensin receptor 1 (NTS 1 ) is a G protein-coupled receptor (GPCR) with promise as a drug target for the treatment of pain, schizophrenia, obesity, addiction, and various cancers. A detailed picture of the NTS 1 structural landscape has been established by X-ray crystallography and cryo-EM and yet, the molecular determinants for why a receptor couples to G protein versus arrestin transducers remain poorly defined. We used 13 C ε H 3 -methionine NMR spectroscopy to show that binding of phosphatidylinositol-4,5-bisphosphate (PIP2) to the receptor's intracellular surface allosterically tunes the timescale of motions at the orthosteric pocket and conserved activation motifs - without dramatically altering the structural ensemble. β-arrestin-1 further remodels the receptor ensemble by reducing conformational exchange kinetics for a subset of resonances, whereas G protein coupling has little to no effect on exchange rates. A β-arrestin biased allosteric modulator transforms the NTS 1 :G protein complex into a concatenation of substates, without triggering transducer dissociation, suggesting that it may function by stabilizing signaling incompetent G protein conformations such as the non-canonical state. Together, our work demonstrates the importance of kinetic information to a complete picture of the GPCR activation landscape., (© 2023. The Author(s).)

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 (Arg B13 ) 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., Competing Interests: The authors declare no competing financial interest., (© 2023 American Chemical Society.)

RXFP4 is a G protein-coupled receptor (GPCR) in the relaxin family. It has recently been recognised that this receptor and its cognate ligand INSL5 may have a role in the regulation of food intake, gut motility, and other functions relevant to metabolic health and disease. Recent data from reporter-mice showed co-location of Rxfp4 and serotonin (5-HT) in the lower gut. We used human single-cell RNA sequence data (scRNASeq) to show that RXFP4 is in a subset of gut enterochromaffin cells that produce 5-HT in humans. We also used RNAScope to show co-location of Rxfp4 mRNA and 5-HT in mouse colon, confirming prior findings. To understand whether RXFP4 might regulate serotonin production, we developed a cell model using Colo320, a human gut-derived immortalised cell line that produces and releases serotonin. Overexpression of RXFP4 in these cells resulted in a constitutive decrease in cAMP levels in both the basal state and in cells treated with forskolin. Treatment of cells with two RXFP4 agonists, INSL5 derived peptide INSL5-A13 and small molecule compound-4, further reduced cAMP levels. This was paralleled by a reduction in expression of mRNA for TPH1, the enzyme controlling the rate limiting step in the production of serotonin. Overexpression of RXFP4 also attenuated the cAMP-induced release of serotonin from Colo320 cells. Together this demonstrates that serotonin producing enterochromaffin cells are the major site of RXFP4 expression in the gut and that RXFP4 can have inhibitory functional impacts on cAMP production as well as TPH1 expression and serotonin release., (© 2023 The Author(s).)

Human relaxin-2 (H2 relaxin) is therapeutically very important due to its strong anti-fibrotic, vasodilatory, and cardioprotective effects. Therefore, relaxin's receptor, relaxin family peptide receptor 1 (RXFP1), is a potential target for the treatment of fibrosis and related disorders, including heart failure. H2 relaxin has a complex two-chain structure (A and B) and three disulfide bridges. Our laboratory has recently developed B7-33 peptide, a single-chain agonist based on the B-chain of H2 relaxin. However, the peptide B7-33 has a short circulation time in vitro in serum (t 1/2 = ~6 min). In this study, we report structure-activity relationship studies on B7-33 utilizing different fatty-acid conjugations at different positions. We have shown that by fatty-acid conjugation with an appropriate spacer length, the in vitro half-life of B7-33 can be increased from 6 min to 60 min. In the future, the lead lipidated molecule will be studied in animal models to measure its PK/PD properties, which will lead to their pre-clinical applications.

The hormone, relaxin (RLX), exerts various organ-protective effects independently of etiology. However, its complex two-chain and three disulphide bonded structure is a limitation to its preparation and affordability. Hence, a single chain-derivative of RLX, B7-33, was developed and shown to retain the anti-fibrotic effects of RLX in vitro and in vivo. Here, we determined whether B7-33 could retain the other cardioprotective effects of RLX, and also compared its therapeutic efficacy to the ACE inhibitor, perindopril. Adult male 129sv mice were subjected to isoprenaline (ISO; 25 mg/kg/day, s.c)-induced cardiomyopathy, then s.c-treated with either RLX (0.5 mg/kg/day), B7-33 (0.25 mg/kg/day; equivalent dose corrected for MW) or perindopril (1 mg/kg/day) from days 7-14 post-injury. Control mice received saline instead of ISO. Changes in animal body weight (BW) and systolic blood pressure (SBP) were measured weekly, whilst cardiomyocyte hypertrophy and measures of vascular dysfunction and rarefaction, left ventricular (LV) inflammation and fibrosis were assessed at day 14 post-injury. ISO-injured mice had significantly increased LV inflammation, cardiomyocyte hypertrophy, fibrosis, vascular rarefaction and aortic contractility in the absence of any changes in BW or SBP at day 14 post-injury. Both B7-33 and RLX equivalently reduced LV fibrosis and normalised the ISO-induced LV inflammation and cardiomyocyte hypertrophy, whilst restoring blood vessel density and aortic contractility. Comparatively, perindopril lowered SBP and the ISO-induced LV inflammation and vascular rarefaction, but not fibrosis or hypertrophy. As B7-33 retained the cardioprotective effects of RLX and provided rapid-occurring anti-fibrotic effects compared to perindopril, it could be considered as a cost-effective cardioprotective therapy., Competing Interests: Conflict of interest statement The authors declare no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Nuclear magnetic resonance (NMR) studies have revealed that fast methyl sidechain dynamics can report on entropically-driven allostery. Yet, NMR applications have been largely limited to the super-microsecond motional regimes of G protein-coupled receptors (GPCRs). We use  13 C ε -methionine chemical shift-based global order parameters to test if ligands affect the fast dynamics of a thermostabilized GPCR, neurotensin receptor 1 (NTS 1 ). We establish that the NTS 1 solution ensemble includes substates with lifetimes on several, discrete timescales. The longest-lived states reflect those captured in agonist- and inverse agonist-bound crystal structures, separated by large energy barriers. We observe that the rapid fluctuations of individual methionine residues, superimposed on these long-lived states, respond collectively with the degree of fast, global dynamics correlating with ligand pharmacology. This approach lends confidence to interpreting spectra in terms of local structure and methyl dihedral angle geometry. The results suggest a role for sub-microsecond dynamics and conformational entropy in GPCR ligand discrimination., Competing Interests: Declaration of interests The authors declare no competing financial interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

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-G i 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., (© 2023. The Author(s).)

The peptide hormone relaxin (RLX), also available as clinical-grade recombinant protein (serelaxin), holds great promise as a cardiovascular and anti-fibrotic agent but is limited by the pharmacokinetic issues common to all peptide drugs. In this study, by a computational modelling chemistry approach, we have synthesized and tested a set of low molecular weight peptides based on the putative receptor-binding domain of the B chain of human H1 RLX isoform, with the objective to obtain RLX analogues with improved pharmacokinetic features. Some of them were stabilized to induce the appropriate 3-D conformation by intra-chain tri-azolic staples, which should theoretically enhance their resistance to digestive enzymes making them suited for oral administration. Despite these favourable premises, none of these H1 peptides, either linear or stapled, revealed a sufficient affinity to the specific RLX receptor RXFP1. Moreover, none of them was endowed with any RLX-like biological effects in RXFP1-expressing THP-1 human monocytic cells and mouse NIH-3T3-derived myofibroblasts in in vitro culture, in terms of significantly relevant cAMP elevation and ERK1/2 phosphorylation, which represent two major signal transduction events downstream RXFP1 activation. This was at variance with authentic serelaxin, which induced a clear-cut, significant activation of both these classical RLX signaling pathways. Albeit negative, the results of this study offer additional information about the structural requirements that new peptide therapeutics shall possess to effectively behave as RXFP1 agonists and RLX analogues., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 D'Ercole, Nistri, Pacini, Carotenuto, Santoro, Papini, Bathgate, Bani and Rovero.)

Cyclic guanosine monophosphate (cGMP) is a second messenger involved in the regulation of numerous physiological processes. The modulation of cGMP is important in many diseases, but reliably assaying cGMP in live cells in a plate-based format with temporal resolution is challenging. The Förster/fluorescence resonance energy transfer (FRET)-based biosensor cGES-DE5 has a high temporal resolution and high selectivity for cGMP over cAMP, so we converted it to use bioluminescence resonance energy transfer (BRET), which is more compatible with plate-based assays. This BRET variant, called CYGYEL (cyclic GMP sensor using YFP-PDE5-Rluc8), was cloned into a lentiviral vector for use across different mammalian cell types. CYGYEL was characterised in HEK293T cells using the nitric oxide donor diethylamine NONOate (DEA), where it was shown to be dynamic, reversible, and able to detect cGMP with or without the use of phosphodiesterase inhibitors. In human primary vascular endothelial and smooth muscle cells, CYGYEL successfully detected cGMP mediated through either soluble or particulate guanylate cyclase using DEA or C-type natriuretic peptide, respectively. Notably, CYGYEL detected differences in kinetics and strength of signal both between ligands and between cell types. CYGYEL remained selective for cGMP over cAMP, but this selectivity was reduced compared to cGES-DE5. CYGYEL streamlines the process of cGMP detection in plate-based assays and can be used to detect cGMP activity across a range of cell types.

Relaxin/insulin-like family peptide receptor 3 (RXFP3) belongs to class A G protein-coupled receptor family. RXFP3 and its endogenous ligand relaxin-3 are mainly expressed in the brain with important roles in the regulation of appetite, energy metabolism, endocrine homeostasis and emotional processing. It is therefore implicated as a potential target for treatment of various central nervous system diseases. Since selective agonists of RXFP3 are restricted to relaxin-3 and its analogs, we conducted a high-throughput screening campaign against 32,021 synthetic and natural product-derived compounds using a cyclic adenosine monophosphate (cAMP) measurement-based method. Only one compound, WNN0109-C011, was identified following primary screening, secondary screening and dose-response studies. Although displayed agonistic effect in cells overexpressing the human RXFP3, it also showed cross-reactivity with the human RXFP4. This hit compound may provide not only a chemical probe to investigate the function of RXFP3/4, but also a novel scaffold for the development of RXFP3/4 agonists.

Our poor understanding of the mechanism by which the peptide-hormone H2 relaxin activates its G protein coupled receptor, RXFP1 and the related receptor RXFP2, has hindered progress in its therapeutic development. Both receptors possess large ectodomains, which bind H2 relaxin, and contain an N-terminal LDLa module that is essential for receptor signaling and postulated to be a tethered agonist. Here, we show that a conserved motif (GDxxGWxxxF), C-terminal to the LDLa module, is critical for receptor activity. Importantly, this motif adopts different structures in RXFP1 and RXFP2, suggesting distinct activation mechanisms. For RXFP1, the motif is flexible, weakly associates with the LDLa module, and requires H2 relaxin binding to stabilize an active conformation. Conversely, the GDxxGWxxxF motif in RXFP2 is more closely associated with the LDLa module, forming an essential binding interface for H2 relaxin. These differences in the activation mechanism will aid drug development targeting these receptors., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Relaxin family peptide receptors (RXFPs) are the potential therapeutic targets for neurological, cardiovascular, and metabolic indications. Among them, RXFP3 and RXFP4 (formerly known as GPR100 or GPCR142) are homologous class A G protein-coupled receptors with short N-terminal domain. Ligands of RXFP3 or RXFP4 are only limited to endogenous peptides and their analogues, and no natural product or synthetic agonists have been reported to date except for a scaffold of indole-containing derivatives as dual agonists of RXFP3 and RXFP4. In this study, a new scaffold of tricyclic derivatives represented by compound 7a was disclosed as a selective RXFP4 agonist after a high-throughput screening campaign against a diverse library of 52,000 synthetic and natural compounds. Two rounds of structural modification around this scaffold were performed focusing on three parts: 2-chlorophenyl group, 4-hydroxylphenyl group and its skeleton including cyclohexane-1,3-dione and 1,2,4-triazole group. Compound 14b with a new skeleton of 7,9-dihydro-4H-thiopyrano[3,4-d][1,2,4]triazolo[1,5-a]pyrimidin-8(5H)-one was thus obtained. The enantiomers of 7a and 14b were also resolved with their 9-(S)-conformer favoring RXFP4 agonism. Compared with 7a, compound 9-(S)-14b exhibited 2.3-fold higher efficacy and better selectivity for RXFP4 (selective ratio of RXFP4 vs. RXFP3 for 9-(S)-14b and 7a were 26.9 and 13.9, respectively)., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The receptor for the neuropeptide relaxin 3, relaxin family peptide 3 (RXFP3) receptor, is an attractive pharmacological target for the control of eating, addictive, and psychiatric behaviors. Several structure-activity relationship studies on both human relaxin 3 (containing 3 disulfide bonds) and its analogue A2 (two disulfide bonds) suggest that the C-terminal carboxylic acid of the tryptophan residue in the B-chain is important for RXFP3 activity. In this study, we have added amide, alcohol, carbamate, and ester functionalities to the C-terminus of A2 and compared their structures and functions. As expected, the C-terminal amide form of A2 showed lower binding affinity for RXFP3 while ester and alcohol substitutions also demonstrated lower affinity. However, while these analogues showed slightly lower binding affinity, there was no significant difference in activation of RXFP3 compared to A2 bearing a C-terminal carboxylic acid, suggesting the binding pocket is able to accommodate additional atoms., Competing Interests: The authors declare no competing financial interest., (© 2020 American Chemical Society.)

The receptor for insulin-like peptide 5 (INSL5), RXFP4, is a potential pharma target for treating human conditions such as constipation, anorexia, and obesity. However, since INSL5 has a complex structure of two chains and three disulfide bonds, its synthesis has proven to be extremely difficult via either chemical or recombinant approaches. Previous studies led to the engineering of a high yielding simplified INSL5 analog, named analog 13 (A13), which retains native INSL5-like activity. The focus of this study is to further simplify the structure of A13 by truncating the N-terminal residues of the B-chain. We have found that the first six residues at the N-terminus of A13 are not important for RXFP4 binding and cAMP potency. The most minimized active structure of INSL5 identified in this study is A13: B7-24 which will be an important research tool to study the physiological role of RXFP4 and a template for further modification to improve its pharmacokinetic properties., Competing Interests: The authors declare no competing financial interest., (© 2020 American Chemical Society.)

Relaxin-3 is a highly conserved two-chain neuropeptide that acts through its endogenous receptor the Relaxin Family Peptide-3 (RXFP3) receptor. The ligand/receptor system is known to modulate several physiological processes, with changes in food intake and anxiety-levels the most well studied in rodent models. Agonist and antagonist analogues based on the native two-chain peptide are costly to synthesise and not ideal drug leads. Since RXFP3 interacting residues are found in the relaxin B-chain only, this has been the focus of analogue development. The B-chain is unstructured without the A-chain support, but in single-chain variants structure can be induced by dicarba-based helical stapling strategies. Here we investigated whether alternative helical inducing strategies also can enhance structure and activity at RXFP3. Combinations of the helix inducing α-aminoisobutyric acid (Aib) were incorporated into the sequence of the relaxin-3 B-chain. Aib residues at positions 13, 17 and 18 partially reintroduce helicity and activity of the relaxin-3 B-chain, but other positions are generally not suited for modifications. We identify Thr21 as a putative new receptor contact residue important for RXFP3 binding. Cysteine residues were also incorporated into the sequence and cross-linked with dichloroacetone or α, α'-dibromo- m -xylene. However, in contrast to previously reported dicarba variants, neither were found to promote structure and RXFP3 activity.

Relaxin/insulin-like family peptide receptor 4 (RXFP4) is a class A G protein-coupled receptor (GPCR), and insulin-like peptide 5 (INSL5) is its endogenous ligand. Although the precise physiological role of INSL5/RXFP4 remains elusive, a number of studies have suggested it to be a potential therapeutic target for obesity and other metabolic disorders. Since selective agonists of RXFP4 are scarcely available and peptidic analogs of INSL5 are hard to make, we conducted a high-throughput screening campaign against 52,000 synthetic and natural compounds targeting RXFP4. Of the 109 initial hits discovered, only 3 compounds were confirmed in secondary screening, with JK0621-D008 displaying the best agonism at human RXFP4. Its S-configuration stereoisomer (JK1) was subsequently isolated and validated by a series of bioassays, demonstrating a consistent agonistic effect in cells overexpressing RXFP4. This scaffold may provide a valuable tool to further explore the biological functions of RXFP4.

Chemogenetics enables manipulation of neuronal activity in experimental animals. While providing information about the transduced neuron expressing a ligand-activated molecule, chemogenetics does not provide understanding about the antecedent circuit that drives that neuron's activity. For current approaches, this is not feasible, because the activating molecules are not genetically encoded. The insect allatostatin/allatostatin receptor system, a highly specific, powerful inhibitory chemogenetic approach, has this advantage, because the ligand, being a peptide, is genetically encoded. We developed viral vector-based systems to express biologically active allatostatin in neurons in vivo and allatostatin receptors in subpopulations of postsynaptic neurons. We demonstrate that activity-dependent release of allatostatin induces inhibition of allatostatin receptor-expressing neurons. We validate the approach in the vagal viscerosensory system where inhibitory, rather than the usual excitatory, viscerosensory input leads to sustained decreases in baroreceptor reflex sensitivity and bodyweight., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Fibrosis is a hallmark of several cardiovascular diseases. The relaxin family peptide receptor 1 (RXFP1) agonist, relaxin, has rapidly occurring anti-fibrotic actions which are mediated through RXFP1 and angiotensin II receptor crosstalk on renal and cardiac myofibroblasts. Here, we investigated whether this would allow relaxin to indirectly activate angiotensin II type 2 receptor (AT 2 R)-specific signal transduction in primary human cardiac myofibroblasts (HCMFs). The anti-fibrotic effects of recombinant human relaxin (RLX; 16.8 nM) or the AT 2 R-agonist, Compound 21 (C21; 1 μM), were evaluated in TGF-β1-stimulated HCMFs, in the absence or presence of an RXFP1 antagonist (1 μM) or AT 2 R antagonist (0.1 μM) to confirm RXFP1-AT 2 R crosstalk. Competition binding for RXFP1 was determined. Western blotting was performed to determine which AT 2 R-specific protein phosphatases were expressed by HCMFs; then, the anti-fibrotic effects of RLX and/or C21 were evaluated in the absence or presence of pharmacological inhibition (NSC95397 (1 μM) for MKP-1; okadaic acid (10 nM) for PP2A) or siRNA-knockdown of these phosphatases after 72 hours. The RLX- or C21-induced increase in ERK1/2 and nNOS phosphorylation, and decrease in α-SMA (myofibroblast differentiation) and collagen-I expression by HCMFs was abrogated by pharmacological blockade of RXFP1 or the AT 2 R, confirming RXFP1-AT 2 R crosstalk in these cells. HCMFs were found to express AT 2 R-dependent MKP-1 and PP2A phosphatases, while pharmacological blockade or siRNA-knockdown of either phosphatase also abolished RLX and/or C21 signal transduction in HCMFs (all P < .05 vs RLX or C21 alone). These findings demonstrated that RLX can indirectly activate AT 2 R-dependent phosphatase activity in HCMFs by signaling through RXFP1-AT 2 R crosstalk, which have important therapeutic implications for its anti-fibrotic actions., (© 2020 Federation of American Societies for Experimental Biology.)

G protein-coupled receptors (GPCRs) use a series of conserved microswitches to transmit signals across the cell membrane via an allosteric network encompassing the ligand-binding site and the G protein-binding site. Crystal structures of GPCRs provide snapshots of their inactive and active states, but poorly describe the conformational dynamics of the allosteric network that underlies GPCR activation. Here, we analyzed the correlation between ligand binding and receptor conformation of the α 1A -adrenoreceptor, a GPCR that stimulates smooth muscle contraction in response to binding noradrenaline. NMR of [ 13 C ϵ H 3 ]methionine-labeled α 1A -adrenoreceptor variants, each exhibiting differing signaling capacities, revealed how different classes of ligands modulate the conformational equilibria of this receptor. [ 13 C ϵ H 3 ]Methionine residues near the microswitches exhibited distinct states that correlated with ligand efficacies, supporting a conformational selection mechanism. We propose that allosteric coupling among the microswitches controls the conformation of the α 1A -adrenoreceptor and underlies the mechanism of ligand modulation of GPCR signaling in cells., (© 2020 Wu et al.)

Background: Insulin-like peptide 5 (INSL5) is a hormone stored in colonic enteroendocrine cells that also contain the unrelated hormones, GLP-1 and PYY. It acts at the relaxin family peptide 4, RXFP4, receptor. RXFP4 is expressed by enteric neurons in the colon, and it has been speculated that INSL5, through its action on enteric neurons, might be involved in the control of colonic contractions. Similar to insulin and relaxin, INSL5 consists of A and B peptide chains linked by three disulfide bonds, two between the chains and one intrinsic to the A chain. Because of its complex structure, it is difficult to synthesize and to prepare peptide analogues to investigate its roles. We have recently developed a potent simplified peptide analogue, INSL5-A13 (INSL5 analogue 13)., Methods: In the present work, we have investigated the actions of INSL5-A13 in mice. We investigated the ability of INSL5-A13 to increase the speed of emptying of a bead from the colon, after expulsion had been slowed by the peripherally restricted opioid agonist, loperamide (1 mg/kg)., Key Results: INSL5-A13 was a full agonist at the mouse RXFP4 expressed in HEK cells, with an EC50 of ~9 nmol/L. INSL5-A13 caused an acceleration of colorectal bead propulsion in mice constipated by loperamide in the dose range 0.2 to 60 µg/kg, with an EC50 of ~6 µg/kg in vivo. It also accelerated bead propulsion in untreated mice. Bead expulsion was not accelerated in RXFP4-/- mice., Conclusion and Inferences: Our data suggest that RXFP4 agonists could be useful in the treatment of constipation., (© 2020 John Wiley & Sons Ltd.)

Cell-cell communication via endogenous peptides and their receptors is vital for controlling all aspects of human physiology and most peptides signal through G protein-coupled receptors (GPCRs). Disordered peptides bind GPCRs through complex modes for which there are few representative crystal structures. The disordered peptide neurotensin (NT) is a neuromodulator of classical neurotransmitters such as dopamine and glutamate, through activation of neurotensin receptor 1 (NTS 1 ). While several experimental structures show how NT binds NTS 1 , details about the structural dynamics of NT during and after binding NTS 1 , or the role of peptide dynamics on receptor activation, remain obscure. Here saturation transfer difference (STD) NMR revealed that the binding mode of NT fragment NT10-13 is heterogeneous. Epitope maps of NT10-13 at NTS 1 suggested that tyrosine 11 (Y11) samples other conformations to those observed in crystal structures of NT-bound NTS 1 . Molecular dynamics (MD) simulations confirmed that when NT is bound to NTS 1 , residue Y11 can exist in two χ 1 rotameric states, gauche plus (g + ) or gauche minus (g - ). Since only the g + Y11 state is observed in all the structures solved to date, we asked if the g - state is important for receptor activation. NT analogues with Y11 replaced with 7-OH-Tic were synthesized to restrain the dynamics of the side chain. P(OH-TIC)IL bound NTS 1 with the same affinity as NT10-13 but did not activate NTS 1 , instead acted as an antagonist. This study highlights that flexibility of Y11 in NT may be required for NT activation of NTS 1 ., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

Relaxin-3 is a neuropeptide with important roles in metabolism, arousal, learning and memory. Its cognate receptor is the relaxin family peptide-3 (RXFP3) receptor. Relaxin-3 agonist and antagonist analogs have been shown to be able to modulate food intake in rodent models. The relaxin-3 B-chain is sufficient for receptor interactions, however, in the absence of a structural support, linear relaxin-3 B-chain analogs are rapidly degraded and thus unsuitable as drug leads. In this study, two different disulfide-stabilized scaffolds were used for grafting of important relaxin-3 B-chain residues to improve structure and stability. The use of both Veronica hederifolia Trypsin inhibitor (VhTI) and apamin grafting resulted in agonist and antagonist analogs with improved helicity. VhTI grafted peptides showed poor binding and low potency at RXFP3, on the other hand, apamin variants retained significant activity. These variants also showed improved half-life in serum from ~5 min to >6 h, and thus are promising RXFP3 specific pharmacological tools and drug leads for neuropharmacological diseases., (Copyright © 2020 Lee, Postan, Song, Clark, Bathgate, Haugaard-Kedström and Rosengren.)

Modern neuroscience utilizes transgenic techniques extensively to study the activity and function of brain neural networks. A key feature of this approach is its compatibility with molecular methods for selective transgene expression in neuronal circuits of interest. Until now, such targeted transgenic approaches have not been applied to the extensive circuitry involving the neuropeptide, relaxin-3. Pharmacological and gene knock-out studies have revealed relaxin-3 signalling modulates interrelated behaviours and cognitive processes, including stress and anxiety, food and alcohol consumption, and spatial and social memory, highlighting the potential of this system as a therapeutic target. In the present study, we aimed to identify a promoter sequence capable of regulating cell-type specific transgene expression from an adeno-associated viral (AAV) vector in relaxin-3 neurons of the rat nucleus incertus (NI). In parallel to relaxin-3 promoter sequences, we also tested an AAV vector containing promoter elements for the tropomyosin receptor kinase A (TrkA) gene, as TrkA is co-expressed with relaxin-3 in rat NI neurons. Stereotaxic injection of an mCherry-expressing AAV vector revealed widespread non-specific TrkA promoter (880 bp) activity in and adjacent to the NI at 8 weeks post-treatment. In contrast, mCherry expression was successfully restricted to relaxin-3 NI neurons with 98% specificity using a 1736 bp relaxin-3 promoter. In addition to detailed anatomical mapping of NI relaxin-3 networks, illustrated here in association with GABAergic medial septum neurons, this method for targeted transgene delivery offers a versatile tool for ongoing preclinical studies of relaxin-3 circuitry., (© 2019 The Author(s).)

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.

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.

Background: Recombinant human relaxin-2 (serelaxin), which has organ-protective actions mediated via its cognate G protein-coupled receptor relaxin family peptide receptor 1 (RXFP1), has emerged as a potential agent to treat fibrosis. Studies have shown that serelaxin requires the angiotensin II (AngII) type 2 receptor (AT 2 R) to ameliorate renal fibrogenesis in vitro and in vivo . Whether its antifibrotic actions are affected by modulation of the AngII type 1 receptor (AT 1 R), which is expressed on myofibroblasts along with RXFP1 and AT 2 R, is unknown., Methods: We examined the signal transduction mechanisms of serelaxin when applied to primary rat renal and human cardiac myofibroblasts in vitro , and in three models of renal- or cardiomyopathy-induced fibrosis in vivo ., Results: The AT 1 R blockers irbesartan and candesartan abrogated antifibrotic signal transduction of serelaxin via RXFP1 in vitro and in vivo . Candesartan also ameliorated serelaxin's antifibrotic actions in the left ventricle of mice with cardiomyopathy, indicating that candesartan's inhibitory effects were not confined to the kidney. We also demonstrated in a transfected cell system that serelaxin did not directly bind to AT 1 Rs but that constitutive AT 1 R-RXFP1 interactions could form. To potentially explain these findings, we also demonstrated that renal and cardiac myofibroblasts expressed all three receptors and that antagonists acting at each receptor directly or allosterically blocked the antifibrotic effects of either serelaxin or an AT 2 R agonist (compound 21)., Conclusions: These findings have significant implications for the concomitant use of RXFP1 or AT 2 R agonists with AT 1 R blockers, and suggest that functional interactions between the three receptors on myofibroblasts may represent new targets for controlling fibrosis progression., (Copyright © 2019 by the American Society of Nephrology.)

Anxiety disorders are highly prevalent in modern society and better treatments are required. Key brain areas and signaling systems underlying anxiety include prefrontal cortex, hippocampus, and amygdala, and monoaminergic and peptidergic systems, respectively. Hindbrain GABAergic projection neurons that express the peptide, relaxin-3, broadly innervate the forebrain, particularly the septum and hippocampus, and relaxin-3 acts via a G i/o -protein-coupled receptor known as the relaxin-family peptide 3 receptor (RXFP3). Thus, relaxin-3/RXFP3 signaling is implicated in modulation of arousal, motivation, mood, memory, and anxiety. Ventral hippocampus (vHip) is central to affective and cognitive processing and displays a high density of relaxin-3-positive nerve fibers and RXFP3 binding sites, but the identity of target neurons and associated effects on behavior are unknown. Therefore, in adult, male rats, we assessed the neurochemical nature of hippocampal RXFP3 mRNA-expressing neurons and anxiety-like and social behavior following chronic RXFP3 activation in vHip by viral vector expression of an RXFP3-selective agonist peptide, R3/I5. RXFP3 mRNA detected by fluorescent in situ hybridization was topographically distributed across the hippocampus in somatostatin- and parvalbumin-mRNA expressing GABA neurons. Chronic RXFP3 activation in vHip increased anxiety-like behavior in the light-dark box and elevated-plus maze, but not the large open-field test, and reduced social interaction with a conspecific stranger. Our data reveal disruptive effects of persistent RXFP3 signaling on hippocampal GABA networks important in anxiety; and identify a potential therapeutic target for anxiety disorders that warrants further investigation in relevant preclinical models., (© 2019 Wiley Periodicals, Inc.)

Relaxin family peptide 1 (RXFP1) is the receptor for relaxin a peptide hormone with important therapeutic potential. Like many G protein-coupled receptors (GPCRs), RXFP1 has been reported to form homodimers. Given the complex activation mechanism of RXFP1 by relaxin, we wondered whether homodimerization may be explicitly required for receptor activation, and therefore sought to determine if there is any relaxin-dependent change in RXFP1 proximity at the cell surface. Bioluminescence resonance energy transfer (BRET) between recombinantly tagged receptors is often used in GPCR proximity studies. RXFP1 targets poorly to the cell surface when overexpressed in cell lines, with the majority of the receptor proteins sequestered within the cell. Thus, any relaxin-induced changes in RXFP1 proximity at the cell surface may be obscured by BRET signal originating from intracellular compartments. We therefore, utilized the newly developed split luciferase system called HiBiT to specifically label the extracellular terminus of cell surface RXFP1 receptors in combination with mCitrine-tagged receptors, using the GABA B heterodimer as a positive control. This demonstrated that the BRET signal detected from RXFP1-RXFP1 proximity at the cell surface does not appear to be due to stable physical interactions. The fact that there is also no relaxin-mediated change in RXFP1-RXFP1 proximity at the cell surface further supports these conclusions. This work provides a basis by which cell surface GPCR proximity and expression levels can be specifically studied using a facile and homogeneous labeling technique such as HiBiT., Competing Interests: The authors declare no competing interests.

Protozoan parasites of the phylum Apicomplexa actively move through tissue to initiate and perpetuate infection. The regulation of parasite motility relies on cyclic nucleotide-dependent kinases, but how these kinases are activated remains unknown. Here, using an array of biochemical and cell biology approaches, we show that the apicomplexan parasite Toxoplasma gondii expresses a large guanylate cyclase (TgGC) protein, which contains several upstream ATPase transporter-like domains. We show that TgGC has a dynamic localization, being concentrated at the apical tip in extracellular parasites, which then relocates to a more cytosolic distribution during intracellular replication. Conditional TgGC knockdown revealed that this protein is essential for acute-stage tachyzoite growth, as TgGC-deficient parasites were defective in motility, host cell attachment, invasion, and subsequent host cell egress. We show that TgGC is critical for a rapid rise in cytosolic [Ca 2+ ] and for secretion of microneme organelles upon stimulation with a cGMP agonist, but these deficiencies can be bypassed by direct activation of signaling by a Ca 2+ ionophore. Furthermore, we found that TgGC is required for transducing changes in extracellular pH and [K + ] to activate cytosolic [Ca 2+ ] flux. Together, the results of our work implicate TgGC as a putative signal transducer that activates Ca 2+ signaling and motility in Toxoplasma ., (© 2019 Yang et al.)