Relaxin-like gonad-stimulating peptide (RGP) regulates final gamete maturation and spawning in starfish through a specific gonadal receptor, the RGP receptor (RGPR). Homologous RGPs have since been identified in sea cucumbers and shown to induce spawning, supporting a conserved reproductive role across echinoderms. However, the molecular basis of RGP-RGPR signalling in sea cucumbers remained unknown, as the sea cucumber RGPR had not previously been identified. Therefore, we systematically searched for G protein-coupled receptors similar to starfish RGPRs using comparative sequence analysis and identified candidate RGPRs in five sea cucumber species: Holothuria leucospilota, Holothuria glaberrima, Holothuria scabra, Apostichopus japonicus, and Isostichopus badionotus. Receptor sequences from Holothuria species shared 77 to 85% identity, while similarity to Apostichopus and Isostichopus sequences was lower, at about 60%. Structural modelling revealed the expected features of relaxin family peptide receptors, including seven transmembrane helices, a large extracellular domain with multiple leucine-rich repeats, and an N-terminal LDLa module. Functional cell-based assays demonstrated that the candidate H. scabra RGPR responded specifically and dose-dependently to recombinant H. scabra RGP, but not to human H2 relaxin, providing the first functional evidence of an RGP receptor in a sea cucumber. An RGPR homologue was detected in the H. leucospilota ovary across all reproductive stages, although expression did not differ significantly among stages. Together, these findings support the conservation of relaxin-like reproductive signalling in echinoderms, while direct functional characterisation of the H. leucospilota RGP-RGPR pair remains necessary.

Insulin-like peptide 5 (INSL5) is a member of the relaxin/insulin peptide family, predominantly produced by enteroendocrine L cells in the distal gut. Although initially characterised based on its structural similarity to insulin and relaxin peptides, INSL5 is now increasingly recognised as an important regulator of gastrointestinal physiology. Its endogenous receptor, relaxin family peptide receptor 4 (RXFP4), is primarily expressed in the gastrointestinal tract and signals mainly through Gi proteins. Recent studies have provided compelling evidence that the INSL5-RXFP4 signalling axis is an important regulator of colonic motility. In this review, we summarise current knowledge of the structure-function relationships of INSL5 and RXFP4, advances in the design of INSL5 analogues, and the signalling mechanisms underlying receptor activation. We also discuss the physiological and pharmacological evidence supporting a role for INSL5-RXFP4 signalling in colonic motility and highlight opportunities for the development of chemical probes and therapeutic leads targeting this system for the treatment of chronic constipation.

Abstract Relaxin family peptide receptor 1 (RXFP1) is a multi-domain GPCR with compelling therapeutic potential, yet uncertainty surrounding the mechanism of its activation by the hormone H2 relaxin has hindered the development of selective modulators. Here, we combine deep learning based structural modelling with de novo protein design to overcome this barrier. We generate a high-confidence structural model of the RXFP1–relaxin complex that is strongly supported by existing biochemical and functional evidence. This model reveals that relaxin engagement stabilises the RXFP1 extracellular linker, thereby triggering receptor activation. Guided by this model, we design mini-protein modulators that either block linker stabilisation or enforce it and induce an active receptor geometry. These molecules act as potent, selective RXFP1 antagonists or agonists, achieving low-nanomolar activity in both engineered and endogenously expressing cell lines despite adopting folds unrelated to relaxin. Together, these findings define the mechanistic basis of RXFP1 signalling, establish the first de novo agonists and antagonists of this receptor, and demonstrate how AI-enabled modelling and design can target structurally complex GPCRs previously inaccessible to structure-guided drug discovery.

BACKGROUND:Relaxins are a family of peptides that regulate reproductive physiology in vertebrates. Evidence that this is an evolutionarily ancient role of relaxins has been provided by the discovery of two relaxin-like gonad-stimulating peptides (RGP1 and RGP2) that trigger spawning in starfish. The main aim of this study was to identify the receptor(s) that mediate(s) the effects of RGP1 and RGP2 in starfish. RESULTS:Here we show that RGP1 and RGP2 belong to a family of peptides that include vertebrate relaxins, Drosophila insulin-like peptide 8 (Dilp8), and other relaxin-like peptides in several protostome taxa. An ortholog of the human relaxin receptors RXFP1 and RXFP2 and the Drosophila receptor LGR3 was identified in starfish (RXFP/LGR3). In Drosophila, but not in humans and other vertebrates, there is a paralog of LGR3 known as LGR4, and here an LGR4-type receptor was also identified in starfish. In vitro pharmacological experiments revealed that both RGP1 and RGP2 act as ligands for RXFP/LGR3 in the starfish Acanthaster cf. solaris and Asterias rubens, but neither peptide acts as a ligand for LGR4 in these species. CONCLUSIONS:Discovery of the RXFP/LGR3-type receptor for RGP1 and RGP2 in starfish provides a new insight into the evolution of relaxin-type signaling as a regulator of reproductive processes. Furthermore, our findings indicate that RXFP/LGR3-type receptors have been lost in several phyla, including urochordates, mollusks, bryozoans, platyhelminthes, and nematodes.

The corallivorous Crown-of-Thorns Starfish (COTS; Acanthaster cf. solaris) has become a major pest across the Indo-Pacific, primarily due to anthropogenic environmental factors that have increased populations beyond sustainable levels. These effects are costly to both the economy and ecology of coral reefs and require human intervention. A better understanding of endogenous reproductive factors could help establish more effective management strategies. Based on information regarding an endogenous spawn-inducing peptide in COTS, known as the relaxin-like gonad-stimulating peptide (RGP), a version of this peptide (called synthetic Aso-RGP; inspired by the sequence from Acanthaster cf. solaris) was chemically synthesized. In this study, we demonstrate that in vivo intra-coelomic injection of reproductively mature COTS with 1 µM synthetic Aso-RGP (100 µl) induced spawning of males and females at 20 ± 6.8 min and 32.3 ± 17.2 min post-injection, respectively. Typical spawning behaviours were also observed, including seeking high-ground, crowning/arching and upwards curling of arms prior to gamete release, leading to >95 % fertilization rate. Subsequent testing of synthetic Aso-RGP at 1 µM on mature COTS ovarian fragments stimulated 100 % oocyte germinal vesicle breakdown (GVBD), similar to that observed for the positive 1-methyladenine (1 and 10 mM) control, confirming activation of the RGP cascade. Synthetic Aso-RGP was not, however, effective in inducing spawning or triggering GVBD outside of the reproductive season, demonstrating it is seasonally functional. Finally, we showed that synthetic Aso-RGP can also stimulate spawning in the COTS sibling species, Acanthaster brevispinus. In summary, this research has established the bioactivity of the synthetic Aso-RGP, which in turn, may prove critical for the development of a novel control approach to manipulate COTS spawning.

The Concise Guide to Pharmacology 2025/26 marks the seventh edition in this series of biennial publications in the British Journal of Pharmacology. Presented in landscape format, the guide provides a comparative overview of the pharmacology of drug target families. The concise nature of the Concise Guide refers to the style of presentation, being clear, accessible, and well-structured, rather than the scope of the content, which spans approximately 500 pages. The Concise Guide summarises the key pharmacological properties of around 1900 human drug targets, and nearly 7000 interactions, involving around 4400 ligands. While the content is a substantially condensed version of the more detailed information and links available at the www.guidetopharmacology.org website, the printed guide serves as a permanent, citable, point-in-time record, that remains stable despite ongoing updates to the online database. The full contents of this publication can be found at https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.70230. The Concise Guides provide expert-curated recommendations of 'Gold Standard' selective pharmacological tools, available either commercially or as donations, which enable the identification of individual drug targets or families of drug targets. While the Concise Guide offers a more streamlined overview, more comprehensive information, including detailed pharmacological profiles and links to multiple online databases, is available through the Guide to Pharmacology website. The 2025/26 edition of the Concise Guide is based on material current as of mid-2025, and supersedes all previous editions, including the 2023/24 Guide, and earlier Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), and as such provides official IUPHAR classification and nomenclature for human drug targets, where applicable. G protein-coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. Each section includes nomenclature guidance, concise summaries, information of the best available pharmacological tools, key references, and suggestions for further reading.

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, A13nR 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.

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.

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.

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 (EC50 = 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.

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.

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 adult rats RSC 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 differences compared 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 showed 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 activation of local inhibitory neurons that provides a stronger memory acquisition which, in turn, retards the extinction process.

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 %. This work presents a comprehensive synthetic strategy for creating relaxin-like peptides characterized by a two-chain structure linked by three disulfide bonds. Utilizing Fmoc solid-phase peptide synthesis, the A- and B-chains are independently assembled with orthogonal cysteine protecting groups (tBu, Acm, Trt) to facilitate the controlled formation of disulfide bonds. The synthesis proceeds through four key steps: (i.) Oxidation of the A-chain to establish the intramolecular disulfide bond; (ii.) Conversion of Cys(tBu) to a more reactive Cys(SPyr) derivative; (iii.) Thiolysis-driven combination of A- and B-chain to form the first inter-chain disulfide bond; and (iv.) Final disulfide bond formation via iodine oxidation, culminating in the production of the fully assembled peptide. image

Background: Preeclampsia (PE) is a hypertensive pregnancy disorder, which occurs in approximately 10% of all gestations. The literature suggests the potential therapeutic role of H2 relaxin in PE. A novel H2 relaxin B-chain-only peptide variant B7-33 (27 amino acids without any disulfide bonds) has recently been developed. Objective: The goal of this study was to test the hypothesis that a novel H2 relaxin B-chain-only peptide variant B7-33 could improve the pathophysiology of placental ischemia in the Reduced Uterine Perfusion Pressure (RUPP) rat model of PE. Methods: The efficacy of B7-33 was evaluated in the RUPP model as described previously. RUPP rats are randomly assigned to 4 groups (N=8/group): 1) vehicle, 2) B733; 3) B733-Fc; and 4) B733-HSA. Rats are dosed twice weekly (i.v.) from GD10 to GD 20. On GD 18, rats are anesthetized with isoflurane, and carotid arterial catheters are inserted into the carotid artery, tunneled under the skin, and externalized at the back of the neck. On the following day, mean arterial pressure (MAP) is monitored with a pressure transducer (Cobe III Tranducer CDX Sema) and recorded continuously for 30 min. On GD18, uterine artery resistance index (UARI) of rats is measured by Doppler sonography. The nitric oxide bioavailability, soluble fms-like tyrosine kinase-1 (sFlt-1), and TNF-α) were measured by commercially available kits. Statistical comparisons were performed using analysis of variance with Duncan’s post hoc test. Results: The RUPP rats have increased MAP (122.2 ± 8.1 mm Hg), plasma TNF-α (223 ±11.4 pg/mL), and plasma sFlt-1(863 ±18.2 pg/mL) along with decreased NO index (14±2 µM) compared to normal pregnancy: MAP (102 ± 5.2 mm Hg); plasma TNF-α (28 ± 4.1 pg/mL); plasma sFlt-1(244 ± 9.4 pg/mL) and NO index (26 ±4.1 µM). Treatment with B733 and B7-33 fusion proteins significantly (*p<0.05) lowers MAP, plasma TNF-α, and plasma sFlt-1 and increased NO index back to that of normal pregnancy. The B7-33 data are consistent with earlier data with serelaxin in the RUPP model. All fusions tested ameliorate hypertension in the RUPP animals. B7-33 normalizes BP and proteinuria in the DOCA rat model of preE. Conclusion: The conclusion of the study is that both B7-33 and B7-33 fusion proteins demonstrate efficacy in attenuating the symptoms of PE including hypertension and inflammation in RUPP model. We conclude that B7-33 is an ideal candidate for development as a novel therapeutic in preE.

α1A-, α1B-, and α1D-adrenoceptors (α1-ARs) are members of the adrenoceptor G protein-coupled receptor family that are activated by adrenaline (epinephrine) and noradrenaline. α1-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 α1-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 α1-ARs, however, and their involvement in disease has been hindered by the lack of sufficiently subtype-selective tool compounds, especially for α1B-AR. Here, we report the discovery of 4-[(2-hydroxyethyl)amino]-6-methyl-2H-chromen-2-one (Cpd1), as an α1B-AR antagonist that has 10-15-fold selectivity over α1A-AR and α1D-AR. Through computational and site-directed mutagenesis studies, we have identified the binding site of Cpd1 in α1B-AR and propose the molecular basis of α1B-AR selectivity, where the nonconserved V19745.52 residue plays a major role, with contributions from L3146.55 within the α1B-AR pocket. By exploring the structure-activity relationships of Cpd1 at α1B-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 α1B-AR. Cpd1 and Cpd24 represent potential leads for α1B-AR-selective drug discovery and novel tool molecules to further study the physiology of α1-ARs.

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 SmBiTR3/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.

The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and about 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at . G protein-coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

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.