Development of a Single-Chain Peptide Agonist of the Relaxin-3 Receptor Using Hydrocarbon Stapling.

Structure-activity studies of the insulin superfamily member, relaxin-3, have shown that its G protein-coupled receptor (RXFP3) binding site is contained within its central B-chain α-helix and this helical structure is essential for receptor activation. We sought to develop a single B-chain mimetic that retained agonist activity. This was achieved by use of solid phase peptide synthesis together with on-resin ruthenium-catalyzed ring closure metathesis of a pair of judiciously placed i,i+4 α-methyl, α-alkenyl amino acids. The resulting hydrocarbon stapled peptide was shown by solution NMR spectroscopy to mimic the native helical conformation of relaxin-3 and to possess potent RXFP3 receptor binding and activation. Alternative stapling procedures were unsuccessful, highlighting the critical need to carefully consider both the peptide sequence and stapling methodology for optimal outcomes. Our result is the first successful minimization of an insulin-like peptide to a single-chain α-helical peptide agonist which will facilitate study of the function of relaxin-3.

International Union of Basic and Clinical Pharmacology. XCV. Recent advances in the understanding of the pharmacology and biological roles of relaxin family peptide receptors 1-4, the receptors for relaxin family peptides.

Relaxin, insulin-like peptide 3 (INSL3), relaxin-3, and INSL5 are the cognate ligands for the relaxin family peptide (RXFP) receptors 1-4, respectively. RXFP1 activates pleiotropic signaling pathways including the signalosome protein complex that facilitates high-sensitivity signaling; coupling to Gα(s), Gα(i), and Gα(o) proteins; interaction with glucocorticoid receptors; and the formation of hetero-oligomers with distinctive pharmacological properties. In addition to relaxin-related ligands, RXFP1 is activated by Clq-tumor necrosis factor-related protein 8 and by small-molecular-weight agonists, such as ML290 [2-isopropoxy-N-(2-(3-(trifluoromethylsulfonyl)phenylcarbamoyl)phenyl)benzamide], that act allosterically. RXFP2 activates only the Gα(s)- and Gα(o)-coupled pathways. Relaxin-3 is primarily a neuropeptide, and its cognate receptor RXFP3 is a target for the treatment of depression, anxiety, and autism. A variety of peptide agonists, antagonists, biased agonists, and an allosteric modulator target RXFP3. Both RXFP3 and the related RXFP4 couple to Gα(i)/Gα(o) proteins. INSL5 has the properties of an incretin; it is secreted from the gut and is orexigenic. The expression of RXFP4 in gut, adipose tissue, and ß-islets together with compromised glucose tolerance in INSL5 or RXFP4 knockout mice suggests a metabolic role. This review focuses on the many advances in our understanding of RXFP receptors in the last 5 years, their signal transduction mechanisms, the development of novel compounds that target RXFP1-4, the challenges facing the field, and current prospects for new therapeutics.

Modulation of feeding by chronic rAAV expression of a relaxin-3 peptide agonist in rat hypothalamus.

Relaxin-3 is a neuropeptide that is abundantly expressed by discrete brainstem neuron populations that broadly innervate forebrain areas rich in the relaxin-3 G-protein-coupled-receptor, RXFP3. Acute and subchronic central administration of synthetic relaxin-3 or an RXFP3-selective agonist peptide, R3/I5, increase feeding and body weight in rats. Intrahypothalamic injection of relaxin-3 also increases feeding. In this study, we developed a recombinant adeno-associated virus 1/2 (rAAV1/2) vector that drives expression and constitutive secretion of bioactive R3/I5 and assessed the effect of intrahypothalamic injections on daily food intake and body weight gain in adult male rats over 8 weeks. In vitro testing revealed that the vector rAAV1/2-fibronectin (FIB)-R3/I5 directs the constitutive secretion of bioactive R3/I5 peptide. Bilateral injection of rAAV1/2-FIB-R3/I5 vector into the paraventricular nucleus produced an increase in daily food intake and body weight gain (P<0.01, ~23%, respectively), relative to control treatment. In a separate cohort of rats, quantitative polymerase chain reaction analysis of hypothalamic mRNA revealed strong expression of R3/I5 transgene at 3 months post-rAAV1/2-FIB-R3/I5 infusion. Levels of mRNA transcripts for the relaxin-3 receptor RXFP3, the hypothalamic 'feeding' peptides neuropeptide Y, AgRP and POMC, and the reproductive hormone, GnRH, were all similar to control, whereas vasopressin and oxytocin (OT) mRNA levels were reduced by ~25% (P=0.051) and ~50% (P<0.005), respectively, in rAAV1/2-FIB-R3/I5-treated rats (at 12 weeks, n=9/8 rats per group). These data demonstrate for the first time that R3/I5 is effective in modulating feeding in the rat by chronic hypothalamic RXFP3 activation and suggest a potential underlying mechanism involving altered OT signalling. Importantly, there was no desensitization of the feeding response over the treatment period and no apparent deleterious health effects, indicating that targeting the relaxin-3-RXFP3 system may be an effective long-term therapy for eating disorders.

Prevention of bleomycin-induced pulmonary fibrosis by a novel antifibrotic peptide with relaxin-like activity.

Pulmonary fibrosis is a progressive and lethal lung disease characterized by accumulation of extracellular matrix and loss of pulmonary function. No cure exists for this pathologic condition, and current treatments often fail to slow its progression or relieve its symptoms. Relaxin was previously shown to induce a matrix-degrading phenotype in human lung fibroblasts in vitro and to inhibit pulmonary fibrosis in vivo. A novel peptide that targets the relaxin RXFP1/LGR7 receptor was recently identified using our computational platform designed to predict novel G protein-coupled receptor peptide agonists. In this study, we examined the antifibrotic properties of this novel peptide, designated CGEN25009, in human cell-based assays and in a murine model of bleomycin-induced pulmonary fibrosis. Similar to relaxin, CGEN25009 was found to have an inhibitory effect on transforming growth factor-ß1-induced collagen deposition in human dermal fibroblasts and to enhance MMP-2 expression. The peptide's biological activity was also similar to relaxin in generating cellular stimulation of cAMP, cGMP, and NO in the THP-1 human cell line. In vivo, 2-week administration of CGEN25009 in a preventive or therapeutic mode (i.e., concurrently with or 7 days after bleomycin treatment, respectively) caused a significant reduction in lung inflammation and injury and ameliorated adverse airway remodeling and peribronchial fibrosis. The results of this study indicate that CGEN25009 displays antifibrotic and anti-inflammatory properties and may offer a new therapeutic option for the treatment of pulmonary fibrosis.