Design, Synthesis, and Characterization of a Single-Chain Peptide Antagonist for the Relaxin-3 Receptor RXFP3

Abstract

Relaxin-3 is a two-chain disulfide-rich peptide that is the ancestral member of the relaxin peptide family and, together with its G protein-coupled receptor RXFP3, is highly expressed in the brain. Strong evolutionary conservation of relaxin-3 suggests a critical biological function and recent studies have demonstrated modulation of sensory, neuroendocrine, metabolic, and cognitive systems. However, detailed studies of central relaxin-3-RXFP3 signaling have until now been severely hampered by the lack of a readily available high-affinity antagonist for RXFP3. Previous studies have utilized a complex two-chain chimeric relaxin peptide, R3(BΔ23-27)R/I5, in which a truncated relaxin-3 B-chain carrying an additional C-terminal Arg residue was combined with the insulin-like peptide 5 (INSL5) A-chain. In this study we demonstrate that, by replacing the native Cys in this truncated relaxin-3 B-chain with Ser, a single-chain linear peptide of 23 amino acids that retains high-affinity antagonism for RXFP3 can be achieved. In vivo studies demonstrate that this peptide, R3 B1-22R, antagonized relaxin-3/RXFP3 induced increases in feeding in rats after intracerebroventricular injection. Thus, R3 B1-22R represents an excellent tool for biological studies probing relaxin pharmacology and a lead molecule for the development of synthetically tractable, single-chain RXFP3 modulators for clinical use.

Synthesis and pharmacological characterization of a europium-labelled single-chain antagonist for binding studies of the relaxin-3 receptor RXFP3

“…Importantly, this K i matches well with that obtained from competition binding studies with Eu-H3/I5 (Haugaard-Kedstrom et al 2011) indicating that the DTPA-cage does not interfere with the ligand binding to RXFP3. Importantly, several other relaxin ligands have been fluorescent labelled using the Eu-DTPA strategy without altered receptor selectivity profile (Belgi et al 2011;Shabanpoor et al 2008Shabanpoor et al , 2012.…”

“…CHO cells stably expressing RXFP3 were plated onto precoated poly-Lysine 96-well viewplates for binding assays as previously described (Haugaard-Kedstrom et al 2011). For saturation binding, media was aspirated off and cells were washed with PBS before incubation with increasing concentrations of Eu-DTPA-R3B1-22R (0.1-50 nM).…”

Minimization of Human Relaxin-3 Leading to High-Affinity Analogues with Increased Selectivity for Relaxin-Family Peptide 3 Receptor (RXFP3) over RXFP1

“…Therefore, in recent study, we created a high-affinity single chain antagonist (H3 B1−22R) by replacing the GGSRW of the H3 relaxin B chain with Arg and mutating the native cysteine (Cys) to Ser. 24 This peptide has a similar RXFP3 affinity to native H3 relaxin for RXFP3 and does not bind to either RXFP1 or RXFP4. The introduction of Arg into the B chain sequence was thought to create an additional contact point between the receptor and the peptide as the peptide without this Arg has very low affinity for RXFP3.…”

A negatively charged transmembrane aspartate residue controls activation of the relaxin-3 receptor RXFP3

“…To further confirm that the RXFP3 mutants retained ligandbinding potency, we carried out competition-binding assays using the agonist relaxin-3 and the small antagonist R3B1-22R, a previously reported relaxin-3 B-chain analogue [27]. As shown in Table 1. wild-type RXFP3 showed typical sigmoidal curves, but with different pIC 50 values (Table 1): relaxin-3 had~18-fold higher binding potency than R3B1-22R.…”