Obesity is the most prevalent metabolic disease worldwide, affecting nearly a quarter of the population and continuing to rise.1 Current therapies often have limited efficacy, adverse side effects, or poor accessibility, highlighting the need for novel and effective weight-loss strategies.2,3 The neuropeptide relaxin-3 and its endogenous receptor, relaxin family peptide receptor 3 (RXFP3), expressed in the brain, are regulators of appetite,4 making this signalling axis a promising therapeutic target. However, relaxin-3, an insulin-like peptide comprising two chains (A and B) and three disulphide bonds, is extremely challenging to synthesise (yield < 5%), limiting its clinical application. To address this, our laboratory recently developed active single-chain relaxin-3-based analogues that are more synthetically accessible (yield > 50%): an agonist5 that stimulates food intake and an antagonist that suppresses it in rats. These effects were demonstrated following intracerebroventricular administration, as the analogues cannot cross the blood-brain barrier (BBB). Although these molecules are valuable tools for investigating RXFP3’s role in appetite regulation, their invasive delivery method limits their translational potential.
Previous research has shown that blood-brain penetrating peptides cross the BBB and can be used for drug delivery, leading to the hypothesis that if the relaxin-3 based analogues are conjugated to BBB penetrating peptides (BPPs), which can cross the BBB while maintaining efficacy, less invasive delivery methods such as intraperitoneal can be used. To achieve this, relaxin-3-based analogues and BPPs were chemically synthesised using solid-phase peptide synthesis and chemically conjugated together using either maleimide-thiol or azide-alkyne reactions. The RXFP3 binding affinity and functional activity of the analogues were assessed using competition binding and cAMP inhibition assays. These experiments identified two novel conjugates with improved binding affinity for RXFP3 while retaining their cAMP signalling potency. Preliminary in vivo testing of the lead analogues in rats was inconclusive, with no observed changes in food or water intake. Further studies are currently underway to evaluate BBB penetration, as well as to conduct detailed behavioural and dose–response experiments to fully characterise their therapeutic potential.