New Tool Peptide Unlocks Drug Discovery for Gut and Brain Relaxin Receptors Linked to Anxiety and Bowel Disorders

SmBiT-conjugated R3/I5 chimeric peptide binds LgBiT-RXFP3 and LgBiT-RXFP4 with nanomolar affinity and minimal non-specific binding, enabling robust saturation and competition binding assays from whole cells and frozen membranes without ligand separation.

Solid-phase peptide synthesis of R3/I5 chimeric peptide and SmBiT-PEG12-R3/I5 conjugate. Stable HEK293T cell lines expressing LgBiT-RXFP3 and LgBiT-RXFP4 were generated and validated for signaling. NanoBiT complementation binding assays were optimized for kinetic, saturation, and competition binding in whole-cell and membrane formats.

Drug discovery for RXFP3 (anxiety, eating disorders) and RXFP4 (irritable bowel, colonic dysmotility) has been stalled by inadequate tools. This assay system removes a major bottleneck, enabling high-throughput screening that could accelerate the development of new treatments for these conditions.

The relaxin family peptides are increasingly recognized as important regulators of brain and gut function, but drug development has lagged behind other peptide receptor systems. This assay tool could catalyze a wave of discovery — similar to how GPCR binding assays accelerated drug development for other receptor families in the 1990s and 2000s.

The assay system was developed and validated in HEK293T cells overexpressing tagged receptors — binding characteristics may differ in native tissues. The R3/I5 chimeric peptide activates both RXFP3 and RXFP4, so receptor-specific selectivity must come from the test compounds, not the tracer. In vivo validation of compounds identified through this screen remains necessary.