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

The takeaway

A synthetic relaxin-3/INSL5 chimeric peptide with NanoBiT conjugation enables robust, high-throughput binding assays for RXFP3 and RXFP4 — receptors targeted for anxiety and colon motility disorders.

Nanomolar affinity, minimal background
The SmBiT-R3/I5 tracer achieves high-quality binding data for both RXFP3 and RXFP4 without the tedious separation steps that limited previous assays

What the researchers found

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.

Why it matters

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 numbers in context

Chimeric peptide designed to interact with both RXFP3 (brain/anxiety) and RXFP4 (gut/motility) receptors.

How the study worked

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.

Who was studied

In vitro receptor binding assays

What this study cannot tell us

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.

How to read the evidence

Preliminary evidence as a tool development study. The assay system is well-validated technically but its ultimate value depends on whether it leads to discovery of therapeutically useful compounds.

When this study was published

Published in 2024, providing a much-needed methodological advance for the relaxin peptide field.

The bigger picture

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.

Questions still open

Will compounds identified through this screening platform show efficacy in animal models of anxiety or bowel dysfunction?
Can the assay system be adapted for other relaxin family receptors (RXFP1, RXFP2)?
Is the NanoBiT approach transferable to other difficult-to-assay peptide GPCR systems?

Common questions

What are relaxin-3 and INSL5 and why do they matter?

Relaxin-3 is a neuropeptide produced in the brain that regulates anxiety, stress responses, appetite, and arousal through its receptor RXFP3. INSL5 is a related peptide produced in the gut that regulates colon motility through RXFP4. Both receptors are considered promising drug targets — RXFP3 for anxiety and eating disorders, RXFP4 for bowel motility problems like IBS — but drug development has been limited by the lack of good screening tools.

What is NanoBiT and why is it useful here?

NanoBiT is a two-part luminescence system where a large piece (LgBiT) and a small piece (SmBiT) produce light only when they come together. By attaching SmBiT to the peptide tracer and LgBiT to the receptor, binding produces a light signal that can be easily measured. The key advantage: the two pieces have such low natural affinity that there's almost no background signal, making it much easier to detect true binding without having to physically separate bound from unbound tracer.

Read the original research

Development of a synthetic relaxin-3/INSL5 chimeric peptide ligand for NanoBiT complementation binding assays.

Biochemical pharmacology, 224, 116238

View on PubMed ↗Publisher’s paper ↗

Citation

Wu, Hongkang; Hoare, Bradley L; Handley, Thomas N G; Akhter Hossain, Mohammed; Bathgate, Ross A D. (2024). Development of a synthetic relaxin-3/INSL5 chimeric peptide ligand for NanoBiT complementation binding assays.. Biochemical pharmacology, 224, 116238. https://doi.org/10.1016/j.bcp.2024.116238