Neuropeptides are essential to the physiological function of the many organs and tissues that make up the human body. They are typically composed of between three to 36 amino acids each, and to date, over one hundred have been discovered. Neuropeptides are produced by neurons to convey signals within the brain or from the brain to specific cells or tissues. Upon their release from the neuron, these messenger molecules bind to corresponding receptor proteins embedded in the membrane of the cell. Activation of the receptor protein subsequently induces the recruitment and activation of other ‘downstream’ signaling molecules already present in the cell. And thus, a signal is propagated through the body, often slowly, and often with long lasting effects.

For neuropeptides to function correctly, their structural integrity must be maintained. Any alteration to their shape or stability may result in weaker or inhibited binding to their receptor protein, and thus reduced potency. Altered neuropeptide-based signalling manifests in a variety of ways, from the onset of disease symptoms to altered feelings or emotions.

Relaxin-3, is one such neuropeptide that, through its interactions with its corresponding receptor RXFP3, plays a role in mediating multiple states and processes. These include stress and anxiety, arousal, food intake and processes involved in aging and age-related diseases.

To better understand the signalling events mediated by relaxin-3, Assoc Prof Gavin Dawe together with colleagues from NUS Pharmacology, Yong Loo Lin School of Medicine, and other collaborators, sought to identify and characterise possible agonists of RXFP3 using ‘stapled’ relaxin-3 variants. Peptide stapling involves replacing specific amino acids in the peptide sequence, to enable the formation of a physical, hydrocarbon-based link between them. Depending on the position of the ‘staple’ linker, peptide function can be retained, altered or lost. When functionality is altered, it is possible for only a subset of downstream signalling events to be activated in what is termed biased signalling. Modified peptides that induce biased signalling are termed biased agonists.

In their work, Dawe and colleagues - including first author Dr Tharindunee Jayakody, an alumna of NUS Pharmacology who is now a lecturer leading a research group at the Department of Chemistry, University of Colombo, Sri Lanka - identified biased signalling whereby the stapled peptide analogs induced activation and coupling of specific G-protein sensors (Gαi1 and Gαo) to RXFP3. The same G-proteins are known to be activated by relaxin-3 and hence it was determined that binding and functionality of the ‘stapled’ peptides was retained, albeit at a lower potency. However, unlike unmodified relaxin-3, these stapled proteins did not stimulate β-arrestin recruitment to RXFP3. This was likely due to conformational changes in RXFP that resulted from its binding to the ‘stapled’ relaxin-3 analogs. β-arrestin recruitment could otherwise induce desensitisation and internalisation of the receptors, as a means to regulate relaxin-3 mediated signalling. Thus, the stapled analogs were identified as biased agonists of RXFP3.

In addition to this key finding, the team also characterised subsequent steps in the signalling cascade, including the inhibition of ERK1/2 activation and reporter gene activation, which was attributed to the lack of β-arrestin at the receptor.

Although the relaxin-3-RXFP3 signalling pathway remains to be fully characterised, the work of Dawe and colleagues has important implications in appreciating the potential of peptides as therapeutic alternatives to traditional small molecule drugs.

Traditionally, peptides are limited in their therapeutic potential due to membrane impermeability and subsequently, their poor uptake into cells. They also exhibit poor stability in vivo. However, ‘stapling’ of peptides has been shown to improve both cellular uptake and stability of the peptide.

For neuropeptides to be viable therapeutic options, characterisation of their target signalling pathways remains a priority. Protein interactions are highly complex, and the inhibition of one step in a signalling pathway can have unforeseen consequences on another. Biased signalling may overcome this if it can be determined that only a subset of signalling events is targeted.

Thus, with further research on the stability and integrity of stapled neuropeptides, and a full characterisation of the signalling pathways that they mediate, it is hoped that new avenues for the treatment of neurological disorders may be on the horizon.

This work was published in Science Signaling in February, 2024.