Targeting of relaxin-3 analogues to the brain for pharmacological modulation of neurosignalling

Abstract

Relaxin-3, a member of the insulin/relaxin superfamily, has been shown to be involved in modulating several neurophysiological process including food intake, stress, addiction, arousal, memory and learning in animal models. Its sequence is highly evolutionary conserved, which strongly suggests an important physiological function. Elucidation of the relaxin-3 three-dimensional structure showed that relaxin-3 adopts a structure similar to insulin, in which the two peptide chains are linked by two inter-chain and one intra-chain disulfides. Residues vital for binding to its cognate receptor, relaxin family peptide 3 receptor (RXFP3), are found in the helical region of the B-chain whereas the C-terminal tail residues of the B-chain are important for activation of the receptor. Truncation of five residues from the C-terminus results in an antagonist, and antagonists and agonists have been important for studying relaxin-3 function. However, the animal studies to date have almost exclusively been conducted via intracerebroventricular injection as these peptides do not efficiently cross the blood-brain barrier (BBB). The BBB is a highly regulated barrier consisting of tight junctions between endothelial cells that prevents paracellular diffusion of most molecules into the brain, and it is decorated with transporters that pump out unwanted products. Many strategies for increasing brain delivery have been utilised over the years, including increasing lipophilicity and positive charge of the drug lead to facilitate membrane permeability and the use of molecular 'Trojan horses'. These 'Trojan horses' can be nonspecific in nature, like cell penetrating peptides, or specifically target receptors expressed on the BBB to enter the brain through receptor-mediated transcytosis. The main aim of this thesis was to design a series of relaxin-3 analogues based solely on the B-chain with increased potential for crossing the BBB when introduced through the systemic circulation, and with an ability to modulate food intake in mice models. In order to achieve this aim, structure-activity relationship studies were first conducted on the single-chain relaxin-3 antagonist (R3 B1-22R) to further understand the binding determinants of this variant. Using Fmoc-peptide synthesis modifications, including an alanine scan, were introduced and there effect on binding evaluated through competition binding assays. There were similarities between the antagonist residues involved in binding to RXFP3 and the relaxin-3 agonist residues, but additional residues are involved in the antagonist and the binding conformations were distinct between the agonist and antagonist. Since linear relaxin-3 B-chain variants are unstructured in solution and easily degraded in serum, we employed several strategies to reintroduce structure into the single-chain analogues. These included substitution with helical promoting residues (Aib), global cyclisation, molecular grafting, and crosslinking of side chains through lactam bonds or hydrocarbon stapling, in order to improve s...