The development of stapled peptides as chemical tools to investigate activin A signalling and as antibacterial agents targeting MsbA-mediated efflux

Peptides are recognised for their ability to disrupt protein-protein interactions (PPIs), which have traditionally been considered ‘undruggable’ with small molecules. However, due to issues such as a susceptibility to degradation by proteases, the use of peptide therapeutics in the clinic has been limited. Peptide stapling, whereby a peptide is constrained into its binding conformation, presents a means by which some of the weaknesses of peptide therapeutics may be overcome. This thesis describes two projects in which stapled peptides were developed to investigate PPIs, and closely related helix-helix packing sites, relevant to human health. 1. The development of stapled peptides as chemical tools to investigate activin A signalling Activin A signalling has been implicated in a wide range of cancers as well as the orphan disease fibrodysplasia ossificans progressiva. In this section, stapled peptides were rationally designed with the intention of inhibiting the PPI between mature activin A and its type I receptor, thus reducing downstream intracellular signalling. The ability of the peptides to bind mature activin A was assessed through biophysical assays, and for the best binder, its ability to inhibit activin A signalling was investigated. These studies ultimately led to the identification of an activin A prodomain-derived stapled peptide that is able to inhibit activin A signalling in vitro. In addition, the effect of stapling on the secondary structures of the peptides was assessed, as was its effect on protease resistance. This work provides a basis for the further development of peptide probes of activin A signalling that may ultimately lead to new therapeutics. 2. The development of stapled peptides as antibacterial agents targeting MsbA- mediated efflux Antimicrobial resistance (AMR) is a significant threat to global health and Gram-negative bacteria are a particularly challenging target. MsbA is an essential transmembrane protein in Gram-negative bacteria, transporting the key outer membrane component lipopolysaccharide (LPS) across the cytoplasmic membrane. In this section, stapled peptides were rationally designed to compete for helix-helix packing sites in the transmembrane region of MsbA. Through disrupting these interactions, it was hoped that the efflux activity of MsbA could be inhibited, compromising cell wall integrity and leading to cell death. Peptides that are able to specifically inhibit MsbA-mediated efflux were identified, and their mechanisms of action were investigated and compared with previously reported small-molecule MsbA inhibitors. Additionally, siderophores were investigated as a means of enabling the peptides to bypass the impermeable outer membrane in order to reach their inner membrane target. This work provides a basis for the development of a novel class of Gram-negative specific antibacterial agents to combat the growing AMR crisis.