A biosynthetic approach to the discovery of novel bioactive peptides
Thesis
Peptides represent a source of novel therapeutics for recalcitrant human diseases, but screening for bioactivity from natural or synthetic sources can be uneconomic. In contrast, in vivo expression of peptides from DNA libraries in a heterologous host such as Escherichia coli may combine production with screening. This dissertation aimed to use such an approach to discover novel bioactive peptides in a high throughput and cost-effective manner, with a focus on antimicrobials and antiaggregants as proof-of-principle. Antimicrobial peptides (AMPs) are innate defence effectors that may combat antibiotic-resistant pathogens. An inducible, autocleaving fusion tag was utilised to produce the model murine cathelicidin K2C18, along with a number of variants, which exhibited varying degrees of antimicrobial activity against a panel of microbes. Importantly, K2C18 also exhibited a bacteriostatic effect in vivo when secreted to the periplasm. This allowed for the implementation of an in vivo whole cell screen for novel AMPs, using genomic DNA libraries as an input. One putative hit, the peptide S-H4, showed similar in vivo behaviour to K2C18 and was active when added exogenously to microbial cultures. A second in vivo screen was constructed to search for inhibitors of Aβ42 aggregation, a process implicated in Alzheimer’s disease. The aggregation state of Aβ42 was coupled to the fluorescence of a chromophore fusion partner, and used to screen co-expressed peptides from a random DNA library for putative antiaggregants. Additionally, the system incorporated an internal fluorescent reference to allow ratiometric comparison between samples. Several hits were identified and further validated using flow cytometry, with work ongoing to assess their activity in vitro. Proof-of-principle of these two screens was achieved, indicating that such in vivo approaches to bioactive peptide discovery could lead to the development of new and useful therapeutics.