Structure function analysis of cyclotide scaffolds as novel antimicrobial agents

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Cyclic peptides such as the polymyxins have considerable potential as antimicrobials:however, they have challenging pharmacodynamic properties and rapidly generateresistance in target bacteria. Plant-derived cyclotides such as cycloviolacin O, havestable cystine knot (ICK) scaffolds formed by head to tail cyclisation and three disulfidebridges. Such scaffolds have been exploited for other therapeutic applications, butonly to a very limited extent for antimicrobials. This project will aim to i) understandthe underlying features that drive the folding of these peptides, ii) modify the scaffoldto maximise antimicrobial and pharmacodynamic properties, iii) characterise themechanism of action through detailed microbiology and membrane interaction studies.Months 10 - 24 Development of synthetic routes to cycloviolacin O and novelderivatives, including methods that ensure the correct folding of the peptide [1]. Thestudent will explore how changes in the loop length and sequence affect the ability ofthe peptide to fold into its active ICK conformation [2], and will develop a focussedpeptide library, incorporating amino acids which will enhance antibacterial activity andminimise toxicity (Tabor). Months 18 - 24 The peptide library will be assessed forantimicrobial activity in a screening cascade developed and used previously by theapplicants (Sutton) [3] to evaluate antimicrobial peptide function, the potential forresistance emergence and mechanisms of action/resistance. Months 24 - 36 Patchclampstudies (Mason) [3] will be used to understand how the cyclotide analoguesinteract with or penetrate through bacterial membranes and how this relates to theirbiological activity. Months 30 - 48 Based on the information from the biological andbiophysical studies, a second library of peptides will be synthesised and screened toenhance antimicrobial and pharmacodynamic properties. In addition, peptides will besynthesised incorporating (i) acyl chains to enhance membrane interactions (ii)fluorophores for confocal microscopy (iii) photoreactive amino acids to modulate theinhibitor activity [4,5].