Structure function analysis of cyclotide scaffolds as novel antimicrobial agents

Cyclic peptides such as the polymyxins have considerable potential as antimicrobials:
however, they have challenging pharmacodynamic properties and rapidly generate
resistance in target bacteria. Plant-derived cyclotides such as cycloviolacin O, have
stable cystine knot (ICK) scaffolds formed by head to tail cyclisation and three disulfide
bridges. Such scaffolds have been exploited for other therapeutic applications, but
only to a very limited extent for antimicrobials. This project will aim to i) understand
the underlying features that drive the folding of these peptides, ii) modify the scaffold
to maximise antimicrobial and pharmacodynamic properties, iii) characterise the
mechanism of action through detailed microbiology and membrane interaction studies.
Months 10 - 24 Development of synthetic routes to cycloviolacin O and novel
derivatives, including methods that ensure the correct folding of the peptide [1]. The
student will explore how changes in the loop length and sequence affect the ability of
the peptide to fold into its active ICK conformation [2], and will develop a focussed
peptide library, incorporating amino acids which will enhance antibacterial activity and
minimise toxicity (Tabor). Months 18 - 24 The peptide library will be assessed for
antimicrobial activity in a screening cascade developed and used previously by the
applicants (Sutton) [3] to evaluate antimicrobial peptide function, the potential for
resistance emergence and mechanisms of action/resistance. Months 24 - 36 Patchclamp
studies (Mason) [3] will be used to understand how the cyclotide analogues
interact with or penetrate through bacterial membranes and how this relates to their
biological activity. Months 30 - 48 Based on the information from the biological and
biophysical studies, a second library of peptides will be synthesised and screened to
enhance antimicrobial and pharmacodynamic properties. In addition, peptides will be
synthesised incorporating (i) acyl chains to enhance membrane interactions (ii)
fluorophores for confocal microscopy (iii) photoreactive amino acids to modulate the
inhibitor activity [4,5].