Synthesis and Structure-Activity Relationship Studies of Battacin Peptides as Potential Therapeutic Agents Against Bacterial Pathogens

Abstract

Antimicrobial peptides (AMPs), produced by various organisms, can counter several multidrug resistant (MDR) pathogens and can be developed as alternatives to conventional antibiotics. However, they have short half-lives in vivo because of susceptibility to proteases. Structural modifications help to overcome protease susceptibility. Lipopeptides are stable against proteases and are therefore better candidates as future antimicrobials. Battacin, a cyclic lipopeptide, has been reported to possess potent in vitro and in vivo activity against MDR P. aeruginosa and E. coli. This thesis summarises investigation on the synthesis and structure-activity relationship studies of battacin peptides, as potential therapeutic agents against Gram-negative and Gram-positive bacteria. The first aim of the thesis was to synthesise battacin. Solid-phase peptide synthesis (SPPS) with acid sensitive 2-chlorotrityl chloride resin was used to generate the linear version with orthogonal protecting groups. This underwent selective side-chain-to-tail cyclisation, under high peptide dilution (10-3 M), followed by protecting group removal in solution phase, to generate synthetic battacin at a crude yield of 33% overall. To identify a superior analogue with broad spectrum activity a series of five cyclic and five linear amidated battacin iii lipopeptides were designed and synthesised, by conjugating different fatty acid chains (Fmoc, 4-methyl-hexanoyl, geranyl and myristoyl) to the N-terminus of the lipopeptide sequence. The second aim of this thesis was to evaluate the antimicrobial activities of these analogues. A minimum inhibitory concentration (MIC) assay against human (P. aeruginosa, S. aureus and E. coli) and plant (E. amylovora and Pseudomonas syringae pv. actinidiae) pathogens revealed that the 4-methyl-hexanoic acid conjugated linear lipopeptide, GZ3.27, showed greater antibacterial activity than battacin, was membrane-lytic and unlike the natural product, was also active against the Gram-positive pathogens S. aureus. The lipopeptide GZ3.27 also showed the ability to disperse mature biofilms of P. aeruginosa, S. aureus and Pseudomonas syringae pv. actinidiae. Further optimisation and structureactivity relationship studies of GZ3.27, using alanine scanning, revealed that the pentapeptide core (GZ3.159) is important for the antibacterial activity. The membrane-lytic action of GZ3.27 could be responsible for the observed antibacterial activity. Structural characterisations using NMR and CD spectroscopies revealed that a well-defined secondary structure formation is not crucial for the observed activity. The final section of the thesis describes the immobilisation of GZ3.27 onto solid surfaces, to study its potential as an antimicrobial coating in medical implants to prevent bacterial colonisation and implant-related infections. Cysteine modified GZ3.27 (GZ3.163) was selectively conjugated to maleimide PEG coated titanium and silicon surfaces, used in medical implants, and was characterised by X-Ray photoelectron spectroscopy, which indicated successful immobilisation. The immobilised peptide retained antibacterial activity and membrane-lytic mechanism of action, as indicated by a Live/Dead staining assay and scanning electron microscopy respectively.