Abstract
The escalating crisis of antimicrobial resistance (AMR) threatens the foundational efficacy of antibiotics. The Gram-negative pathogen Pseudomonas aeruginosa (P. aeruginosa) is a critical priority in this crisis, notorious for its intrinsic and acquired resistance mechanisms, including low membrane permeability and potent efflux pumps, which leads to clinically difficult-to-treat infections. This thesis addresses this challenge by developing a targeted antibiotic delivery strategy designed to circumvent resistance and minimise non-selective antimicrobial pressure that drives AMR.
A prodrug platform based on a ‘Trojan horse’ hypothesis, was designed to exploit the bacterial dipeptide permease (Dpp) transporter for pathogen-specific delivery. The model antibiotic, ciprofloxacin, was conjugated to various dipeptides via a bioreducible disulfide linker. This strategy was designed to circumvent efflux-mediated resistance through a two-step mechanism: first, active import of the prodrug via the Dpp system, and second, intracellular release of the active ciprofloxacin payload via reduction of the disulfide linker by GSH.
A systematic synthetic approach generated three distinct generations of prodrugs to investigate the structure-activity relationships. Generation 1, comprising of non-cleavable conjugates, confirmed that masking the C3 carboxylate of ciprofloxacin effectively abolished its antibacterial activity, validating the prodrug design at this position. Generations 2 and 3 incorporated a disulfide linker, differing in their attachment to the N- or C-terminus of the peptide, respectively. High-Performance Liquid Chromatography (HPLC) assays confirmed the GSH-dependent release of ciprofloxacin from these cleavable conjugates, though stability studies identified hydrolytic instability in longer peptide analogues as a key limitation for future optimisation.
Biological evaluation revealed that the cleavable prodrugs exhibited restored partial antibacterial activity against P. aeruginosa. A critical finding was the discrepancy between minimum inhibitory concentration (MIC) values determined by standard visual turbidity readings and those from triphenyltetrazolium chloride (TTC) metabolic staining, indicating that conventional assays may underestimate the potency of intracellularly activated prodrugs, providing a new outlook on established literature. Simultaneous measurements of optical density and luminescence in time-kill assays revealed a delayed bactericidal onset for the prodrugs. This kinetic profile is consistent with a mechanism dependent on transporter-mediated uptake and intracellular activation, contrasting with the immediate, diffusion-driven action of free ciprofloxacin. The major multidrug resistant (MDR) efflux pump MexAB-OprM was found to play a minimal role in the efflux of the cleavable prodrug conjugates, suggesting that other MDR efflux pumps are responsible.
This thesis provides proof-of-concept for a prodrug strategy that enhances the specificity of antibiotic action against P. aeruginosa. The findings validate the potential of peptide-based conjugates to overcome efflux-mediated resistance and underscore the importance of linker stability for future development. This work lays a foundational synthetic and biological framework for the continued development of pathogen-specific therapies, contributing a valuable approach to the global effort against AMR.