Abstract:
Rise in antimicrobial resistance has become a growing global threat. This has led to an ever-increasing need for new and effective antibiotics. However, development of newer antibiotics has seen limited progress. Moreover, this challenge is increased against Gram-negative bacteria, whose extra outer membrane effectively acts as a permeability barrier. Innovative targeting strategies are urgently required, exploiting endogenous bacterial uptake systems aiming to reduce permeability challenge. This project involves characterizing two sets of novel cleavable antibiotic conjugates designed against Gram-negative bacteria. The first set includes gold-cleavable linezolid-based conjugates (SH592/SH594). The second set involves murepavadin conjugates, targeting Pseudomonas aeruginosa surface lectins. Both strategies are designed to limit antibiotic activity to the target site upon conjugate cleavage, reducing off-target cytotoxic effects associated with free murepavadin and linezolid-based antibiotics. Optimization of gold-mediated cleavage conditions, combined with pre-feeding experiments in E. coli ΔentA, demonstrated significantly reduced MIC values even for the conjugated form. Additionally, quantitative uptake data for SH592 obtained by label-free mass spectrometry experiments suggested that MIC lowering was mediated by improved uptake of the conjugate though the siderophore pathway. Secondly, biofilm assays were designed to investigate biofilm eradication and accumulation ability of the lectin-targeting conjugates. These conjugates incorporate multiple galactose-based sugars and were predicted to enhance overall avidity of the molecules to biofilms. The assays demonstrated comparable anti-biofilm activity of the conjugates to free drug. Additionally, the multivalent conjugate precursors exhibited comparable accumulation within the biofilm matrix to compounds containing one and two galactose moieties. This finding suggests that conjugate design requires further optimization to improve activity. Overall, this study explored two targeted antibiotic delivery strategies, through optimization of unique cleavable conjugates, designed against Gram-negative bacteria.
