Identifying novel antimicrobial potentiating agents for the treatment of chronic Pseudomonas aeruginosa infection

Abstract

The rapid emergence of multidrug-resistant (MDR) Pseudomonas aeruginosa poses a serious public health challenge, driving the need for new antimicrobial strategies. This project investigates macrocyclic compounds as potential adjuvants to existing antibiotics, focusing on chronic P. aeruginosa infections. Owing to their structural complexity and ability to engage difficult biological targets, macrocycles are promising candidates for enhancing antibiotic efficacy.

A high-throughput screen of 3,360 macrocycles from the ChemBridge library identified 84 compounds with potentiating activity: 41 with tobramycin, 43 with colistin. Computational tools, including SwissADME, were used to assess drug-likeness and prioritize lead compounds for further evaluation.

The therapeutic potential of macrocycle-antibiotic combinations was explored through in vitro and in vivo models. While no compounds disrupted established biofilms, several inhibited biofilm formation and potentiated antibiotics against clinical isolates. In the Galleria mellonella infection model, selected combinations significantly improved larval survival compared to antibiotics alone.

Mechanistic insights were gained through cell membrane integrity assays and proteomics. One lead compound, Ndkg-Z, was shown to disrupt both outer and inner membranes, confirmed by increased NPN uptake and cytoplasmic membrane depolarization. Proteomic analysis of P. aeruginosa PAO1 exposed to Ndkg-Z revealed downregulation of 51 proteins, including those linked to membrane structure, ribosomal function, and virulence. Upregulation of stress response proteins suggested an adaptive but insufficient bacterial response.

This thesis highlights macrocycles as dual-action antimicrobial adjuvants capable of enhancing existing therapies and targeting multiple resistance mechanisms. By disrupting membranes and impairing protein synthesis, macrocycles offer a novel approach to tackling MDR P. aeruginosa and may contribute to the development of next-generation antimicrobials.