The Trojan Horse strategy and metallodrugs to combat antimicrobial resistance

Abstract

In the face of the growing threat of antimicrobial resistance, the repurposing of traditional antibiotics has gained attention as a potential strategy. One way to increase the antibacterial activity of known antibiotics is the Trojan Horse strategy, by which drugs can be delivered into the bacterial cells through nutrient uptake pathways to bypass the membrane barrier. Siderophores, produced by microorganisms to scavenge iron from the environment, are promising drug carriers through iron uptake pathways. Sideromycins are investigated to combat the outer membranei barrier of Gram-negative bacteria. Another potent weapon against antimicrobial resistance is the metallodrug, which has been used for centuries. Metal ions are now attracting more interests from scientific communities in the fight against AMR owing to their unique mode of action. In chapter 2, two nonnative bi-catechol and mixed-ligand siderophores were designed and conjugated with ampicillin/ciprofloxacin to investigate their potential as drug carriers. The result demonstrated that both siderophores can broaden the antibacterial spectrum of ampicillin to resistant strains, within 2-AMP exhibiting the most potent antibacterial activity with a MIC value of 0.5 μg/mL against PAO1, which is comparable to the recent clinically approved sideromycin drug cefiderocol (CEF). The intracellular concentration of iron increased in the presence of 2-AMP, indicating this nonnative bi-catechol can functioned as a siderophore and promote the cellular iron absorption. We further validated the transporters involved in the 2-AMP uptake and PAO1ΔPiuA demonstrated lower sensitivity to 2-AMP, indicating the PiuA plays an important role in bi- catechol siderophore transport. In chapter 3, we firstly proposed a dual-Trojan Horse strategy through metallo-sideromycins, which utilize sideromycins to deliver antibacterial metal ions Bi3+ into the bacterial cells simultaneously. We demonstrated a strong synergy between Bi3+ compounds and CEF against PAO1 by a 64-fold reduction in the MIC of CEF. Importantly, CBS could enhance the CEF efficacy against biofilm formation, suppress the development of high-level bacterial resistance to CEF, and restore the efficacy of CEF against CEF-resistant P. aeruginosa clinical isolates. The co- therapy significantly increases the survival rate of mice and decreases bacterial loads in the lung in vivo. The observed phenomena are partially attributable to the competitive binding of Bi3+ to cefiderocol with Fe3+, leading to enhanced uptake of Bi3+ and reduced levels of Fe3+ in cells. In chapter 4, we demonstrated that gallium (III) drugs could also enhance the efficacy of CEF against P. aeruginosa strains. 3D heatmaps confirm the synergy between CEF and gallium nitrate (GaN) with a Bliss score of 6.187. The combination synergistically disrupted over 75% of biofilms. UV/NMR titration, and mass spectrometry confirm the formation of a 1:1 complex of Ga-CEF. Intracellular gallium concentrations significantly increase in the presence of CEF, suggesting that ii CEF enhances gallium uptake by transporting the Ga-CEF complex through iron-siderophore channels. The co-therapy exhibits low cytotoxicity to mammalian cells and high potency in a murine lung infection model compared to monotherapy, which supports the potential translation of the co-therapy into clinical use as they are already clinically approved drugs with established safety profiles and clear mechanisms of action.