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Supporting data for “THE TROJAN HORSE STRATEGY AND METALLODRUGS TO COMBAT ANTIMICROBIAL RESISTANCE”

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posted on 2024-06-24, 02:51 authored by Chenyuan WangChenyuan Wang

The rapid emergence of antimicrobial resistance (AMR) pathogens highlights the urgent need to approach this global burden with alternative strategies. 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. Inspired by natural antimicrobial agents salmycin and albomycin, siderophore-antibiotic conjugates, also named as sideromycins, are investigated to combat the outer membrane barrier of Gram-negative bacteria. Another potent weapon against antimicrobial resistance is the metallodrug, which has been used for centuries. Metal ions have a long history to serve as antimicrobial agents and metal-based compounds 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 them with ampicillin/ciprofloxacin to investigate their potential as drug carriers. The result demonstrated that bi-catechol and mixed-ligand 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. 2-AMP also demonstrated a dose-dependent antibiofilm formation ability against PAO1. 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 by construct PAO1ΔPirA and PAO1ΔPiuA, and importantly, 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 such as Bi3+ into the bacterial cells simultaneously. Using cefiderocol (CEF) as a showcase, we demonstrated a strong synergy between Bi3+ compounds and CEF against P. aeruginosa strains 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. Notably, the co-therapy of CBS and CEF significantly increases the survival rate of mice and decreases bacterial loads in the lung in a murine acute pneumonia model. 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. Time kill assays and 3D heatmaps confirm the strong synergy between CEF and gallium nitrate (GaN) with a Bliss score of 6.187. The combination of CEF and GaN synergistically disrupted over 75% of biofilms. UV-vis spectroscopy, NMR titration, and mass spectrometry confirm the formation of a 1:1 complex of gallium-cefiderocol (Ga-CEF). Intracellular gallium concentrations significantly increase in the presence of CEF, suggesting that CEF enhances gallium uptake by transporting the Ga-CEF complex through iron-siderophore channels. Importantly, the combination of GaN and CEF exhibits low cytotoxicity to mammalian cells and high potency in a murine lung infection model compared to monotherapy. These findings support the potential translation of the combination therapy of CEF and GaN into clinical use, as both drugs are already clinically approved with established safety profiles and clear mechanisms of action.

Funding

the Research Grants Council of Hong Kong SAR (R7070-18, 17308921, 2122-7S04)

the Health and Medical Research Fund of the Health Bureau of Hong Kong SAR (CID HKU1-13)

the University of Hong Kong (URC (202107185074) and Norman & Cecilia Yip Foundation)

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