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

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.

抗菌药物耐药性（AMR）病原体的迅速出现凸显了以替代策略应对这一全球负担的紧迫需求。面对日益增长的抗菌药物耐药性威胁，传统抗生素的再利用作为一种潜在策略引起了广泛关注。提升已知抗生素的抗菌活性的一种途径是采用特洛伊木马策略，通过营养摄取途径将药物递送至细菌细胞中，从而绕过细胞膜屏障。铁载体，由微生物产生以从环境中获取铁元素，通过铁摄取途径成为有希望的药物载体。受天然抗菌剂萨氏霉素和阿尔博霉素的启发，铁载体-抗生素偶联物，也被称为铁霉素，被研究以对抗革兰氏阴性菌的外膜屏障。对抗抗菌药物耐药性的另一强力武器是金属药物，其使用已有数百年历史。金属离子作为抗菌剂有着悠久的历史，而基于金属的化合物因其独特的药理作用模式，现在正吸引科学界更多关注，以对抗AMR。在第2章中，我们设计并合成了两种非天然双儿茶酚和混合配体铁载体，并将它们与氨苄西林/环丙沙星偶联，以研究其作为药物载体的潜力。结果显示，双儿茶酚和混合配体铁载体可以扩大氨苄西林对耐药菌株的抗菌谱，其中2-AMP表现出最强大的抗菌活性，对PAO1的最低抑菌浓度（MIC）为0.5 μg/mL，与近期临床批准的铁霉素药物头孢吡肟相当。2-AMP还显示出对PAO1的生物膜形成的剂量依赖性抑制作用。在2-AMP存在的情况下，细胞内铁浓度增加，表明这种非天然双儿茶酚可以作为一种铁载体，促进细胞对铁的吸收。我们进一步通过构建PAO1ΔPirA和PAO1ΔPiuA验证了参与2-AMP摄取的转运蛋白，并且重要的是，PAO1ΔPiuA对2-AMP的敏感性较低，表明PiuA在双儿茶酚铁载体运输中起着重要作用。在第3章中，我们首先提出了一种双重特洛伊木马策略，通过金属铁霉素，利用铁霉素同时将抗菌金属离子如Bi3+递送至细菌细胞中。以头孢吡肟（CEF）为例，我们通过CEF对铜绿假单胞菌菌株的最低抑菌浓度（MIC）降低64倍，证明了Bi3+化合物与CEF之间存在着强大的协同作用。重要的是，CBS可以增强CEF对生物膜形成的抑制作用，抑制对CEF产生的高级别细菌耐药性的发展，并恢复CEF对头孢吡肟耐药的铜绿假单胞菌临床分离株的疗效。值得注意的是，CBS和CEF的联合治疗显著提高了小鼠的存活率，并降低了肺部的细菌负荷，在急性肺炎小鼠模型中。观察到的现象部分归因于Bi3+与Fe3+对头孢吡肟的竞争性结合，导致Bi3+的细胞内摄取增加和Fe3+水平降低。在第4章中，我们证明了镓（III）药物也能增强CEF对铜绿假单胞菌菌株的疗效。时间-杀灭实验和3D热图证实了CEF与硝酸镓（GaN）之间存在着强烈的协同作用， Bliss得分为6.187。CEF与GaN的结合协同破坏了超过75%的生物膜。紫外-可见光谱、核磁滴定和质谱证实了镓-头孢吡肟（Ga-CEF）1:1复合物的形成。在CEF存在的情况下，细胞内镓浓度显著增加，表明CEF通过铁-铁载体通道将Ga-CEF复合物运输，从而增强了镓的摄取。重要的是，与单药治疗相比，GaN和CEF的联合对哺乳动物细胞具有低细胞毒性，并在小鼠肺感染模型中表现出高活性。这些发现支持了CEF和GaN联合治疗策略向临床应用的转化，因为这两种药物均已获得临床批准，并具有确立的安全性和明确的药理作用机制。