The disulfide bond as a chemical tool in cyclic peptide antibiotics: engineering disulfide polymyxins and murepavadin

耐多药（MDR）病原体的增加已成为全球关注的问题。据最近估计，2019年全球有495万人死于耐药细菌感染。其中，127万例死亡直接归因于抗菌素耐药性。在欧洲，欧洲疾病预防和控制中心（ECDC）报告说，耐抗生素细菌感染造成33 000人死亡（欧洲经济区数据）。同样，在美国，疾病预防控制中心估计，每年发生超过280万例抗生素耐药性感染，导致超过3.5万人死亡。世卫组织和美国疾病控制与预防中心已将革兰氏阴性病原体，如鲍曼不动杆菌耐碳青霉烯菌株、肠杆菌科（包括大肠杆菌和肺炎克雷伯菌）和铜绿假单胞菌（也是多重耐药菌株）确定为紧急或严重威胁。关于与耐药性相关的死亡人数，2019年，六种主要病原体（大肠杆菌、金黄色葡萄球菌、肺炎克雷伯菌、肺炎链球菌、鲍曼假杆菌和铜绿假单胞菌）造成了约92.9万例AMR死亡。特别是，2015年，在欧盟和欧洲经济区，耐粘菌素、耐碳青霉烯或耐多药铜绿假单胞菌显示出与抗生素耐药细菌感染引起的死亡和残疾调整生命年相关的一些最大影响。因此，治疗这些铜绿假单胞菌引起的感染显然是该领域未满足的医疗需求。在这方面，抗菌肽，特别是那些环状的，非核糖体生物合成的（或核糖体合成和翻译后修饰）提供了一个被探索作为潜在药物的机会。在这类课程中，我们发现了众所周知的临床使用的药物，如达托霉素和多粘菌素（多粘菌素B和多粘菌素E/粘菌素，用于前药形式粘菌素酸酯）或新型化合物，如达巴actin或teixobactin。达托霉素主要用于治疗革兰氏阳性金黄色葡萄球菌引起的感染，而多粘菌素已成为治疗多重耐药（MDR）革兰氏阴性菌（GNB）引起的感染的最后手段。由于多粘菌素的肾毒性和神经毒性，当没有其他治疗选择时，多粘菌素被用作最后的抗生素。因此，无此类不良反应的新型多粘菌素的可用性将是治疗多重耐药（MDR）革兰氏阴性菌引起的感染的一大进步。该项目提出了一种创新的化学工具，可应用于已知的环肽抗生素，包括临床使用或处于临床开发阶段的抗生素，以减少其肾毒性。该设计的基本原理包括维持抗生素的整体结构以保持抗菌活性，同时在肽骨架内的化学工具的存在将促进抗生素在肾脏中最终积累时通过解毒酶的初始代谢。该项目遵循一个概念验证方案，包括必要的化学制备模型化合物，体外和体内分析，以评估活性和低毒性，并估计治疗窗口。最后，提出了在膜水平上验证设计假设和作用机制的实验。

The increase of multidrug-resistant (MDR) pathogens has become a matter of global concern. It has been recently estimated that 4.95 million people died worldwide due to drug-resistant bacterial infections in 2019. Of those, 1.27 million deaths were directly attributed to antimicrobial resistance (AMR). In Europe, the European Centre for Disease Prevention and Control (ECDC) has reported that antibiotic-resistant bacterial infections caused 33,000 deaths (data for the European Economic Area). Similarly, in the US, the CDC estimates that more than 2.8 million antibiotic-resistant infections take place annually with more than 35,000 dead people as a consequence. The WHO and CDC have identified Gram-negative pathogens such as carbapenem-resistant strains of Acinetobacter baumannii, Enterobacteriaceae (which including Escherichia coli and Klebsiella pneumoniae), and Pseudomonas aeruginosa (also multi-drug resistant strains) as urgent or serious threats. Regarding the number of deaths associated with resistance, six leading pathogens (E. coli, S. aureus, K. pneumoniae, Streptococcus pneumoniae, A. baumannii, and P. aeruginosa) were responsible for ca 929,000 deaths attributable to AMR in 2019.In particular, colistin-resistant, carbapenem-resistant, or multidrug-resistant P. aeruginosa shows some of the highest impact related to deaths and disability-adjusted life-years caused by infections with antibiotic-resistant bacteria in the EU and the European Economic Area in 2015. Hence, the treatment of these P. aeruginosa caused infections is a clear unmet medical need in the field. In this regards, antimicrobial peptides, particularly those that are cyclic, non-ribosomally biosynthesized (or ribosomally synthesized and post-translationally modified) offer an opportunity to be explored as potential drugs. In this class we find well-known clinically-used drugs such as daptomycin and polymyxins (polymyxin B and polymyxin E/colistin, used in the prodrug form colistimethate) or novel compounds such as darobactin or teixobactin. Daptomycin is mostly indicated to treat Gram-positive Staphylococcus aureus caused infections, whereas polymyxins have become a last resort therapeutic option to treat multi-drug resistant (MDR) Gram-negative bacteria (GNB) caused infections. Polymyxins are used as last resort antibiotics when no other therapeutic option is available due to their nephrotoxicity and neurotoxicity. Hence, the availability of novel polymyxins devoid of such adverse effects would be a great advance for the treatment of infections caused by multi-drug resistant (MDR) Gram-negative bacteria. The project presents an innovative chemical tool to be applied to known cyclic peptide antibiotics, including those in clinical use or in a clinical development phase, to reduce their nephrotoxicity. The rationale of the design consists of maintaining the overall structure of the antibiotic to preserve the antibacterial activity while the presence of the chemical tool within the peptide backbone would facilitate the initial metabolization by detoxifying enzymes upon eventual accumulation of the antibiotic in the kidney. The project follows a proof-of-concept scheme involving the necessary chemistry to prepare the model compounds, the in vitro and in vivo assays to assess activity and low toxicity, and estimation the therapeutic window. Finally, tests to prove the design hypothesis and the mechanism of action at the membrane level are also proposed.