Nature inspired treatments for persistent C. difficile infections

描述（由申请人提供）：由于高毒力菌株的增加，难辨梭菌感染（CDI）变得更加难以治疗，这些菌株增加了发病率和死亡率，并可能在感染患者中持续和复发。为了解决对新型抗艰难梭菌药物的迫切需求，我们探索了自然利用包括乳酸杆菌在内的有益细菌（即益生菌）通过产生新型抗菌剂来抑制肠道病原体（如艰难梭菌）的前提，这些抗菌剂通过进化对肠道微环境进行优化。在这方面，我们探索了从罗伊氏乳杆菌中开发罗伊霉素作为天然抗艰难梭菌剂的潜力。益生菌正在成为CDI的替代治疗方法。然而，关于是否可以使用活益生菌治疗严重CDI，特别是免疫功能低下患者，目前仍存在争议。因此，我们假设开发由益生菌产生的抗菌剂将利用其天然治疗特性之一来局部杀死肠道中的艰难梭菌，提供更可靠和有效的治疗策略。将这一概念应用于我们对红霉素的研究表明，它具有令人印象深刻的抗菌和治疗CDI的药理学特性。这些包括：快速杀死不生长的、产生毒素的固定相艰难梭菌；细菌膜特异作用机制的研究狭窄的活动范围；对肠道上皮缺乏细胞毒性；蛋白质水解稳定性；能够在肠道中达到高的非吸收浓度以杀死；低分子量和易于合成，将允许先进的化学优化。重要的是，目前处方的抗生素万古霉素和甲硝唑不能杀死产生毒素的固定相细胞，这两种抗生素只能杀死活跃生长的艰难梭菌。我们认为，开发益生菌衍生的罗伊霉素衍生物治疗CDI的建议具有高度创新性，并将通过三个迭代目标来实现：(i)合成一组扩展的罗伊霉素类似物，以优化抗艰难梭菌活性并增加对膜靶位的亲和力；㈡通过三个阶段的测试，包括抗菌剂评估、药代动力学测试、毒理学和体内功效实验，逐步开发和鉴定化合物。符合试验选择标准的化合物将进入下一阶段，从而获得具有有效体内疗效和良好安全性的主要候选物；（iii）作用方式研究，探索靶向梭状菌膜的基本抗菌作用。该项目的长期目标是开发优化的益生菌衍生的reuteric环素的先导类似物，这些类似物在膜靶点上表现出新的作用模式，并具有抗生素特性，使其能够作为治疗CDI的候选药物进入高级临床前研究。

DESCRIPTION (provided by applicant): Clostridium difficile infections (CDI) have become more difficult to treat due to the rise of hypervirulent strains that have increased morbidity as well as mortality and the likelihood of persistence and relapse in infected patients. To address the dire need for new anti-difficile agents, we explored the premise that nature utilizes good bacteria (i.e. probiotics), including Lactobacillus spp., to suppress gut pathogens such as C. difficile by producing novel antimicrobials, which were optimized by evolution to the microenvironment of the gut. In this regard, we explored the potential for developing reutericyclin from Lactobacillus reuteri as a natural anti- difficile agent. Probiotics are emerging as alternate treatments for CDI. However, there is ongoing debate on whether live probiotics may be used treat severe CDI, especially in immunocompromised patients. We therefore hypothesize that developing the antimicrobial produced by the probiotic specie would harness one of its natural therapeutic properties for localized killing of C. difficile in the gut, providing a more reliable and efficacious treatment strategy. Application of this concept to our studies on reutericyclin revealed that it has impressive antimicrobial and pharmacological properties for treating CDI. These include: rapid killing of nongrowing, toxin- producing stationary phase C. difficile; a novel mechanism of action specific to the bacterial membrane; a narrow spectrum of activity; lack of cytotoxicity against gut epithelia; stability to proteolysis; ability to achieve high non- absorbed concentrations in gut for killing; a low molecular weight and ease of synthesis that will allow advanced chemical optimization. Importantly, the killing of toxin-producing stationary phase cells is not shown by currently prescribed antibiotics vancomycin and metronidazole, which only kill actively growing C. difficile. We believe that this proposal for developing probiotic-derived reutericylcin derivatives to treat CDI is highly innovative and will be achieved through three iterative aims: (i) Synthesis of an expanded sets of reutericyclin analogs to optimize anti-difficile activity and increase affinity for the membrane target site ; (ii) Lead development and characterization involving the stepwise progression of compounds through three stages of tests that include antimicrobial assessment, pharmacokinetic testing, toxicologic and in vivo efficacy experiments. Compounds meeting the selection criteria of the tests will move onto the next stage such that a lead candidate is obtained with potent in vivo efficacy and excellent safety profile; (iii) Mode of action studies to explore the fundamental antibacterial effects of targeting the clostridial membrane. The long-term goal of this project is to develop optimized lead analogs of probiotic derived reutericyclin that exhibit novel modes of action at the membrane target and have characterized antibiotic properties that would allow their progression into advanced preclinical studies as candidates for treating CDI.