Development of Chemical Tools to Manipulate Copper at the Host/Pathogen Interface

描述（由申请人提供）：免疫系统的巨噬细胞将铜（Cu）浓缩到吞噬体中以加强微生物杀灭，而微生物通过上调Cu抗性途径进行抵消。有一个未满足的机会，创造创新的抗菌剂，操纵铜沿着这个主机/病原体接口，并在理解铜的免疫和微生物毒性的机制仍然存在显着的差距。长期目标是开发化学工具来操纵生物金属离子的位置，形态和反应性，以获得潜在的治疗益处。本申请的总体目标是使用可降解的金属结合剂（称为前螯合剂）来操纵先天免疫细胞中的Cu以杀死感染的微生物。核心假设是，可以被触发以响应感染而选择性地动员Cu的小分子可以促进免疫系统对杀菌Cu的使用，逃避病原体的Cu抗性途径，并避免破坏宿主的整体金属状态。该假设是基于来自申请人实验室的初步体外数据来制定的，该数据显示选择的前螯合剂由与活化的巨噬细胞相关的反应物触发，以将无毒前药转化为有效的Cu依赖性杀真菌剂。该假设将在真菌病原体新型隐球菌中通过解决三个具体目标来进一步测试：1）鉴定增强Cu刺激的微生物杀伤但避免哺乳动物细胞毒性的螯合剂/前螯合剂对; 2）描绘Cu依赖性微生物杀伤的作用模式;和3）开发多响应荧光探针以可视化响应于巨噬细胞活化的金属再分布。在第一个目标下，将测定小分子的Cu依赖性杀微生物活性，并合成前螯合剂版本并测定哺乳动物细胞活力。将测试有前景的化合物通过巨噬细胞的感染清除，并表征前螯合剂性质。初步结果证明了申请人的这些试验和前螯合剂合成/表征策略的可行性。第二个目标受益于一个既定的合作，联合收割机结合生化，遗传和分析测试，以阐明真菌病原体如何响应，适应，并屈服于铜提供的潜在治疗剂。第三个目的建立在申请人在设计荧光探针以产生能够响应于由巨噬细胞活化诱导的变化的化学环境而感测金属离子的荧光前螯合剂方面的经验的基础上。整体方法是创新的，因为它利用了独特的化学环境中创建的主机响应感染动员内源性铜加剧微生物的杀伤。这项研究意义重大，因为它代表了开发基于Cu生物学的广谱抗菌剂的第一步，同时阐明了Cu诱导的微生物毒性机制。

DESCRIPTION (provided by applicant): Macrophage cells of the immune system concentrate copper (Cu) into phagosomes to intensify microbial killing, while microbes counteract by upregulating Cu resistance pathways. There is an unmet opportunity to create innovative antimicrobial agents that manipulate Cu along this host/pathogen interface, and there remain significant gaps in understanding the mechanisms of Cu in immunity and microbial toxicity. The long-term goal is to develop chemical tools to manipulate biological metal ion location, speciation, and reactivity for potential therapeutic benefit. The overall objective of the current application is to use triggerable metal-binding agents, called prochelators, to manipulate Cu in innate immune cells to kill infecting microbes. The central hypothesis is that small molecules that can be triggered to mobilize Cu selectively in response to infection can boost the immune system's use of bactericidal Cu, evade the Cu resistance pathways of the pathogen, and avoid disrupting the overall metal status of the host. This hypothesis is formulated based on preliminary in vitro data from the applicant's laboratory showing that select prochelators are triggered by reactants associated with activated macrophages to convert non-toxic prodrugs into potent Cu-dependent fungicides. The hypothesis will be further tested in the fungal pathogen Cryptococcus neoformans by addressing three specific aims: 1) Identify chelator/prochelator pairs that enhance Cu-stimulated microbial killing but avoid mammalian cell toxicity; 2) Delineate mode of action of Cu-dependent microbial killing; and 3) Develop multiresponsive fluorescent probes to visualize metal redistribution in response to macrophage activation. Under the first aim, small molecules will be assayed for Cu-dependent microbicidal activity and prochelator versions will be synthesized and assayed for mammalian cell viability. Promising compounds will be tested for infection clearance by macrophages and characterized with respect to prochelator properties. Preliminary results demonstrate feasibility of these assays and prochelator synthesis/characterization strategies by the applicant. The second aim benefits from an established collaboration to combine biochemical, genetic, and analytical testing to elucidate how a fungal pathogen responds, adapts, and succumbs to Cu delivered by a potential therapeutic agent. The third aim builds on the applicant's experience in designing fluorescent probes to create fluorescent prochelators capable of sensing metal ions in response to the changing chemical environment induced by macrophage activation. The overall approach is innovative because it exploits the unique chemical milieu created by the host in response to infection to mobilize endogenous Cu to exacerbate microbial killing. The proposed research is significant because it represents the first step in developing broad-spectrum antimicrobial agents based on Cu biology while elucidating mechanisms of Cu-induced microbial toxicity.