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

开发在宿主/病原体界面操纵铜的化学工具

• 批准号: 8505953
• 负责人: Katherine J. Franz
• 金额: $ 29.42万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8505953
• 关键词: Address; Antifungal Agents; Antifungal Therapy; Binding; Biochemical Genetics; Biological; Biological Assay; Biology; Breathing; Cell Death; Cell Survival; Cells; Chelating Agents; Chemicals; Collaborations; Copper; Cryptococcus neoformans; Data; Detox; Development; Elements; Environment; Fluorescent Probes; Funding; Goals; Health; Human; Hydrogen Peroxide; Immune; Immune system; Immunity; Immunocompromised Host; In Vitro; Individual; Industrial fungicide; Infection; Invaded; Ionophores; Ions; Iron; Laboratories; Location; Macrophage Activation; Mammalian Cell; Measures; Meningoencephalitis; Metal Ion Binding; Metals; Microbe; Modeling; Nutrient; Organism; Oxidative Stress; Pathway interactions; Peroxonitrite; Phagosomes; Prodrugs; Property; Proteins; Race; Research; Resistance; Respiratory Burst; Role; Solubility; Testing; Therapeutic; Therapeutic Agents; Toxic effect; Toxin; Virulence; Work; antimicrobial; antimicrobial drug; arm; bactericide; base; design; experience; fighting; fungus; in vitro testing; innovation; interest; killings; macrophage; microbial; microbicide; microorganism; mutant; pathogen; public health relevance; resistance mechanism; response; small molecule; tool; weapons

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.

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的杀死，而微生物则通过上调铜抗性途径来抵消。这是一个未被满足的机会来创造创新的抗菌剂，操纵铜沿着宿主/病原体界面，在理解铜在免疫和微生物毒性中的机制方面仍然存在重大差距。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，以获得潜在的治疗效益。当前应用的总体目标是使用可触发的金属结合剂，称为前螯合剂，来操纵先天免疫细胞中的铜来杀死感染的微生物。核心假设是，可以触发小分子选择性地动员Cu以应对感染，可以增强免疫系统对杀菌Cu的使用，逃避病原体的Cu抗性途径，避免破坏宿主的整体金属状态。该假设是基于申请人实验室的初步体外数据制定的，该数据表明，与活化的巨噬细胞相关的反应物触发选择性前螯合剂，将无毒的前药物转化为有效的cu依赖性杀菌剂。这一假设将在真菌病原体新隐球菌中进一步验证，通过解决三个具体目标：1)确定螯合物/前螯合物对，增强cu刺激下的微生物杀灭，但避免哺乳动物细胞毒性；2)描述cu依赖性微生物杀灭的作用模式；3)开发多响应荧光探针，可视化巨噬细胞活化后金属再分配的过程。在第一个目标下，小分子将被用于检测cu依赖的杀微生物活性，前螯合物将被合成并用于检测哺乳动物细胞的活力。有希望的化合物将被测试用于巨噬细胞的感染清除和表征有关的前螯合剂性质。初步结果证明了这些检测和申请人的促螯合剂合成/表征策略的可行性。第二个目标得益于已建立的合作，将生化、遗传和分析测试结合起来，阐明真菌病原体如何对潜在治疗剂递送的铜作出反应、适应和屈服。第三个目标建立在申请人设计荧光探针的经验基础上，以创建能够感知金属离子的荧光促剂，以响应巨噬细胞激活引起的化学环境的变化。整体方法是创新的，因为它利用宿主在响应感染时产生的独特化学环境来调动内源性Cu来加剧微生物的杀死。该研究的重要意义在于，它代表了基于Cu生物学开发广谱抗菌药物的第一步，同时阐明了Cu诱导微生物毒性的机制。