Methods to study Cryptosporidium drug resistance

研究隐孢子虫耐药性的方法

• 批准号: 10591168
• 负责人: CHRISTOPHER D HUSTON
• 金额: $ 19.5万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-11-14 至 2024-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10591168
• 关键词: Acceleration; Acquired Immunodeficiency Syndrome; Address; Amino Acids; Amino Acyl-tRNA Synthetases; Animals; Antimalarials; Antimicrobial Resistance; Backcrossings; Biological Assay; Cells; Chemicals; Child; Chloroquine; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Combination Drug Therapy; Combined Modality Therapy; Communicable Diseases; Computer Simulation; Cryptosporidiosis; Cryptosporidium; Cryptosporidium parvum; DNA; Dairying; Data; Defect; Development; Diarrhea; Disease Outbreaks; Dose; Drug Combinations; Drug resistance; Drug usage; Engineering; Evolution; Failure; Future; Genes; Genetic; Genetic Epistasis; Genomics; Glean; Goals; Grant; Growth; Immunocompromised Host; In Vitro; Infant; Infection; Iowa; Knowledge; Lead; Life; Link; Malaria; Measurement; Measures; Methionine-tRNA Ligase; Methods; Modeling; Mus; Mutation; Parasite resistance; Parasites; Patients; Pharmaceutical Preparations; Pharmacology; Pharmacotherapy; Piperazines; Placebos; Positioning Attribute; Predisposition; Public Health; Recrudescences; Resistance; Resources; Risk; Series; Speed; Stress; Suicide; Testing; Time; Transgenic Organisms; Tuberculosis; United States; Work; curative treatments; diarrheal disease; disorder risk; drug development; drug efficacy; drug resistance development; efficacy study; efficacy trial; experimental study; fitness; improved; in vivo; inhibitor; lead series; microbial; models and simulation; mutant; nitazoxanide; novel; parasite genome; prevent; reproductive fitness; resistance mutation; resistant strain; response; vector; waterborne

PROJECT SUMMARY: Cryptosporidiosis, the diarrheal disease caused by Cryptosporidium parasites, is amongst the most important causes of life-threatening diarrhea in children globally and causes incurable diarrhea in AIDS patients. The only approved treatment is just over 50% effective for children and equivalent to a placebo for AIDS patients. Improved anticryptosporidials are in development, but there is a concern that drug resistance will emerge if they are not used carefully. Stressing the risk of resistance, drug-resistant mutants with amino acid-coding target mutations emerged in under a week of drug treatment in a recent dairy calf study. The Cryptosporidium drug development field is in some ways reminiscent of that for the related malaria parasite in the 1940s when chloroquine became available and the inevitability of drug resistance and need for strategies to prevent it were poorly understood. Based on the hard lessons from malaria and antimicrobial resistance in general, combination therapy is now the standard for treating malaria and many other infectious diseases where the risk of resistance is high (e.g., AIDS, tuberculosis). This R21 grant is based on the premise that a basic understanding of anticryptosporidial resistance and its evolution is critical to maximize the benefits of newly developed drugs. The current goal is to develop the essential methods to study Cryptosporidium drug resistance that are needed to facilitate drug lead prioritization and, should combination therapy be needed, to identify optimal drug-drug combinations. The strategy exploits knowledge gleaned from the dairy calf experiment noted above regarding numbers of parasites and time required to select for Cryptosporidium resistance, and knowledge of resistance- conferring mutations for two positive control compounds. Aim 1 begins with optimizing a method to select drug- resistant parasites in immunocompromised mice by directly comparing results with wildtype C. parvum and a `mutator' C. parvum line, with or without backcrossing resistant strains against wildtype to identify relevant mutations. The optimized method will then be used to determine the relative genetic barrier to resistance for three promising anticryptosporidial lead series, and resistance-linked mutations will be studied using CRISPR. In aim 2, the effect of three known resistance-conferring mutations on parasite growth and in vivo reproductive fitness will be determined when they are introduced alone or in combinations of two, which will provide methods to formally measure fitness and identify epistasis between different resistance-conferring mutations. This application addresses a time-sensitive and critical issue: Along with key data for three promising anticryptosporidial lead chemical series, these studies will yield the methods needed to incorporate drug- resistance testing into early stages of anticryptosporidial development and will lay the groundwork for future development of a computer simulation model to guide animal work and begin selecting the most promising drug combinations.

项目概要： 隐孢子虫病是由隐孢子虫寄生虫引起的腹泻病，是最常见的疾病之一。 导致全球儿童危及生命的腹泻的重要原因，并导致艾滋病患者无法治愈的腹泻。 唯一获批的治疗方法对儿童有效率略高于 50%，相当于艾滋病患者的安慰剂。 改进的抗隐孢子虫药物正在开发中，但有人担心，如果它们被使用，就会出现耐药性。 不小心使用。强调具有氨基酸编码靶标的耐药性、耐药突变体的风险 在最近的一项奶牛研究中，在药物治疗不到一周的时间内出现了突变。隐孢子虫药物 发展领域在某些方面让人想起 20 世纪 40 年代相关疟疾寄生虫的发展领域。 随着氯喹的出现，耐药性不可避免，需要制定预防策略。 不太了解。根据疟疾和一般抗生素耐药性的惨痛教训，结合 疗法现已成为治疗疟疾和许多其他传染病的标准，这些疾病存在耐药性风险 高（例如艾滋病、结核病）。这项 R21 资助的前提是对 抗隐孢子虫耐药性及其进化对于最大限度地发挥新开发药物的益处至关重要。这 当前的目标是开发研究隐孢子虫耐药性所需的基本方法 促进药物先导优先排序，如果需要联合治疗，确定最佳药物 组合。该策略利用了从上述奶牛实验中收集到的知识 选择隐孢子虫抗性所需的寄生虫数量和时间以及抗性知识- 赋予两种阳性对照化合物突变。目标 1 首先优化选择药物的方法 通过直接将结果与野生型 C. parvum 和 a “突变体”C. parvum 系，有或没有针对野生型的回交抗性菌株以鉴定相关 突变。然后，优化的方法将用于确定耐药性的相对遗传障碍。 将使用 CRISPR 研究三个有前景的抗隐孢子虫先导系列和耐药性相关突变。 在目标 2 中，三种已知的抗性突变对寄生虫生长和体内繁殖的影响 当它们单独引入或两者组合引入时，将确定适合度，这将提供方法 正式测量适应性并识别不同的抗性突变之间的上位性。这 应用程序解决了一个时间敏感且关键的问题：以及三个有前途的关键数据 抗隐孢子虫先导化学系列，这些研究将产生结合药物所需的方法 将抗性测试纳入抗隐孢子虫开发的早期阶段，并将为未来奠定基础 开发计算机模拟模型来指导动物工作并开始选择最有前途的药物 组合。