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属寄生虫引起的一种妇科疾病， 这是全球危及儿童生命的腹泻的重要原因，也是艾滋病患者无法治愈的腹泻的原因。 唯一批准的治疗方法对儿童的有效性略高于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，但人们担心，如果它们被使用， 没有被小心使用。强调耐药风险，耐药突变体与氨基酸编码靶点 在最近的一项奶牛研究中，在药物治疗不到一周的时间里，突变就出现了。隐孢子虫药物 发展领域在某种程度上让人想起20世纪40年代相关的疟疾寄生虫， 随着氯喹的出现，抗药性的不可避免性和预防抗药性的策略的必要性被消除。 不太了解。基于疟疾和抗生素耐药性的惨痛教训， 治疗现在是治疗疟疾和许多其他传染病的标准， 是高的（例如，艾滋病、结核病）。这个R21补助金是基于一个前提，即基本了解 抗隐孢子虫抗性及其演变对于最大化新开发药物的益处至关重要。的 目前的目标是开发研究隐孢子虫抗药性的基本方法， 促进药物优先级排序，如果需要联合治疗， 组合。该策略利用了从上述奶牛实验中收集的知识， 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从优化药物筛选方法开始- 通过直接与野生型C. parvum和a 'mutator' C. parvum系，有或没有针对野生型的回交抗性菌株，以鉴定相关的 突变。然后，将使用优化的方法来确定对以下抗性的相对遗传屏障： 将使用CRISPR研究三个有前途的抗隐孢子虫先导系列和抗性连锁突变。 在目的2中，研究了三种已知的赋予抗性的突变对寄生虫生长和体内生殖的影响。 当它们单独引入或两种组合引入时，将确定适合性，这将提供方法 来正式测量适应性并识别不同抗性赋予突变之间的上位性。这 应用程序解决了一个时间敏感和关键问题：沿着三个有希望的关键数据 抗隐孢子虫铅化学系列，这些研究将产生所需的方法，将药物， 抗隐孢子虫发展的早期阶段的抗性测试，并将奠定基础，为未来 开发一个计算机模拟模型来指导动物实验，并开始选择最有前途的药物 组合。