IMPDH inhibitors for the treatment of Cryptosporidium infections

项目摘要/摘要 隐孢子虫种类，如微小隐孢子虫，是原生动物寄生虫，对人类健康构成重大威胁。 幼儿和免疫功能受损的成年人。这些生物是潜在的生物恐怖分子，可能 也会使大量人口丧失工作能力。目前隐孢子虫感染的治疗选择有限。 常用的抗寄生虫药物无效，FDA唯一批准的药物疗效不佳，以及 目前还没有疫苗。隐孢子虫依赖肌苷5‘-单磷酸脱氢酶 (IMPDH)，如基因组分析所揭示的，产生鸟嘌呤核苷酸，因此，生存。这个 寄生虫的impdh基因可能是从ε变形杆菌中获得的，所以微小隐孢子虫的impdh基因(CpIMPDH)具有很高的致病性。 与人类同源物背道而驰。因此，选择性CpIMPDH抑制剂可能会提供一种有效的 对患者毒性最小的隐孢子虫病的治疗策略。到目前为止，我们已经确定 几类结构不同的CpIMPDH抑制剂，已显示出良好的酶促作用 细胞培养模型中的效力、对人IMPDH的选择性、抗寄生虫活性以及一种化合物 系列，在急性隐孢子虫病小鼠模型中的体内疗效。这项研究的总体目标是进一步 改进CpIMPDH抑制剂以实现对急性和慢性小鼠隐孢子虫病模型的疗效 支持选择性抑制CpIMPDH是治疗微小隐孢子虫的可行策略的假设 感染，为研究界提供关于最佳化合物性能的指导 体内疗效和临床前候选药物的选择有待进一步开发。这些目标将通过以下方式实现 追求三个具体目标：1)CpIMPDH抑制剂在CpIMPDH检测中的设计和体外评价 抑制力和选择性、微小弧菌细胞培养感染模型和ADME特性(包括 溶解性、肠微粒体稳定性、细胞摄取、外排、毒性和UGT介导的葡萄糖醛酸化作用)； 评估CpIMPDH抑制剂的体内药代动力学和急性毒性特性，包括体内 正常小鼠和微小弧菌感染小鼠的吸收和组织分布模型；3)评价优化 CpIMPDH抑制剂在急、慢性隐孢子虫病动物模型中的作用 荧光寄生虫，粪便卵囊排泄和胃肠道大体和组织病理学。此外， 将检查CpIMPDH抑制剂对共生细菌种群的影响，以更全面地评估 药效学。该项目还将制定一项肠肝循环战略，以最大限度地 胃肠道浓度，同时减少全身暴露和毒性风险。 1

PROJECT SUMMARY/ABSTRACT Cryptosporidium species, such as C. parvum, are protozoan parasites that present a significant health threat to young children and immunocompromised adults. These organisms are potential bio-terrorism agents that could incapacitate large populations, as well. Current treatment options for Cryptosporidium infections are limited. Commonly used anti-parasitic drugs are ineffective, the only FDA-approved drug is poorly efficacious, and vaccines are unavailable. Cryptosporidium species rely on inosine 5’-monophosphate dehydrogenase (IMPDH), as revealed by genomic analysis, for producing guanine nucleotides and, hence, survival. The parasite likely obtained its IMPDH gene from ε-proteobacterium, so C. parvum IMPDH (CpIMPDH) is highly diverged from the human orthologs. Consequently, selective CpIMPDH inhibitors may provide an effective strategy for the treatment of cryptosporidiosis with minimum toxicity to patients. To date, we have identified several structurally distinct classes of CpIMPDH inhibitors that have demonstrated excellent enzymatic potency, selectivity over human IMPDH, anti-parasitic activity in a cell culture model and, for one compound series, in vivo efficacy in an acute cryptosporidiosis mouse model. The overall goals of this study are to further refine CpIMPDH inhibitors to achieve efficacy in both acute and chronic mouse models of cryptosporidiosis to support the hypothesis that selective CpIMPDH inhibition is a viable treatment strategy for C. parvum infections, provide guidance for the research community with respect to optimal compound properties for in vivo efficacy and selection of pre-clinical candidates for further development. These goals will be achieved by pursuing three specific aims: 1) design and in vitro evaluation of CpIMPDH inhibitors in assays of CpIMPDH inhibitory potency and selectivity, a C. parvum cell culture infection model and ADME properties (including solubility, intestinal microsomal stability, cellular uptake, efflux, toxicity, and UGT-mediated glucuronidation); 2) assess the in vivo pharmacokinetic and acute toxicity properties of CpIMPDH inhibitors, including in vivo models of absorption and tissue distribution in both normal and C. parvum infected mice; 3) evaluate optimized CpIMPDH inhibitors in acute and chronic animal models of cryptosporidiosis assessing efficacy via imaging of fluorescent parasites, fecal oocyst excretion and gastrointestinal gross and histopathology. In addition, the impact of CpIMPDH inhibitors on commensal bacteria populations will be examined to more fully evaluate pharmacodynamics. This project will also develop a strategy for enterohepatic recycling in order to maximize gastrointestinal concentrations, while reducing systemic exposure and toxicity risk. 1