Optimizing IMPDH inhibitors for the treatment of cryptosporidiosis

描述(申请人提供)：隐孢子虫种类，包括微小隐孢子虫和人隐孢子虫，是原生动物寄生虫，尤其对免疫功能低下的个人和幼儿构成健康威胁。这些生物也是潜在的生物恐怖分子，可能会使大量人口丧失能力。目前的治疗选择有限。常用的抗寄生虫药物无效，FDA唯一批准的药物疗效不佳，疫苗也不可用。基因组分析表明，隐孢子虫依赖于肌苷5‘-单磷酸脱氢酶(IMPDH)来产生鸟嘌呤核苷酸，从而生存。这种寄生虫的impdh基因似乎是通过侧向转移的方式从ε变形杆菌中获得的，因此微小隐孢子虫的impdh基因与人类同源基因有很大的差异。因此，选择性CpIMPDH抑制剂可能为治疗隐孢子虫病提供一种有效的策略，且对患者的毒性最小。到目前为止，已经确定了几类结构不同的CpIMPDH抑制剂，它们在细胞培养模型中显示出优异的酶活性、对人IMPDH的选择性、抗寄生虫活性，对于一个化合物系列，在急性隐孢子虫病小鼠模型中显示出体内疗效。这项拟议研究的总体目标是进一步提炼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，以支持选择性抑制CpIMPDH是这种原虫感染的可行治疗策略的假设，为研究界提供关于体内疗效的最佳化合物性质的指导，并提供CpIMPDH作为分子靶标的进一步临床前验证。这些目标将通过追求三个特定目标来实现：1)设计、合成和体外评价CpIMPDH抑制剂，包括CpIMPDH抑制效力和选择性的检测、微小隐孢子虫细胞培养感染模型和ADME特性(包括小鼠和人肠道和肝脏微生物体的稳定性、细胞摄取、外排和毒性以及UGT介导的葡萄糖醛酸化作用)；2)评估CpIMPDH抑制剂在体内的药代动力学和急性毒性特性，包括在未感染和微小隐孢子虫感染的小鼠体内的口服吸收和组织分布模型；3)在急性和慢性隐孢子虫病动物模型中评价优化的CpIMPDH抑制剂，评估粪便排泄、胃肠道和大体组织病理学。此外，还将研究CpIMPDH抑制剂对共生细菌种群的影响，以更全面地评估药效学效应，因为许多细菌具有类似于CpIMPDH的IMPDH。该项目还将制定将药物分配限制在胃肠道的策略。由于这一策略可能适用于其他胃肠道疾病， 例如肠易激疾病和结肠癌，这项工作具有广泛的意义，超越了隐孢子虫病的治疗。

DESCRIPTION (provided by applicant): Cryptosporidium species, including C. parvum and C. hominis, are protozoan parasites that present a health threat particularly to immunocompromised individuals and young children. These organisms are also potential bio-terrorism agents that could incapacitate large populations. Current treatment options are limited. Commonly used antiparasitic drugs are ineffective, the only FDA-approved drug is poorly efficacious, and vaccines are unavailable. Genomic analysis has revealed that Cryptosporidium species rely on inosine 5'- monophosphate dehydrogenase (IMPDH) for producing guanine nucleotides and, hence, survival. It also appears that the parasite obtained its IMPDH gene from an ε-proteobacterium by lateral transfer, so C. parvum IMPDH (CpIMPDH) is highly diverged from the human orthologs. Therefore, selective CpIMPDH inhibitors may provide an effective strategy for the treatment of cryptosporidiosis with minimum toxicity to the patient. To date, several structurally distinct classes of CpIMPDH inhibitors have been identified 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 proposed 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 this protozoan infection, provide guidance for the research community with respect to optimal compound properties for in vivo efficacy and provide further pre-clinical validation of CpIMPDH as a molecular target. These goals will be achieved by pursuing three specific aims: 1) design, synthesis 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 mouse and human intestinal and liver microsome stability, cellular uptake, efflux and toxicity, and UGT-mediated glucuronidation); 2) assess the in vivo pharmacokinetic and acute toxicity properties of CpIMPDH inhibitors, including in vivo models of oral absorption and tissue distribution in both non-infected and C. parvum infected mice; 3) evaluate optimized CpIMPDH inhibitors in acute and chronic animal models of cryptosporidiosis assessing fecal oocyst excretion and gastrointestinal gross and histopathology. In addition, the impact of CpIMPDH inhibitors on commensal bacteria populations will also be examined to more fully evaluate pharmacodynamics effects, since many bacteria have IMPDHs that are similar to CpIMPDH. This project will also develop strategies for limiting drug distribution to the gastrointestinal tract. Since this strategy could be applicable to other gastrointestinal diseases, such as irritable bowel disease and colon cancer, this work has broad implications beyond the treatment of cryptosporidiosis.