PROTOZOAN PURINE PHOSPHORIBOSYLTRANSFERASES AS TARGETS TO TREAT MALARIA, AFRICAN TRYPANOSOMIASIS AND CHAGAS'S DISEASE

项目摘要/摘要 寄生原生动物依赖嘌呤抢救途径，次黄嘌呤-鸟嘌呤-黄嘌呤 磷酸核糖基转移酶(HGXPRT，以下简称嘌呤磷酸核糖基转移酶，PPRT)是必需的。这个 锥虫属导致人类疾病发病率高，使人衰弱。疫苗既没有用，也没有用 治疗方法是存在的。克氏锥虫在中南美洲引起恰加斯病，超过40人 德克萨斯州最近报告了病例。罗多西布氏锥虫和冈比亚布氏锥虫 导致非洲昏睡病。包括克鲁兹毛滴虫和布鲁氏毛滴虫在内的寄生原虫不能产生嘌呤。 通过嘌呤回收途径重新合成核苷酸和核苷。PPRT催化了 由5-磷酸核糖-1-焦磷酸(PRPP)和各自的碱基形成GMP、IMP和XMP， 鸟嘌呤、次黄嘌呤和黄嘌呤。恶性疟原虫，是最致命的疟疾形式，是 也是一种嘌呤营养缺乏症，其PPRT的作用是次黄嘌呤的唯一生理途径 整合到核苷酸池中。基于过渡态的过渡态类似物(TSAI) 因为相关的磷酸核糖转移酶是恶性疟原虫PPRT的抑制因子。细胞通透性前药受阻 恶性疟原虫在培养中的增殖和对人HGPRT的选择性，尽管有很高的 酶的结构相似性和活性部位的保守性。 这项研究将在体外和体内设计、合成和表征新型的PPRTS抑制剂 恶性疟原虫T.brucei SSP.和克氏锥虫。过渡态方法论，量子计算 缓蚀剂将采用化学、基于X射线结构的缓蚀剂设计和专家化学合成 发展。现有的对恶性疟原虫PPRT具有亚纳摩尔效力的先导化合物将启动和 通知我们的抑制程序。已经报道了克鲁兹毛滴虫PPRT的晶体结构，但没有有效的抑制剂 已经被定义了。目前还没有关于布鲁氏毛滴虫的抑制剂开发的报道，PPRT也没有被报道 从布氏毛滴虫的运动学特征。新的先导化合物的产生预计将继续进行 恶性疟原虫PPRT和锥虫PPRT一旦过渡状态结构 这些酶已经被解决了。将评估与每个靶PPRT结合的优化的抑制剂 通过X射线结晶学分析，为化学提纯提供指导。强大且有选择性(与人类 PPRT的抑制剂将在恶性疟原虫、克鲁兹疟原虫和布氏疟原虫的细胞培养中进行评估。 用于杀寄生虫的活动。其中最有效的将在疟疾的小鼠模型中进行评估，Chagas的 疾病和非洲昏睡病。重要的是，这项提案的成功结果可能会带来新的 治疗三种疾病的治疗剂，这些疾病包括未得到满足或未得到满足的医疗需求。这 开发计划使用过渡态理论来实现NIH缩短药物提前期的目标 发展。

PROJECT SUMMARY/ABSTRACT Parasitic protozoa rely on purine salvage pathways for which the enzyme hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT; hereafter, purine phosphoribosyltransferase (PPRT)) is essential. The genus Trypanosoma causes debilitating human diseases of high morbidity. Neither vaccines nor useful therapies exist. Trypanosoma cruzi causes Chagas’s Disease in Central and South America, with over 40 cases recently reported in Texas. Trypanosoma brucei rhodosiense and Trypanosoma brucei gambiense cause African sleeping sickness. Parasitic protozoa including T. cruzi and T. brucei are incapable of purine biosynthesis de novo and make nucleotides and nucleosides by purine salvage pathways. PPRT catalyzes the formation of GMP, IMP, and XMP from 5-phospho-ribose 1-pyrophosphate (PRPP) and the respective bases, guanine, hypoxanthine, and xanthine. Plasmodium falciparum, causes the most virulent form of malaria, and is also a purine auxotroph for which the action of PPRT is the only physiological path for hypoxanthine incorporation into the nucleotide pool. Transition-state analogue inhibitors (TSAIs) based on transition states for related phosphoribosyltransferases are inhibitors of P. falciparum PPRT. Cell-permeable prodrugs blocked the proliferation of P. falciparum in culture and showed selectivity vs. human HGPRT, despite the high structural similarity and active-site conservation of the enzymes. This research will design, synthesize, and characterize both in vitro and in vivo novel inhibitors of the PPRTs from P. falciparum, T. brucei ssp. and T. cruzi. Transition-state methodology, quantum computational chemistry, X-ray structure-based inhibitor design and expert chemical synthesis will be used for inhibitor development. Existing lead compounds of sub-nanomolar potency for PPRT from P. falciparum will initiate and inform our inhibitor program. Crystal structures have been reported for T. cruzi PPRT, but no potent inhibitors have been defined. No inhibitor development for T. brucei has been reported, and PPRTs have not been kinetically characterized from T. brucei. The generation of new lead compounds is anticipated to proceed rapidly for P. falciparum PPRT and emerge for Trypanosoma PPRTs once the transition-state structures of these enzymes have been solved. Optimized inhibitors which bind each of the target PPRTs will be evaluated by X-ray crystallography, to provide a refinement guide for chemistry. Potent and selective (vs. human HGPRT) inhibitors of the PPRTs will be evaluated in cell cultures of P. falciparum, T. cruzi, and T. brucei ssp for parasiticidal activity. The most effective of these will be evaluated in murine models of Malaria, Chagas’s disease and African sleeping sickness. Importantly, a successful outcome from this proposal could lead to new therapeutic agents to treat three diseases which comprise unmet or under-met medical needs. This development plan uses transition state theory to meet the NIH goals of reducing the lead time for drug development.