Purine Pathways and Inhibitor Design in Plasmodium

描述(申请人提供)：恶性疟原虫是疟疾的主要死亡原因，每年夺走100多万儿童的生命，并导致3亿至5亿人临床疾病。这种寄生虫已经对大多数抗疟疾药物产生了抗药性，需要新的药物。恶性疟原虫是一种嘌呤营养缺陷型生物，需要从人的红细胞中回收嘌呤才能生存。利用过渡态分析的前沿技术，解决了恶性疟原虫嘌呤核苷磷酸酶(PNP)和腺苷脱氨酶(ADA)的过渡态结构，并用于设计与其过渡态相匹配的过渡态类似物。这些抑制剂阻断了各自的途径，杀死了培养在人类红细胞中的寄生虫，但不能治愈约氏疟原虫在小鼠身上的感染，小鼠是这种疾病的动物模型。代谢物标记模式和小鼠研究已经确定，新的途径仍有待发现和靶向。解决恶性疟原虫次黄嘌呤-鸟嘌呤-黄嘌呤磷酸核糖转移酶(HGXPRT)的过渡态结构将有助于针对两个关键靶点的抑制剂设计。HGXPRT是嘌呤合成的最关键步骤。在疟疾新生的嘧啶生物合成的基本途径中，转位磷酸核糖转移酶(OPRT)是形成所有嘧啶核苷酸的重要第一步。这些过渡态结构将通过结合动力学同位素效应和量子化学的前沿方法来解决。新一代的抑制剂将在这些过渡态上形成模式，并在人类红细胞和约氏疟原虫感染的小鼠模型中测试对寄生虫的抑制作用。将用带有特定放射性同位素标记的嘌呤前体来研究寄生虫中的嘌呤挽救和合成途径以及它们与抑制剂的阻断。加速器质谱仪(AMS)的超灵敏方法将被用来跟踪正常的嘌呤回收途径，而不会扰乱培养细胞和小鼠感染的正常池。AMS方法揭示了嘌呤回收的未知途径，这些途径将在代谢、酶和抑制剂方法中定义。阻断嘌呤挽救或嘧啶合成的抗疟药可能是有用的治疗药物，作为单一药物或与针对其他途径的药物联合使用。同时阻断两个靶标会降低寄生虫的突变逃逸能力。与公共卫生相关疟疾是一种传染病，由蚊子在发展中国家热带地区传播的寄生虫引起。每年约有100万儿童死于这种疾病，由于寄生虫获得的抗生素耐药性，目前的药物正在失去效力。这项研究提出了治疗疟疾的新方法，方法是发现在不损害人类宿主的情况下杀死寄生虫的新方法，并探索新的药物。

DESCRIPTION (provided by applicant): Plasmodium falciparum is the leading cause of death from malaria, taking the lives of over a million children and causing clinical illness in 300 to 500 million people each year. The parasite has acquired resistance against most antimalarials and new drugs are required. P. falciparum is a purine auxotroph, requiring purine salvage from human erythrocytes for survival. Using the frontier technology of transition state analysis, the transition state structures of P. falciparum purine nucleoside phosphorylase (PNP) and adenosine deaminase (ADA) have been solved and used to design transition state analogue inhibitors to match a their transition states. These inhibitors block their respective pathways and kill parasites cultured in human erythrocytes, but do not cure infections of Plasmodium yoelii in mice, an animal model of the disease. Metabolite labeling patterns and mouse studies have established that new pathways remain to be discovered and targeted. Inhibitor design against two critical targets will be assisted by solving the transition state structures of P. falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT), the most critical step in purine synthesis. In the essential pathway of malarial de novo pyrimidine biosynthesis, orotate phosphoribosyltransferase (OPRT) is the essential first step to form all pyrimidine nucleotides. These transition state structures will be solved by frontier methods coupling kinetic isotope effects and quantum chemistry. A new generation of inhibitors will be patterned on these transition states and tested against parasites cultured in human erythrocytes and in the mouse model of P. yoelii infection. Purine salvage and synthetic pathways in parasites and their interruption with inhibitors will be investigated with purine precursors with specific radioisotope labels. The ultrasensitive method of accelerator mass spectrometry (AMS) will be used to follow normal pathways of purine salvage without perturbing normal pools in cultured cells and in mouse infections. The AMS approach has revealed uncharacterized pathways of purine salvage and these will be defined in metabolic, enzymatic and inhibitor approaches. Antimalarials that block purine salvage or pyrimidine synthesis may be useful therapeutics as single agents or in combination with agents targeted against other pathways. Simultaneous blocking of two targets decreases the ability of mutational escape by the parasite. PUBLIC HEALTH RELEVANCE Malaria is an infectious disease cause by parasites spread by mosquitoes in tropical regions of the developing world. Approximately one million children die each year from the disease and current drugs are losing their efficiency because of acquired antibiotic resistance by the parasites. This research proposes new ways to treat malaria by discovered new ways to kill the parasites without harming the human host and by exploring new drugs.