Purine Pathways and Inhibitor Design in Plasmodium

描述（由申请人提供）：恶性疟原虫是导致疟疾死亡的主要原因，每年夺去100多万儿童的生命，使3亿至5亿人患上临床疾病。这种寄生虫已对大多数抗疟药产生耐药性，因此需要新的药物。恶性疟原虫是一种嘌呤缺乏症，需要从人红细胞中提取嘌呤来维持生存。利用前沿的过渡态分析技术，解决了恶性疟原虫嘌呤核苷磷酸化酶（PNP）和腺苷脱氨酶（ADA）的过渡态结构，并设计了过渡态类似物抑制剂来匹配它们的过渡态。这些抑制剂阻断各自的途径并杀死在人红细胞中培养的寄生虫，但不能治愈小鼠（该病的动物模型）的约氏疟原虫感染。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定位。针对两个关键靶点的抑制剂设计将通过解决恶性疟原虫次黄嘌呤-鸟嘌呤-黄嘌呤磷酸核糖基转移酶（HGXPRT）的过渡态结构来辅助，HGXPRT是嘌呤合成的最关键步骤。在疟疾从头合成嘧啶生物合成的基本途径中，羊角酸磷酸核糖基转移酶（OPRT）是形成所有嘧啶核苷酸的基本第一步。这些过渡态结构将通过前沿方法耦合动力学同位素效应和量子化学来求解。新一代抑制剂将根据这些过渡状态进行设计，并在人红细胞和小鼠约氏疟原虫感染模型中对培养的寄生虫进行试验。寄生虫中的嘌呤回收和合成途径及其抑制剂的中断将用具有特定放射性同位素标记的嘌呤前体进行研究。超灵敏的加速器质谱法（AMS）将用于跟踪正常的嘌呤回收途径，而不干扰培养细胞和小鼠感染中的正常池。AMS方法揭示了嘌呤回收的未表征途径，这些途径将在代谢，酶和抑制剂方法中定义。阻断嘌呤回收或嘧啶合成的抗疟药物可作为单一药物或与靶向其他途径的药物联合使用。同时阻断两个靶标降低了寄生虫突变逃逸的能力。疟疾是发展中国家热带地区由蚊子传播的寄生虫引起的一种传染病。每年大约有100万儿童死于这种疾病，由于寄生虫获得抗生素耐药性，目前的药物正在失去效力。这项研究通过发现在不伤害人类宿主的情况下杀死寄生虫的新方法和探索新药，提出了治疗疟疾的新方法。

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.