Identification of G6PD inhibitors for the development of novel antimalarial drugs

描述（由申请人提供）：热带疟疾，由寄生虫恶性疟原虫引起，每年造成多达300万人死亡。由于寄生虫对大多数临床可用药物产生耐药性，因此迫切需要新型抗疟药物。葡萄糖-6-磷酸脱氢酶（G6 PD）是抗疟疾药物设计的一个新靶点，该靶点是基于观察到这种酶遗传缺陷的人可以免受疟疾的影响。G6 PD催化戊糖磷酸途径的初始步骤，产生NADPH，这是一种对红细胞（RBC）中的氧化应激解毒的重要还原等价物。疟原虫在红细胞阶段易受氧化应激的影响。天然存在的G6 PD缺乏导致还原当量的缺乏、氧化应激的增加、寄生虫感染的RBC的吞噬作用的增强，并且因此导致针对疟疾的保护。寄生虫感染的RBC中的NADPH由人G6 PD产生，但也由具有G6 PD活性的寄生虫酶产生，称为恶性疟原虫葡萄糖-6-磷酸脱氢酶-6-磷酸葡萄糖内酯酶（PfGluPho）。我们推测，抑制PfGluPho和在一定程度上抑制人G6 PD可以降低患疟疾的风险。到目前为止，探索G6 PD和PfGluPho作为抗疟疾药物靶标受到缺乏重组PfGluPho的限制。最近，我们的德国团队生产了第一个完整且功能性的重组PfGluPho。我们现在的目标是使用这种重组的纯蛋白来鉴定抑制剂，这些抑制剂可能是新型和创新抗疟药物的潜在候选者。在AIM 1中，我们将以mg的量生产人G6 PD和PfGluPho。然后，AIM 2在已经建立的高通量筛选试验中使用这些蛋白质来鉴定酶抑制剂。在低微摩尔至纳摩尔浓度范围内具有活性的化合物将对分离的酶进行详细的动力学分析。AIM 3跟踪所鉴定的抑制剂并评估它们是否影响恶性疟原虫生长和寄生虫血症而在哺乳动物细胞中没有细胞毒性。由于G6 PD缺陷是针对疟疾寄生虫的经证实的原理，并且由于寄生虫酶在结构上和机制上不同于人类宿主酶，PfGluPho是极好的药物靶标。我们的目标是确定2-3个先导化合物，这些化合物在纳摩尔范围内具有活性，没有显著的细胞毒性，可用于进一步的药物开发。这可能是一种高风险的方法，因为不能保证高通量筛选和后续测定能够识别命中。然而，我们已经鉴定了164种抑制细菌G6 PD的化合物，我们预计PfGluPho和人类G6 PD的命中率相似。这种方法有可能产生高影响力的结果。疟疾是全世界最致命的疾病。疟疾寄生虫对大多数现有药物产生了抗药性。因此，迫切需要新型和创新的抗疟药物。公共卫生相关性：热带疟疾每年造成多达300万人死亡。疟疾寄生虫恶性疟原虫对大多数临床可用药物产生抗药性。迫切需要新的抗疟疾药物。葡萄糖-6-磷酸脱氢酶是抗疟疾药物设计的一个新靶点，其基础是观察到这种酶缺乏的人可以免受疟疾的侵害。我们的目标是确定化合物，抑制这种酶在疟疾寄生虫，并在一定程度上，在人类。我们的研究结果可能为开发新的抗疟药物铺平道路。

DESCRIPTION (provided by applicant): Tropical malaria, caused by the parasite Plasmodium falciparum, is responsible for up to three million deaths each year. Since the parasite develops resistance against most clinically available drugs, novel antimalarial drugs are urgently needed. Glucose-6-phosphate dehydrogenase (G6PD) is a novel target for antimalarial drug design based on observations that humans with a genetic deficiency in this enzyme are protected against malaria. G6PD catalyses the initial step of the pentose phosphate pathway, yielding NADPH, an essential reducing equivalent to detoxify oxidative stress in red blood cells (RBCs). The malaria parasite is susceptible to oxidative stress in the RBC stage. Naturally occurring G6PD deficiency leads to a lack of reducing equivalents, an increase in oxidative stress, enhanced phagocytosis of parasite-infected RBCs, and, as a consequence, to a protection against malaria. NADPH in parasite-infected RBCs is generated by human G6PD but also by a parasite enzyme with G6PD activity, called P. falciparum glucose-6-phosphate-dehydrogenase-6- phosphogluconolactonase (PfGluPho). We hypothesize that inhibiting PfGluPho and, to a certain extent, human G6PD reduces the risk of developing malaria. So far, exploring G6PD and PfGluPho as antimalarial drug targets was limited by a lack of recombinant PfGluPho. Very recently our German team produced the first complete and functional recombinant PfGluPho. We now aim at using this recombinant, pure protein to identify inhibitors which could be potential candidates for novel and innovative antimalarial drugs. In AIM 1 we will produce human G6PD and PfGluPho in mg quantities. AIM 2 then uses these proteins in already established high-throughput screening assays to identify enzyme inhibitors. Compounds active in the low micromolar to nanomolar concentration range will be subject to detailed kinetic analyses on isolated enzymes. AIM 3 follows up on the identified inhibitors and assesses whether they impact P. falciparum growth and parasitemia without being cytotoxic in mammalian cells. Since G6PD deficiency is a proven principle against malarial parasites and since the parasite enzyme differs structurally and mechanistically from the human host enzyme, PfGluPho is an excellent drug target. We aim at identifying 2-3 lead compounds which are active in the nanomolar range without significant cytotoxicity, which can be used for further drug development. This may be a high risk approach since it is not guaranteed that high-throughput screening and follow-up assays identify a hit. However, we have already identified 164 compounds that inhibit bacterial G6PD, and we anticipate a similar hit rate for PfGluPho and human G6PD. This approach has the potential to generate high impact results. Malaria is the most deadly disease worldwide. The malaria parasite develops resistance to most of the currently available drugs. Thus, novel and innovative antimalarial drugs are desperately needed. PUBLIC HEALTH RELEVANCE: Tropical malaria is responsible for up to three million deaths annually. The malaria parasite Plasmodium falciparum develops resistances against most clinically available drugs. Novel antimalarial drugs are urgently needed. Glucose-6-phosphate dehydrogenase is a novel target for antimalarial drug design based on observations that humans with a deficiency in this enzyme are protected from malaria. We aim at identifying compounds that inhibit this enzyme both in malaria parasites and, to a certain extent, in humans. Our results may pave the way for the development of novel antimalarial drugs.