Selective Inhibitors of Plasmodium Falciparum G6PD as Novel Antimalarials

描述（由申请人提供）：热带疟疾，由寄生虫恶性疟原虫引起，每年造成近100万人死亡。由于寄生虫对大多数临床相关药物产生耐药性，因此迫切需要新型抗疟药物。葡萄糖-6-磷酸脱氢酶（G6PD）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。G6PD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G6PD缺乏导致还原当量的缺乏，氧化应激的增加，并且因此，对疟疾的保护。寄生虫感染的RBC中的NADPH由人G6PD产生，但也由具有G6PD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）。PfGluPho敲低和敲除导致寄生虫的生长停滞和死亡。因此，我们的总体目标是开发PfGluPho抑制剂来杀死寄生虫并治疗疟疾。我们 第一个克隆和表达重组PfGluPho，建立了高通量筛选试验，进行了药物化学，体外ADME和啮齿动物药代动力学研究，并确定了两个探针ML276和ML304，选择性抑制PfGluPho（IC 50 <1 μ M），但不抑制人G6 PD，这对于避免溶血毒性至关重要。这两种探针都能抑制氯喹敏感和抗性寄生虫的生长，IC 50在低μ M范围内，但在微粒体稳定性和啮齿动物药代动力学方面表现出局限性。基于这些有希望的和广泛的初步结果，我们现在的目标是继续我们成功的团队方法，以推动这两种探针开发新型抗疟药物。Specific Aim 1设计并合成了ML 276和ML 304的新型类似物。具体目标2确定了它们的体外效力和选择性，测试了它们的细胞内活性和毒性，并评价了它们的理化性质。具体目标3确定其啮齿动物药代动力学和潜在脱靶效应。具体目标4评估其在人类红细胞中的潜在溶血风险，并在疟疾小鼠模型中进行概念验证研究。特定目标2至4中的试验结果将反馈到化学设计和合成工艺中，以进一步优化特定目标1中所述的化合物。互补的技能组合、已建立的合作关系和无与伦比的资源预示着拟议目标的熟练执行，目标是产生一种有效、选择性和稳定的PfGluPho抑制剂，具有体外、细胞内和体内抗疟原虫活性。这种方法有可能产生 高影响力的结果是开发新的和迫切需要的抗疟疾药物，并帮助治疗和消除世界上最致命的疾病之一。

DESCRIPTION (provided by applicant): Tropical malaria, caused by the parasite Plasmodium falciparum, is responsible for nearly one million deaths each year. Since the parasite develops resistance against most clinically relevant 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 catalyzes the initial step in 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, 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). PfGluPho knockdown and knockout leads to growth arrest and death of the parasite. Therefore, our overall objective is to develop PfGluPho inhibitors to kill the parasite and treat malaria. We were the first to clone and express recombinant PfGluPho, established a high-throughput screening assay, conducted medicinal chemistry, in vitro ADME and rodent pharmacokinetic studies and identified two probes, ML276 and ML304, that selectively inhibit PfGluPho (IC50 <1 �M), but not human G6PD, which is critical for avoiding hemolytic toxicity. Both probes inhibit the growth of chloroquine-sensitive and -resistant parasites with IC50s in the low �M range, but show limitations in microsomal stability and rodent pharmacokinetics. Based on these promising and extensive preliminary results, we now aim to continue our successful team approach to advance the two probes towards the development of novel antimalarial drugs. Specific Aim 1 designs and synthesizes novel analogs of ML276 and ML304. Specific Aim 2 determines their in vitro potency and selectivity, tests their in cellulo activity and toxicity, and evaluates their physicochemical properties. Specific Aim 3 determines their rodent pharmacokinetics and potential off-target effects. Specific Aim 4 assesses their potential hemolytic risk in human RBCs and executes proof-of-concept studies in a malaria mouse model. Results from the assays in Specific Aims 2 to 4 will feedback into the chemical design and synthesis process for further compound optimization described in Specific Aim 1. Complimentary skillsets, established collaborations and unparalleled resources foreshadow a proficient execution of the proposed aims with the goal to generate a potent, selective and stable PfGluPho inhibitor with in vitro, in cellulo and in vivo activity against malaria parasites. This approach has the potential to generate high impact results towards developing novel and desperately needed antimalarial drugs and to help treat and eradicate one of the most deadly diseases in the world.