ASSESSING IN VITRO/VIVO EFFICACY OF ANTIMALARIAL ENR INHIBITORS

在热带地区，由恶性疟原虫感染引起的疟疾对人类健康造成了巨大的损失。目前的抗疟治疗正受到恶性疟原虫菌株的传播的严重影响，这些菌株对氯喹（干扰血红素解毒）和乙胺嘧啶-磺胺嘧啶（抑制叶酸生物合成）具有抗药性。几个研究小组最近将恶性疟原虫II型脂肪酸合成（FAS-II）（一种人类缺乏的顶质体途径）定义为独特的药物靶点。FAS-II酶烯酰ACP还原酶（ENR）是几种广泛使用的抗菌剂（包括三氯生、异烟肼和乙硫异烟胺）的靶标。我们的计划代表葛兰素史克，德克萨斯A&M大学和阿尔伯特爱因斯坦医学院之间的公共/私人合作伙伴关系，建议对纯化的恶性疟原虫ENR（PfENR）进行高通量筛选，并随后进行整合化学，生物化学，结构生物学，分子生物学和寄生虫学的Hits to Leads计划。在本项目的目标1中，我们将评估测试ENR抑制剂对耐药细胞的有效性。 和药物敏感的恶性疟原虫。目的2将评估有前途的 PfENR抑制剂，使用非转化伯氏疟原虫以及表达来自恶性疟原虫或间日疟原虫（第二重要的人类疟疾物种）的烯酰ACP还原酶的转基因伯氏疟原虫克隆代替啮齿动物疟疾酶（转基因伯氏疟原虫系将作为项目3的一部分产生）。目的3提出开发低表达PfENR或ENR活性功能互补的转基因“作用模式”恶性疟原虫系，以评估抑制剂特异性。目的4建立间日疟原虫enr基因替代pfenr基因的恶性疟原虫转基因株系，作为测定间日疟原虫体外抗疟活性的替代模型。该项目提供了关键的资源和试剂， 有效的口服生物可利用的抑制剂，可以沿着关键路径进入临床前开发，作为新的候选抗疟疾药物。

Malaria caused by infection with Plasmodium falciparum exerts an enormous toll on human health in tropical regions. Current antimalarial treatments are being severely compromised by the spread of P. falciparum strains resistant to chloroquine (that interferes with heme detoxification) and pyrimethamine-sulfadoxine (that inhibits folate biosynthesis). Several groups have recently defmed P. falciparum type II fatty acid synthesis (FAS-II), an apicoplast pathway that is lacking in humans, as a unique drug target. The FAS-II enzyme enoyl ACP reductase (ENR) is the target of several widely used antimicrobial agents including triclosan, isoniazid and ethionamide. Our Program, representing a public/private partnership between GlaxoSmithKline, Texas A&M University and the Albert Einstein College of Medicine, proposes to implement a high throughput screen against purified P. falciparum ENR (PfENR) and follow this with a Hits to Leads program that integrates chemistry, biochemistry, structural biology, molecular biology and parasitology. In Aim 1 of this project, we will evaluate the efficacy of test ENR inhibitors against drug-resistant and drug-sensitive P. falciparum in vitro. Aim 2 will assess the in vivo efficacy of promising PfENR inhibitors, using non-transformed P. berghei as well as transgenic P. berghei clones expressing the enoyl ACP reductase from P. falciparum or P. vivax (the second most important human malaria species) in the place of the rodent malarial enzyme (the transgenic P. berghei lines will be generated as part of Project 3). Aim 3 proposes to develop transgenic, "mode of action" P. falciparum lines that underexpress PfENR or are functionally complemented for ENR activity, to assess inhibitor specificity. Aim 4 will develop a transgenic P. falciparum line expressing the P. vivax enr in the place of pfenr, as a surrogate model to measure in vitro activity against P. vivax. This project provides key resources and reagents to identify potent, orally bioavailable inhibitors that can move down the critical path and enter preclinical development as new candidate antimalarial drugs.