ASSESSING IN VITRO/VIVO EFFICACY OF ANTIMALARIAL ENR INHIBITORS

由恶性疟原虫感染引起的疟疾对热带地区的人类健康造成了巨大的损害。目前的抗疟疾治疗正受到抗氯喹(干扰血红素解毒)和乙胺嘧啶-磺胺多辛(抑制叶酸生物合成)的恶性疟原虫菌株的传播的严重影响。最近，几个小组已经将恶性疟原虫II型脂肪酸合成(Fas-II)作为一个独特的药物靶点进行了研究，Fas-II是人类缺乏的一种质外体途径。Fas-II酶Enoyl ACP还原酶(ENR)是几种广泛使用的抗菌剂的靶标，包括三氯生、异烟肼和乙硫酰胺。我们的计划代表了葛兰素史克、德克萨斯A&M大学和阿尔伯特·爱因斯坦医学院之间的公私合作伙伴关系，建议实施针对纯化的恶性疟原虫ENR(PfENR)的高通量筛查，并在此之后实施HITS到LEADS计划，该计划整合了化学、生物化学、结构生物学、分子生物学和寄生虫学。在本项目的目标1中，我们将评估测试的ENR抑制剂对耐药的疗效 以及体外对药物敏感的恶性疟原虫。目标2将评估Promising的体内疗效 PfENR抑制剂，使用未转化的伯氏疟原虫以及表达恶性疟原虫或间日疟原虫(人类第二大疟疾物种)的Enoyl ACP还原酶的转基因伯氏疟原虫克隆，以取代啮齿动物疟疾酶(转基因伯氏疟原虫株将作为项目3的一部分产生)。目的3建议建立PfENR低表达或ENR活性功能互补的恶性疟原虫转基因“作用模式”品系，以评估抑制物的特异性。目的建立表达间日疟原虫enR基因的恶性疟原虫转基因株系，作为检测间日疟原虫体外抗恶性疟原虫活性的替代模型。该项目提供关键资源和试剂以确定 有效的、口服生物可用的抑制剂，可以沿着关键的道路前进，作为新的候选抗疟疾药物进入临床前开发。

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.