A Cell-based HTS for Delayed Death Inhibitors of the Malarial Parasite Plastid

描述（由申请人提供）：恶性疟原虫是一种顶复门寄生虫，是严重疟疾的病原体，每年导致5亿多人患病，100多万儿童死亡。现有的药物存在寄生虫抗药性，成本高或毒性大，临床前的管道少得可怜。最近的抗疟药物发现的努力集中在顶质体，一个重要的细胞器与蓝藻质体的遗传相关。这个细胞器是脂肪酸、类异戊二烯和血红素生物合成的场所，并且包含估计400种核编码蛋白质，许多功能未知。针对顶质体核糖体的抗生素（如阿奇霉素）的详细研究表明，顶质体抑制剂表现出独特的延迟死亡表型，只有药物处理的寄生虫的后代死亡。为什么顶质体对于恶性疟原虫是必不可少的，以及什么因素介导其关键的生物学功能是有趣的问题，可以用新的顶质体特异性探针进行实验解决。我们建议开发检测，确定抑制剂的顶质体发展，利用这种延迟死亡表型。我们的初步筛选将培养的恶性疟原虫暴露于化合物一代或两代（48或96小时）。使用dsDNA嵌入SYBR绿色染料测量寄生虫生长。首先在96小时试验中检测化合物，仅在48小时试验中复检活性化合物。仅在第二代中表现出增加的效力的化合物将被保留用于进一步筛选。这将包括重复测定、剂量-反应测定以鉴定性能良好的抑制剂，以及反筛选以针对一般细胞毒性抑制剂进行选择。第二次筛选将使用[3 H]-次黄嘌呤作为生长抑制的替代指标。另一个基于全细胞成像，将使用表达GFP标记的顶质体的转基因系来视觉识别顶质体发育的抑制剂。试验开发将使用参比化合物阿奇霉素、氯喹和异烟肼（分别代表延迟死亡、速效和非活性化合物）进行指导，并使用400种FDA批准的药物库进行HTS配置。后续研究将包括与哥伦比亚大学分子库筛选中心网络中心的Tecan HTS自动化平台的优化。除了它们的治疗潜力，顶质体抑制剂将是无价的，使我们能够直接解决有关关键顶质体过程和蛋白质的问题。我们建议开发一种高通量的检测方法，以筛选化学库的化合物，抑制发展的一个隔间称为尖质体恶性疟原虫疟疾寄生虫。顶质体的抑制的特征在于延迟死亡类型，由此化合物仅杀死受感染的红细胞内的药物处理的寄生虫的后代。我们打算确定新的抑制剂，可用于了解顶质体生物学和这种延迟死亡的原因，并有可能开发新的治疗方法。

DESCRIPTION (provided by applicant): Plasmodium falciparum, an Apicomplexan parasite and the causal agent of severe malaria, causes disease in over 500 million individuals and kills over one million children yearly. Existing drugs suffer from parasite resistance, high cost or toxicity, and the pre-clinical pipeline is woefully thin. Recent antimalarial drug discovery efforts have focused on the apicoplast, an essential organelle phylogenetically related to cyanobacterial plastids. This organelle is the site of fatty acid, isoprenoid and heme biosynthesis, and contains an estimated 400 nuclear-encoded proteins, many of unknown function. Detailed studies with antibiotics that target apicoplast ribosomes (such as azithromycin) reveal that apicoplast inhibitors manifest a unique delayed death phenotype, whereby only the progeny of drug-treated parasites die. Why the apicoplast is essential for P. falciparum and what factors mediate its critical biological functions are intriguing questions that can be experimentally addressed with novel apicoplast-specific probes. We propose to develop assays that identify inhibitors of apicoplast development by taking advantage of this delayed death phenotype. Our primary screen will expose cultured P. falciparum to compounds for one or two generations (48 or 96 hr). Parasite growth will be measured using the dsDNA-intercalating SYBR Green dye. Compounds will be tested first in 96 hr assays, and only active compounds will be retested in 48 hr assays. Compounds that manifest increased potency only in the second generation will be retained for further screening. This will include repeat assays, dose-response assays to identify well- behaved inhibitors, and counterscreens to select against generally cytotoxic inhibitors. One secondary screen will use [3H]-hypoxanthine as an alternative measure of growth inhibition. Another, based on whole cell imaging, will use a transgenic line expressing GFP-labeled apicoplasts to visually identify inhibitors of apicoplast development. Assay development will be guided using the reference compounds azithromycin, chloroquine and isoniazid (representing delayed death, fast-acting and inactive compounds respectively) and configured for HTS using a library of 400 FDA-approved drugs. Later studies will include optimization on the Tecan HTS automation platform at the Molecular Libraries Screening Center Network Center at Columbia University, in collaboration with members of this center. In addition to their therapeutic potential, apicoplast inhibitors will be invaluable in allowing us to directly address questions about key apicoplast processes and proteins. We propose to develop a high throughput assay to screen chemical libraries for compounds that inhibit the development of a compartment called the apicoplast in Plasmodium falciparum malaria parasites. Inhibition of the apicoplast is characterized by a type of delayed death whereby compounds only kill the progeny of drug-treated parasites inside infected red blood cells. We intend to identify new inhibitors that can be used to understand apicoplast biology and the reasons for this delayed death, and that have potential for the development of new therapeutics.