Defining the targets of broad intervention antimalarial agents

描述（由申请人提供）：尽管表型细胞筛选最近已被用于驱动抗疟药物的发现，但仍然明显需要合理的靶向药物发现。当缺乏适当的高通量细胞测定时，尤其如此。这就是药物发现工作的情况，其目的是提供伯氨喹的替代品，伯氨喹是一种具有已知毒性的药物，是消除间日疟原虫肝脏阶段感染并阻断配子体传播的唯一药剂。目前，还没有已知的化学验证的寄生虫蛋白质的目标，是至关重要的复制或催眠虫肝脏阶段以及无性的血液和配子体阶段，并可用于大型化合物库的生化筛选。疟原虫寄生虫物种的基因组编码超过5，500种蛋白质，其中许多蛋白质尚未鉴定。我们的中心假设是，大量新颖的、经过化学验证的多阶段抗疟靶点仍有待发现。为了测试这一点，我们建议集中在五个最近发现的，化学上不同的支架，对寄生虫的生命周期，包括肝脏和无性血液阶段的广泛活动。我们将通过确定哪些化合物对来自多药耐药菌株、配子体和间日疟原虫肝型的恶性疟原虫无性血阶段有活性来进一步优先考虑化合物。为了产生低水平的抗性，我们将使恶性疟原虫寄生虫（三个独立的选择，针对每个支架系列的三个成员）暴露于亚致死浓度的化合物。耐药克隆的基因组将通过下一代测序与配对末端读取（产生约100 X覆盖率）进行检查，以确定化学选择期间每个克隆中出现的完整遗传变化。基于我们的初步数据，当考虑每个支架家族产生的所有9种耐药菌株的全基因组序列时，我们预计每个支架仅在一个基因中发现统计学显著的新出现突变富集。候选靶标的重要性将通过将遗传变化引入药物敏感的恶性疟原虫寄生虫并测试抗性的获得，或相反地去除候选突变并测试抗性的丧失来验证。转染实验将包括锌指核酸酶，这构成了它们在恶性疟原虫中介导高效基因编辑能力的重大突破。我们还将通过详细的分子表征来确定所鉴定的基因是小分子的靶基因还是参与抗性的基因。这项工作将为疟疾社区提供恶性疟原虫如何获得耐药性的系统图片，并有望产生几个新的经验证的目标，可用于药物开发工作，重点是寻找新的根治剂。这项研究，这是符合NIAID的使命声明，承诺利用化学支架活性抗疟原虫寄生虫，以确定新的目标，发展广泛的干预战略的基础上化学预防和治疗疟疾感染。

DESCRIPTION (provided by applicant): Although phenotypic cellular screening has recently been used to drive antimalarial drug discovery, there continues to be a clear need for rational target-based drug discovery. This is especially true when appropriate high-throughput cellular assays are lacking. Such is the case for drug discovery efforts that aim to provide a replacement for primaquine, a drug with known toxicity that is the only agent that eliminates Plasmodium vivax liver stage infection and blocks gametocyte transmission. At present, there are no known chemically validated parasite protein targets that are critical to replicating or hypnozoite liver stages as well as asexual blood and gametocyte stages, and that could be used in biochemical screens of large compound libraries. The genomes of Plasmodium parasite species encode over 5,500 proteins, many of which remain uncharacterized. Our central hypothesis is that a wealth of novel, chemically validated multi-stage antimalarial targets still remain to be discovered. To test this we propose to focus on five recently discovered, chemically distinct scaffolds with broad ranging activity against the parasite lifecycle, including liver and asexual blood stages. We will further prioritize compounds by determining which of these are active against P. falciparum asexual blood stages from multidrug-resistant strains, gametocytes, and P. vivax hepatic forms. To generate low-level resistance, we will expose P. falciparum parasites (three independent selections, against three members of each scaffold series) to sublethal concentrations of compound. The genomes of drug-resistant clones will be examined by next-generation sequencing with paired-end reads (yielding ~100X coverage) to identify the complete suite of genetic changes that have emerged in each clone during chemical selection. Based on our preliminary data, we expect to find a statistically significant enrichment of newly emerged mutations in only one gene for each scaffold, when considering the whole-genome sequence for all nine resistant strains created per scaffold family. The importance of the candidate targets wil be verified by introducing genetic changes into drug-sensitive P. falciparum parasites and testing for gain of resistance, or conversely removing candidate mutations and testing for loss of resistance. Transfection experiments will include zinc finger nucleases, which constitute a major breakthrough in their ability to mediate highly efficient gene editing in P. falciparum. We will alo seek to determine whether the identified gene is the target of the small molecule or a gene involved in resistance through a detailed molecular characterization. The work will provide the malaria community with a systematic picture of how P. falciparum acquires drug resistance, and is expected to yield several new validated targets that can be used in drug development efforts focused on finding new radical-cure agents. This research, which is consistent with NIAID's mission statement, promises to leverage chemical scaffolds active against Plasmodium parasites to define new targets for the development of broad intervention strategies based on chemoprophylaxis and curative treatment of malarial infections.