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被用于推动抗疟药物的发现，但仍然明显需要合理的基于靶标的药物发现。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，旨在提供一种替代伯氨喹的药物，这种药物具有已知的毒性，是唯一一种消除间日疟原虫肝期感染并阻止配子体传播的药物。目前，还没有已知的经过化学验证的寄生虫蛋白靶点，这些靶点对复制或诱导子体肝脏阶段以及无性血液和配子细胞阶段至关重要，并且可以用于大型化合物文库的生化筛选。疟原虫的基因组编码超过5500种蛋白质，其中许多仍未被表征。我们的中心假设是，大量新的、经过化学验证的多阶段抗疟靶点仍有待发现。为了验证这一点，我们建议将重点放在最近发现的五种化学上不同的支架上，这些支架具有广泛的抗寄生虫生命周期活性，包括肝脏和无性血液阶段。我们将进一步确定化合物的优先级，确定哪些化合物对恶性疟原虫无性血期（来自多药耐药菌株、配子体细胞和间日疟原虫肝型）有活性。为了产生低水平耐药性，我们将恶性疟原虫（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中。耐药克隆的基因组将通过下一代测序与成对末端读取（产生约100倍的覆盖率）来检查，以确定在化学选择过程中每个克隆中出现的完整的遗传变化。根据我们的初步数据，当考虑到每个支架家族产生的所有9个耐药菌株的全基因组序列时，我们期望在每个支架中发现仅在一个基因中新出现的突变的统计学显著富集。候选靶点的重要性将通过将基因改变引入对药物敏感的恶性疟原虫并检测获得耐药性，或相反地去除候选突变并检测失去耐药性来验证。转染实验将包括锌指核酸酶，这是其介导恶性疟原虫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。这项工作将为疟疾界提供恶性疟原虫如何获得耐药性的系统图像，并有望产生几个新的经过验证的靶点，这些靶点可用于专注于寻找新的根治药物的药物开发工作。这项研究与NIAID的使命宣言一致，有望利用对疟原虫具有活性的化学支架，为基于化学预防和根治性治疗疟疾感染的广泛干预策略的发展确定新的目标。

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.