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Defining the targets of broad intervention antimalarial agents

确定广泛干预抗疟药物的目标

基本信息

批准号：
8581051
负责人：
Elizabeth A Winzeler
金额：
$ 64.81万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-08-14 至 2017-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8581051
关键词：
Address Anopheles Genus Antimalarials Artemisinins Binding Sites Biochemical Bioinformatics Biological Biological Assay Biology Blood Body Weight Cell Death Cellular Assay Chemical Evolution Chemicals Chemoprophylaxis Combined Modality Therapy Communities Data Development Drug resistance Drug-sensitive Evolution Failure Family Gene Exchanges Genes Genetic Genetic Engineering Genome Genomics Hepatic Human In Vitro Infection Inhibitory Concentration 50 Intervention Knock-out Libraries Liver Malaria Mediating Medicine Methods Mission Mitochondria Molecular Multi-Drug Resistance Mutation Nucleic Acids Parasites Pattern Pharmaceutical Preparations Physiology Plasmodium Plasmodium falciparum Plasmodium vivax Primaquine Probability Property Protein Biosynthesis Proteins Reading Recombinants Research Research Proposals Resistance Risk Role Series Single Nucleotide Polymorphism Speed Staging Testing Toxic effect Transfection Validation Variant Work Zinc Fingers antimicrobial drug artemisinine asexual base drug candidate drug development drug discovery genome sequencing inhibitor/antagonist innovation member mutant next generation next generation sequencing novel nuclease parasite genome pathogen pressure prevent public health relevance public-private partnership research study resistance mechanism resistant strain scaffold screening small molecule transmission process vector mosquito

项目摘要

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.

描述（由申请人提供）：尽管表型细胞筛选最近已被用于推动抗疟药物的发现，但仍然明显需要基于合理靶标的药物发现。当缺乏适当的高通量细胞检测时尤其如此。旨在提供伯氨喹替代品的药物发现工作就是这种情况，伯氨喹是一种具有已知毒性的药物，是消除间日疟原虫肝期感染并阻止配子体传播的唯一药物。目前，还没有已知的化学验证的寄生虫蛋白靶标对复制或催眠肝脏阶段以及无性血液和配子体阶段至关重要，并且可以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，其中许多蛋白质仍未被表征。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，我们建议重点关注最近发现的五种化学性质不同的支架，它们对寄生虫生命周期具有广泛的活性，包括肝脏和无性血液阶段。我们将通过确定哪些化合物对来自多重耐药菌株、配子体和间日疟原虫肝型的恶性疟原虫无性血液阶段具有活性，进一步确定化合物的优先顺序。为了产生低水平的耐药性，我们将恶性疟原虫寄生虫（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中。耐药克隆的基因组将通过新一代测序与双端读取（产生~100X覆盖率）进行检查，以识别化学选择过程中每个克隆中出现的完整遗传变化。根据我们的初步数据，当考虑每个支架家族创建的所有九种抗性菌株的全基因组序列时，我们预计每个支架的一个基因中新出现的突变会出现统计上显着的富集。候选靶标的重要性将通过将基因变化引入药物敏感的恶性疟原虫寄生虫中并测试耐药性的获得，或相反地去除候选突变并测试耐药性的丧失来验证。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寻求确定所识别的基因是小分子的靶标还是与耐药性有关的基因。这项工作将为疟疾界提供关于恶性疟原虫如何获得耐药性的系统图景，并有望产生几个新的经过验证的靶标，这些靶标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，承诺利用对疟原虫寄生虫具有活性的化学支架来确定新目标，以制定基于化学预防和疟疾感染治疗的广泛干预策略。