Development of a cell-based method for high-throughput drug screening in malaria

开发基于细胞的疟疾高通量药物筛选方法

• 批准号: 7555778
• 负责人: LIWANG CUI
• 金额: $ 16.6万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-15 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7555778
• 关键词: Antimalarials; Artemisinins; Automation; Back; Biological; Biological Assay; Cells; Cessation of life; Chloroquine; Combined Modality Therapy; Condition; Detection; Developed Countries; Developing Countries; Development; Dimethyl Sulfoxide; Disasters; Dose; Drug resistance; Erythrocytes; Ethanolamines; Falciparum Malaria; Firefly Luciferases; Future; Growth; Hematocrit procedure; Hypoxanthine; Hypoxanthines; In Vitro; Libraries; Luciferases; Malaria; Measures; Methodology; Methods; Molecular; Multi-Drug Resistance; Noise; Numbers; One-Step dentin bonding system; Parasite resistance; Parasitemia; Parasites; Pharmaceutical Preparations; Phase; Plasmodium falciparum; Preclinical Drug Evaluation; Public Health; Purpose; Pyrimethamine-Sulfadoxine; Radiolabeled; Range; Reporter; Resistance development; Resources; Screening procedure; Signal Transduction; Snow; Southeastern Asia; Staging; Standards of Weights and Measures; System; Techniques; Testing; Transgenic Organisms; Validation; Wood material; abstracting; artemisinine; asexual; assay development; base; drug discovery; drug testing; ethanolamine; genetically modified cells; high throughput screening; improved; luminescence; novel; radiotracer; research study; response; small molecule libraries; success; tool; uptake

DESCRIPTION (provided by applicant): Abstract The global burden of malaria and the widespread drug resistance in Plasmodium falciparum demand novel and improved therapy. The availability of large libraries of small molecular compounds provides a rich resource for the discovery of antimalarial drug candidates. Screening of these structurally diverse small-molecule libraries requires the development of a robust, high-throughput, and highly reproducible assay. Luminescence-based assays are highly preferred and widely used in high-throughput screening (HTS) in many biological systems as they have better signal-to-noise ratios, are as sensitive as radiolabel-based assays and easy to automate. We propose to develop a malaria parasite cell-based assay using the firefly luciferase reporter system. So far, we have developed a luciferase-expressing P. falciparum line and optimized a luciferase assay for 96-well microtiter plates. In the first phase of the project, we will validate and optimize this assay in a 384-well format and develop another luciferase- expressing line for complementary drug screening. In the second phase, we propose to perform a pilot HTS of a preexisting 3,000-compound library representing a structurally- diverse 100,000 compound library. Positive hits from the primary screening will be confirmed by dose-ranging experiments using the standard [H3] hypoxanthine incorporation assay. This high-throughput luminescence-based assay system will be a very useful tool for future HTS efforts in search for novel antimalarial therapies. Narrative With an annual death toll exceeding one million, malaria is a significant public health problem in many developing countries. With the emergence and spread of parasites resistant to many antimalarial drugs, there is a pressing need for the discovery of novel and improved drugs for malaria control. To facilitate antimalarial drug discovery, we propose to develop a highly sensitive, cell-based, one-step luminescent drug screen assay for high throughput screening of large chemical libraries.

描述（由申请人提供）：摘要疟疾的全球负担和恶性疟原虫的广泛耐药性需要新的和改进的治疗。小分子化合物的大型库的可用性为发现抗疟候选药物提供了丰富的资源。筛选这些结构多样的小分子文库需要开发一种稳健、高通量和高度可重复的测定方法。基于发光的测定是高度优选的，并且广泛用于许多生物系统中的高通量筛选（HTS），因为它们具有更好的信噪比，与基于放射性标记的测定一样灵敏并且易于自动化。我们建议使用萤火虫荧光素酶报告系统开发一种基于疟原虫细胞的检测方法。到目前为止，我们已经开发了一种表达荧光素酶的恶性疟原虫株系，并优化了96孔微量滴定板的荧光素酶测定。在项目的第一阶段，我们将在384孔格式中验证和优化该测定，并开发另一种荧光素酶表达系用于互补药物筛选。在第二阶段，我们建议对预先存在的3，000种化合物文库进行试验HTS，该文库代表结构多样的100，000种化合物文库。将使用标准[H3]次黄嘌呤掺入试验通过剂量范围实验确认初步筛选的阳性命中。这种高通量的发光检测系统将是一个非常有用的工具，为未来的HTS努力寻找新的抗疟疗法。疟疾每年的死亡人数超过一百万，是许多发展中国家的一个重大公共卫生问题。随着寄生虫对许多抗疟药物产生抗药性并扩散，迫切需要发现新的和改进的疟疾控制药物。为了促进抗疟药物的发现，我们建议开发一种高灵敏度的，基于细胞的，一步发光药物筛选测定，用于大化学库的高通量筛选。