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

描述(由申请人提供)：全球疟疾负担和恶性疟原虫广泛的耐药性需要新的和改进的治疗方法。大量小分子化合物的可获得性为发现抗疟疾候选药物提供了丰富的资源。筛选这些结构不同的小分子文库需要开发一种强大的、高通量和高度重复性的分析方法。基于发光的分析方法具有更好的信噪比，与基于放射性标记的分析方法一样灵敏，并且易于自动化，因此在许多生物系统的高通量筛选(HTS)中得到了高度的首选和广泛的应用。我们建议开发一种基于疟疾寄生虫细胞的检测方法，使用萤火虫荧光素酶报告系统。到目前为止，我们已经建立了表达荧光素酶的恶性疟原虫品系，并优化了适用于96孔板的荧光素酶检测方法。在该项目的第一阶段，我们将以384孔的形式验证和优化这种检测方法，并开发另一种用于补充药物筛选的荧光素酶表达株。在第二阶段，我们建议对现有的3,000个化合物库进行试点HTS，代表结构多样化的100,000个化合物库。初步筛选的阳性结果将通过使用标准的[H3]次黄嘌呤掺入试验的剂量范围实验来确认。这种高通量的基于发光的检测系统将是未来HTS努力寻找新的抗疟疾疗法的一个非常有用的工具。每年死亡人数超过100万人的疟疾是许多发展中国家的一个重大公共卫生问题。随着对许多抗疟疾药物产生抗药性的寄生虫的出现和传播，迫切需要发现新的和改进的疟疾控制药物。为了促进抗疟疾药物的发现，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试验，用于高通量筛选大型化学库。

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.