Developing High-Throughput Assays for DRACO Broad-Spectrum Antiviral Molecules

描述（由申请人提供）：该项目的最终目标是开发安全的广谱抗病毒治疗/预防药物，因为目前针对病毒的治疗药物相对较少，而且大多数存在的治疗药物都是高度病毒特异性的或有不良副作用。该项目已证实的双链RNA激活Caspase Oligomerizer （DRACO）方法选择性地诱导含有任何病毒双链RNA （dsRNA）的细胞凋亡（细胞自杀），迅速杀死感染细胞而不伤害未感染细胞。我们之前已经创建了一种蛋白质DRACO，它与细胞procaspase-9 caspase募集域（CARD）结合，在dsRNA存在的情况下诱导细胞凋亡。我们已经证明该蛋白对细胞中的15种不同病毒和小鼠中的3种病毒无毒且有效。我们现在提议与哈佛医学院生物防御和新发传染病卓越区域中心国家筛选实验室（NSRB）合作，以开发高通量筛选分析，以识别与大DRACO蛋白具有相似特性但更容易制造、储存和使用的小化学分子。我们的具体目标是：1。开发一种简单、可靠的procaspase-9 CARD结合活性的初级检测方法，适用于小分子的高通量筛选。在与NSRB实验室协商后，我们将开发与高通量筛选设备和化合物文库完全兼容的检测方法。2. 开发一种反筛选试验，以区分与procaspase-9 CARD结合的分子与与Apaf-1 CARD结合的分子。同样的反筛选试验可以用来筛选非特异性结合蛋白质的分子。3. 与NSRB实验室合作，使用他们的化合物库和机器人高通量筛选设备进行初级和反筛选分析的测试运行。我们将使用这些测试运行的结果来迭代优化分析试剂、条件和方案。拟议的工作将推动draco作为安全、广谱抗病毒治疗的最终用途，填补现有治疗方法的巨大空白。

DESCRIPTION (provided by applicant): This project's ultimate objective is to develop safe broad-spectrum antiviral therapeutics/prophylactics, since there are currently relatively few therapeutics for viruses, and most which do exist are highly virus- specific or have undesirable side effects. The project's proven Double-stranded RNA Activated Caspase Oligomerizer (DRACO) approach selectively induces apoptosis (cell suicide) in cells containing any viral double-stranded RNA (dsRNA), rapidly killing infected cells without harming uninfected cells. We have previously created a protein DRACO that binds to the cellular procaspase-9 caspase recruitment domain (CARD) to induce apoptosis in the presence of dsRNA. We have demonstrated that this protein DRACO is nontoxic and effective against 15 different viruses in cells and 3 viruses in mice. We now propose to collaborate with the National Screening Laboratory for the Regional Centers of Excellence in Biodefense and Emerging Infectious Diseases (NSRB) at Harvard Medical School in order to develop high-throughput screening assays to identify small chemical molecules that would have similar properties to the large DRACO protein but would be easier to manufacture, store, and use. Our specific aims are to: 1. Develop a simple, reliable primary assay for procaspase-9 CARD binding activity suitable for high- throughput screening of small molecules. In consultation with the NSRB lab, we will develop assays that are fully compatible with high-throughput screening equipment and compound libraries. 2. Develop a counter-screening assay to distinguish molecules that bind to procaspase-9 CARD from molecules that bind to Apaf-1 CARD. This same counter-screening assay can be used to screen out molecules that nonspecifically bind to proteins. 3. Collaborate with the NSRB lab to conduct test runs of both the primary and counter-screening assays using their compound library and robotic high-throughput screening equipment. We will use the results of those test runs to iteratively optimize the assay reagents, conditions, and protocols. The proposed work should advance DRACOs toward ultimate utility as safe, broad-spectrum antiviral therapeutics, filling a large gap in existing therapeutics.