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

描述（由申请人提供）：本项目的最终目标是开发安全的广谱抗病毒治疗剂/抗病毒药物，因为目前针对病毒的治疗剂相对较少，并且大多数确实存在的治疗剂是高度病毒特异性的或具有不良副作用。该项目的双链RNA激活半胱天冬酶寡聚化剂（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），快速杀死感染的细胞，而不伤害未感染的细胞。我们先前已经创建了一种蛋白DRACO，其与细胞的半胱天冬酶前体-9半胱天冬酶募集结构域（CARD）结合，以在dsRNA存在下诱导细胞凋亡。我们已经证明，这种蛋白质DRACO对细胞中的15种不同病毒和小鼠中的3种病毒是无毒和有效的。我们现在提议与位于哈佛医学院的生物防御和新发传染病区域卓越中心（NSRB）的国家筛选实验室合作，以开发高通量筛选测定法，以鉴定与大型DRACO蛋白质具有相似性质但更容易制造、储存和使用的小化学分子。我们的具体目标是：1.开发适用于小分子高通量筛选的procaspase-9 CARD结合活性的简单、可靠的初步测定。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出非特异性结合蛋白质的分子。3.与NSRB实验室合作，使用其化合物库和机器人高通量筛选设备进行初级和反筛选检测的测试运行。我们将使用这些测试运行的结果来迭代优化检测试剂、条件和方案。拟议的工作应该推进DRACO作为安全，广谱抗病毒治疗的最终用途，填补了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.