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

描述(申请人提供)：该项目的最终目标是开发安全、广谱的抗病毒疗法/预防药物，因为目前治疗病毒的药物相对较少，而且大多数确实存在的药物是高度针对病毒的或具有不良副作用。该项目已证实的双链RNA激活的Caspase寡聚体(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)，快速杀死受感染的细胞，而不损害未受感染的细胞。我们先前已经创造了一种蛋白Draco，它与细胞内的proaspase-9半胱氨酸酶招募结构域(CARD)结合，在dsRNA存在的情况下诱导细胞凋亡。我们已经证明，这种蛋白Draco是无毒的，对细胞内的15种不同病毒和小鼠的3种病毒有效。我们现在提议与哈佛医学院生物防御和新发传染病区域卓越中心(NSRB)国家筛查实验室合作，开发高通量筛查分析方法，以识别与大Draco蛋白具有相似性质但更易于制造、存储和使用的小化学分子。我们的具体目标是：1.建立一种简单、可靠的原天冬氨酸氨基转移酶-9卡片结合活性的初步检测方法，适用于高通量筛选小分子。与NSRB实验室协商，我们将开发与高通量筛选设备和化合物文库完全兼容的分析方法。2.建立一种反筛选方法来区分与原天冬氨酸蛋白酶-9卡片结合的分子和与APAF-1卡片结合的分子。同样的反筛选试验也可以用来筛选出与蛋白质非特异性结合的分子。3.与NSRB实验室合作，使用他们的化合物文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反复优化分析试剂、条件和方案。拟议的工作应该会推动Dracos作为安全、广谱的抗病毒治疗药物的最终实用，填补现有治疗药物的一大空白。

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.