Elucidating the Mechanism of Novel Dengue Antivirals

描述（由申请人提供）：目前尚无对抗登革热病毒（DENV）感染的抗病毒药物或疫苗。这是一个严重的失败，因为DENV是公认的a类生物恐怖主义制剂。此外，登革热病毒每年造成约150万登革热临床病例，其中50万感染者出现严重出血热。为了解决缺乏治疗方法来对抗这种高优先级和广泛的病原体的问题，我们使用“虚拟筛选”来发现假定的DEN2V NS3蛋白酶抑制剂。这种计算密集型的方法系统地检查了一个小分子数据库，以确定可能与DEN2V NS3蛋白酶的催化位点或P1位点相互作用的化合物。我们的计算研究预测了大约170种低分子量化合物可以作为DEN2V NS3蛋白酶抑制剂。初步的体外实验使用了一小部分计算机推荐的抑制剂和重组DEN2V NS3蛋白酶。值得注意的是，大多数被测化合物降低了DEN2V NS3蛋白酶的活性。两种化合物在初步细胞培养实验中进一步检测；两种化合物均表现出对DEN2V病毒的抗病毒活性。这些化合物的EC50值为亚微摩尔，无明显的细胞毒性，选择性指数为>150，对胰蛋白酶水解活性无影响。鉴于我们最初成功地从计算机预测的抑制剂中鉴定出有希望的登革热抗病毒先导物，我们假设，通过彻底分析我们的计算研究推荐的整套化合物，将发现额外的新型抗病毒先导物。为了验证这一假设，我们将完成DEN2V NS3蛋白酶的体外测定，以确定每种计算机推荐化合物的Ki和抑制机制（Specific Aim 1）。低Ki值的化合物将在培养细胞中进行抗病毒活性测试（Specific Aim 2），并通过一种新的基于细胞的NS3蛋白酶报告试验（Specific Aim 3）验证其抗病毒机制。新发现的抑制剂的特异性将进一步使用相关丝氨酸蛋白酶胰蛋白酶和凝乳胰蛋白酶以及血液凝固试验进行测试。由于DENV感染常伴有出血性并发症，因此凝血试验应谨慎，以确保登革热蛋白酶抑制剂不干扰凝血级联反应（Specific Aim 4）。这一项目的成功完成将确定并确定新的抗病毒先导物的特征，以便进一步开发登革热病毒感染的治疗方法。此外，确定这些先导抗病毒药物的机制基础和特异性将为未来基于结构的抗病毒优化研究提供坚实的基础，从而产生对相关黄病毒（包括西尼罗河病毒、黄热病病毒和丙型肝炎病毒）有效的治疗方法。

DESCRIPTION (provided by applicant): No antivirals or vaccines exist to combat dengue virus (DENV) infection. This is a serious failing since DENV is a recognized Category A bioterrorist agent. In addition, annually DENV causes about1.5 million clinical cases of dengue fever with >500,000 infected individuals developing severe hemorrhagic fever. To address the lack of therapeutics to combat this high-priority and widespread pathogen, we used "virtual screening" to discover putative DEN2V NS3 protease inhibitors. This computationally intensive approach systematically examined a database of small molecules to identify compounds that might interact with either the catalytic or P1 sites of DEN2V NS3 protease. Our computational studies predicted approximately 170 low molecular weight compounds would function as DEN2V NS3 protease inhibitors. Preliminary in vitro assays were done using a small subset of computer-recommended inhibitors and recombinant DEN2V NS3 protease. Significantly, the majority of tested compounds reduced DEN2V NS3 protease activity. Two compounds were further examined in preliminary cell culture experiments; both compounds demonstrated antiviral activity against DEN2V challenge. These compounds had sub-micromolar EC50 values, no apparent cytotoxicity, selectivity indices >150, and no impact on trypsin proteolytic activity. Given our initial success at identifying promising dengue antiviral leads from computer-predicted inhibitors, we hypothesize that additional novel antiviral leads will be found by thoroughly analyzing the entire set of compounds recommended by our computational studies. To test this hypothesis, we will complete in vitro DEN2V NS3 protease assays to determine the Ki and inhibition mechanism of each computer-recommended compound (Specific Aim 1). Compounds with low Ki values will be tested for antiviral activity in cultured cells (Specific Aim 2), and their antiviral mechanism validated using a novel cell-based NS3 protease reporter assay (Specific Aim 3). The specificity of newly discovered inhibitors will be further tested using related serine proteases trypsin and chymotrypsin, and blood-clotting assays. Since hemorrhagic complications often accompany DENV infection, the blood-clotting assay is prudent to ensure dengue protease inhibitors do not interfere with the blood coagulation cascade (Specific Aim 4). Successful completion of this project will identify and characterize new antiviral leads for further development as therapeutics for dengue virus infection. In addition, determining the mechanistic basis and specificity of these lead antivirals will provide a solid foundation for future structure-based antiviral optimization studies to produce therapeutics effective against related flaviviruses, including West Nile, Yellow Fever, and Hepatitis C viruses.