Elucidating the Mechanism of Novel Dengue Antivirals

描述（由申请人提供）：目前没有对抗登革热病毒（DENV）感染的抗病毒药或疫苗。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，每年DENV引起约150万例登革热临床病例，其中> 500，000名感染个体发展为严重出血热。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，我们使用“虚拟筛选”来发现推定的DEN 2 V NS 3蛋白酶抑制剂。这种计算密集型方法系统地检查了小分子数据库，以识别可能与DEN 2 V NS 3蛋白酶的催化或P1位点相互作用的化合物。我们的计算研究预测了大约170种低分子量化合物将作为DEN 2 V NS 3蛋白酶抑制剂发挥作用。使用一小部分计算机推荐的抑制剂和重组DEN 2 V NS 3蛋白酶进行初步体外测定。值得注意的是，大多数测试的化合物降低了DEN 2 V NS 3蛋白酶活性。在初步细胞培养实验中进一步检查了两种化合物;这两种化合物均表现出针对DEN 2 V攻击的抗病毒活性。这些化合物具有亚微摩尔EC 50值，无明显细胞毒性，选择性指数>150，并且对胰蛋白酶蛋白水解活性无影响。鉴于我们在从计算机预测的抑制剂中识别有希望的登革热抗病毒线索方面取得的初步成功，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，将发现其他新的抗病毒线索。为了检验这一假设，我们将完成体外DEN 2 V NS 3蛋白酶测定，以确定每种计算机推荐化合物的Ki和抑制机制（特异性目的1）。将在培养的细胞中测试具有低Ki值的化合物的抗病毒活性（特异性目标2），并且使用新型基于细胞的NS 3蛋白酶报告基因测定（特异性目标3）验证其抗病毒机制。新发现的抑制剂的特异性将进一步测试使用相关的丝氨酸蛋白酶胰蛋白酶和胰凝乳蛋白酶，和凝血试验。由于出血性并发症通常伴随DENV感染，因此凝血测定是谨慎的，以确保登革热蛋白酶抑制剂不会干扰凝血级联反应（具体目标4）。该项目的成功完成将确定和表征新的抗病毒先导物，以进一步开发作为登革热病毒感染的治疗药物。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，以产生有效对抗相关黄病毒（包括西尼罗河病毒、黄热病和丙型肝炎病毒）的治疗药物。

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.