Development of West Nile Virus/Broad Spectrum Flavivirus Protease Inhibitors

西尼罗病毒/广谱黄病毒蛋白酶抑制剂的开发

• 批准号: 8771658
• 负责人: Radhakrishnan Padmanabhan
• 金额: $ 19.96万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8771658
• 关键词: Address; Adopted; Animal Model; Antiviral Agents; Aprotinin; Arbovirus Infections; Binding; Biochemical; Biological Assay; Biology; Caco-2 Cells; Cells; Centers for Disease Control and Prevention (U.S.); Cessation of life; Characteristics; Chemicals; Collaborations; Complex; Computer Simulation; Crystallization; Culicidae; Cytochrome P450; Dengue Virus; Development; Disease; Disease Outbreaks; Docking; Doctor of Medicine; Drug Kinetics; Enzymes; Epidemic; Escherichia coli; Exhibits; Family; Flaviviridae; Flavivirus; Fluorescence; Funding; Future; Goals; HIV-1; Hepatitis C virus; Human; Hypersensitivity; Immunology; In Vitro; Inhibitory Concentration 50; Institutes; Kidney; Laboratories; Lead; Letters; Libraries; Ligand Binding; Ligands; Light; Mammalian Cell; Metabolic; Methodology; Modeling; Molecular Immunology; Molecular Models; Molecular Weight; Monkeys; Mus; National Institute of Allergy and Infectious Disease; Neurologic; Pathogenesis; Patients; Peptide Hydrolases; Peptides; Permeability; Pharmaceutical Chemistry; Pharmaceutical Preparations; Phase; Plasma Proteins; Play; Property; Protease Inhibitor; Protein Binding; Proteins; Renilla Luciferases; Replicon; Reporter; Research; Resolution; Role; Scheme; Serotyping; Structure; Structure-Activity Relationship; Symptoms; System; Testing; United States National Institutes of Health; Vaccines; Vero Cells; Viral; Virus; Virus Diseases; Virus-like particle; West Nile virus; X-Ray Crystallography; analog; base; biosafety level 3 facility; chemical synthesis; cytotoxicity; design; drug discovery; drug market; flu; high throughput screening; in vitro Assay; in vivo; inhibitor/antagonist; innovation; medical schools; member; molecular modeling; mortality; mouse model; neurovirulence; process optimization; programs; public health relevance; scaffold; small molecule; three dimensional structure; virology

DESCRIPTION (provided by applicant): West Nile virus (WNV), mosquito-borne member of the Flaviviridae family was first introduced in 1999 epidemic and quickly spread throughout the US. After a brief decline in WNV infections, there was a sudden surge in 2012, the largest seen in the US, with 5,387 cases in 48 states with 243 deaths (CDC). Of these, 2,734 (51%) were classified as neuroinvasive disease (CDC). WNV infections in humans are generally asymptomatic or exhibit mild flu-like symptoms. However, in some hospitalized patients, WNV infections lead to severe neurological sequelae resulting in higher percent of mortality. Extensive research has shed light on the biology and pathogenesis of WNV. However, currently, there are no vaccines or antiviral drugs available for human use. This multi-disciplinary R21 proposal addresses this critical need. Using high throughput screen (HTS), we have identified five lead compounds with IC50 values in the low to submicromolar range inhibiting both WNV and dengue virus proteases. The strengths of our proposal are: (1) Our lab was the first to establish a sensitive fluorescence-based in vitro assay for dengue virus (DENV) protease which is now adopted in various HTS campaigns by several groups. (2) Our HTS campaign of ~120,000 compounds at NSRB at Harvard Medical School resulted in 73 drug-like compounds representing five distinct chemical scaffolds that exhibited 51-90% inhibition of DENV2 and WNV proteases at 10 mM. (3) The five selected compounds from this list all have molecular weights ranging between 338-392 Da, cLogP: 1.8-4.4, and ligand efficiency: 0.25-0.32. These values are well within Lipinski's rule of five. (4) Since the lead compounds were found to inhibit both WNV and all four serotypes of DENV proteases, optimization of one or more of these compounds could lead to broad spectrum inhibitor(s) for related viruses. We propose the following Specific Aims. In Aim 1.1, we propose to optimize the two of the hits identified in the HTS and selected for this proposal into potent WNV and DENV2 protease inhibitors by iterative medicinal chemistry, biochemical virology, molecular modeling and X-ray crystallography. The potencies (IC50s) of the derivatives of the two lead compounds will be determined in inhibition of the viral protease in vitro and a structure activity relationship (SAR) will be established. In im 1.2, we propose to optimize the WNV and DENV2 protease expression systems in E. coli to produce mg quantities of purified proteases suitable for crystallization and determine the structures of ligand-bound complexes. In Aim 2, we propose to determine the EC50 values, the efficacy of these compounds in inhibition of WNV and DENV2 replication by 50% using WNV and DENV2 Renilla luciferase (Rluc) reporter replicon-expressing monkey kidney (Vero) and BHK-21 cells, respectively. In addition, we propose to validate EC50 values for two compounds using WNV Reporter replicon Virus-like Particles (RVPs) and infectious WNV in collaboration with Dr. Ted Pierson (NIAID) in the BSL-3 facility. The cytotoxicity (CC50) of the lead compounds which show the greatest potencies (IC50 and EC50 values) will be determined. In the R21 phase, we propose to achieve the following milestone: We will identify 1-3 compounds with IC50 and EC50s values in the range of ~500 nM in inhibiting WNV and DENV2 viral replication in Vero and BHK-21 cells with low cytotoxicity (e 200 mM). We will perform P450 inhibition, mouse metabolic stability, and Caco-2 cell permeability for the two chosen lead compounds in Year 1 and for the five optimized compounds in year 2. In addition, we will perform plasma protein binding for the five compounds and mouse PK studies for the two of the most promising compounds. As a future goal (beyond the R21 phase), Two optimized compounds that have desirable drug-like characteristics will be tested in a mouse model for WNV through collaboration with Dr. Phil Murphy, M.D., Chief, Laboratory of Molecular Immunology, NIAID/NIH as well as in the AG129 mouse model for dengue virus in collaboration with Dr. Sujan Shresta, La Jolla Institute of Allergy and Immunology, La Jolla, CA.

描述（由申请人提供）：西尼罗河病毒（WNV）是黄病毒科蚊媒成员，于 1999 年首次引入并迅速传播到整个美国。西尼罗河病毒感染率短暂下降后，2012 年突然激增，为美国最大的一次，48 个州出现 5,387 例病例，其中 243 人死亡 (CDC)。其中，2,734 例 (51%) 被归类为神经侵袭性疾病 (CDC)。人类感染西尼罗河病毒通常无症状或表现出轻微的流感样症状。然而，在一些住院患者中，西尼罗河病毒感染会导致严重的神经系统后遗症，导致死亡率更高。广泛的研究揭示了西尼罗河病毒的生物学和发病机制。然而，目前还没有可供人类使用的疫苗或抗病毒药物。这项多学科 R21 提案解决了这一关键需求。使用高通量筛选 (HTS)，我们鉴定了五种 IC50 值在低至亚微摩尔范围内的先导化合物，可抑制西尼罗河病毒和登革热病毒蛋白酶。我们提案的优点是：（1）我们的实验室是第一个建立敏感的基于荧光的登革热病毒（DENV）蛋白酶体外测定方法的实验室，该方法现已被多个团体在各种 HTS 活动中采用。 (2) 我们在哈佛医学院 NSRB 进行的约 120,000 种化合物的 HTS 活动产生了 73 种药物样化合物，代表五种不同的化学支架，在 10 mM 浓度下对 DENV2 和 WNV 蛋白酶表现出 51-90% 的抑制作用。 (3) 从该列表中选择的五种化合物的分子量均在338-392 Da之间，cLogP：1.8-4.4，配体效率：0.25-0.32。这些值完全符合利平斯基的五法则。 (4) 由于发现先导化合物可以抑制 WNV 和所有四种血清型的 DENV 蛋白酶，因此对这些化合物中的一种或多种进行优化可以产生针对相关病毒的广谱抑制剂。 我们提出以下具体目标。在目标 1.1 中，我们建议通过迭代药物化学、生化病毒学、分子建模和 X 射线晶体学来优化 HTS 中确定的两个命中，并为本提案选择为有效的 WNV 和 DENV2 蛋白酶抑制剂。将确定两种先导化合物的衍生物在体外抑制病毒蛋白酶的效力（IC50），并建立结构活性关系（SAR）。在 im 1.2 中，我们建议优化大肠杆菌中的 WNV 和 DENV2 蛋白酶表达系统，以产生毫克量的适合结晶的纯化蛋白酶并确定配体结合复合物的结构。在目标 2 中，我们建议使用表达 WNV 和 DENV2 海肾荧光素酶 (Rluc) 报告复制子的猴肾 (Vero) 和 BHK-21 细胞确定 EC50 值，即这些化合物抑制 WNV 和 DENV2 复制 50% 的功效， 分别。此外，我们建议与 BSL-3 设施中的 Ted Pierson 博士 (NIAID) 合作，使用 WNV 报告复制子病毒样颗粒 (RVP) 和传染性 WNV 来验证两种化合物的 EC50 值。将确定显示出最大效力（IC50 和 EC50 值）的先导化合物的细胞毒性（CC50）。 在R21阶段，我们建议实现以下里程碑：我们将鉴定1-3种IC50和EC50值在~500 nM范围内的化合物，以低细胞毒性抑制Vero和BHK-21细胞中的WNV和DENV2病毒复制（ e 200 毫米）。我们将在第一年对两种选定的先导化合物进行 P450 抑制、小鼠代谢稳定性和 Caco-2 细胞通透性测试，并在第二年对五种优化化合物进行测试。此外，我们还将对这五种化合物和小鼠进行血浆蛋白结合测试两种最有前途的化合物的 PK 研究。 作为未来的目标（超越 R21 阶段），将通过与 NIAID/NIH 分子免疫学实验室主任 Phil Murphy 博士合作，在 WNV 小鼠模型中测试两种具有所需药物样特性的优化化合物以及与加利福尼亚州拉霍亚过敏和免疫学研究所的 Sujan Shresta 博士合作的登革热病毒 AG129 小鼠模型。