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），Flaviviridae家族的蚊子传播成员首次于1999年引入，并迅速遍及美国。在WNV感染短暂下降之后，2012年突然激增，是美国最大的，有5387例死亡人数为243例（CDC）。其中，有2,734（51％）被归类为神经侵袭性疾病（CDC）。人类中的WNV感染通常是无症状的或表现出轻度流感样症状。但是，在一些住院的患者中，WNV感染导致严重的神经后遗症，导致死亡率较高。广泛的研究阐明了WNV的生物学和发病机理。但是，目前，尚无可供人类使用的疫苗或抗病毒药物。这个多学科的R21提案满足了这一关键需求。使用高吞吐量屏幕（HTS），我们已经确定了五种具有IC50值的五种铅化合物，在低至近极范围内抑制了WNV和登革热病毒蛋白酶。我们提案的优势是：（1）我们的实验室是第一个建立基于敏感荧光的体外测定的登革热病毒（DENV）蛋白酶的实验室，该测定法现已在各种HTS运动中通过几个组采用。 （2）我们在哈佛医学院NSRB举行的HTS运动大约120,000种化合物，导致73种类似药物的化合物，代表了五种不同的化学支架，在10 mm处表现出51-90％的DENV2和WNV蛋白酶的抑制作用。 （3）此列表中的五种选定化合物的分子量范围在338-392 DA，Clogp：1.8-4.4和配体效率之间：0.25-0.32。这些价值在Lipinski的五个规则范围内很好。 （4）由于发现铅化合物抑制了WNV和DENV蛋白酶的所有四种血清型，因此对其中一种或多种化合物的优化可能会导致相关病毒的频谱抑制剂（S）。 我们提出以下特定目标。在AIM 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中鉴定出的两个命中，并为该建议选择为有效的WNV和DENV2蛋白酶抑制剂。将确定两种铅化合物的衍生物的效力（IC50），以抑制病毒蛋白酶在体外抑制，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，以产生适合结晶的纯蛋白酶，并确定配体结合配合物的结构。在AIM 2中，我们建议确定EC50值，这些化合物在抑制WNV和DENV2复制方面的功效分别分别使用WNV和DENV2 RENILLA荧光素酶（RLUC）replorter replorter replicon表达表达猴子肾脏（VERO）和BHK-21细胞。此外，我们建议使用BSL-3设施中的Ted Pierson（NIAID）合作，使用WNV报告的复制品样本样颗粒（RVP）和感染性WNV来验证两种化合物的EC50值。将确定显示最大效力（IC50和EC50值）的铅化合物的细胞毒性（CC50）。 在R21阶段，我们提议实现以下里程碑：在抑制Vero和BHK-21细胞中，〜500 nm的IC50和EC50值的1-3种化合物在〜500 nm的范围内，具有低细胞毒性（E 200 mm）。我们将对1年级选择的两种铅化合物以及2年级的五种优化化合物进行P450抑制作用，小鼠代谢稳定性和CACO-2细胞渗透性。此外，我们还将针对最有利的两种化合物的五种化合物和小鼠PK进行血浆蛋白结合。 作为未来的目标（超出R21阶段），将通过与分子免疫学/NIH的分子免疫学/NIH实验室的负责人Phil Murphy博士合作，在WNV的鼠标模型中进行两种优化的化合物，并在WNV的鼠标模型中进行测试。乔拉，加利福尼亚。