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(申请人提供)：西尼罗河病毒(WNV)，由蚊媒传播的黄病毒科家族成员，于1999年首次流行，并迅速在美国传播。西尼罗河病毒感染人数在短暂下降后，在2012年突然激增，这是美国有史以来最大的一次，48个州的5387例病例，243人死亡(CDC)。其中2734例(51%)被列为神经侵袭性疾病(CDC)。人类感染西尼罗河病毒通常没有症状或表现出轻微的流感样症状。然而，在一些住院患者中，WNV感染会导致严重的神经后遗症，导致更高的死亡率。广泛的研究揭示了西尼罗河病毒的生物学和致病机理。然而，目前还没有疫苗或抗病毒药物可供人类使用。这项多学科的R21提案满足了这一关键需求。利用高通量筛选(HTS)，我们已经鉴定出五种先导化合物，其IC50值在低到亚微摩尔范围内，对西尼罗河病毒和登革病毒蛋白水解酶都有抑制作用。我们建议的优点是：(1)我们的实验室首次建立了一种灵敏的基于荧光的登革病毒(DENV)蛋白水解酶的体外检测方法，该方法目前已被多个HTS活动小组采用。(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种类药物化合物，代表5种不同的化学支架，在10 mM时对DENV2和WNV蛋白酶显示出51-90%的抑制。(3)5个化合物的相对分子质量均在338-392Da之间，cLogP为1.8-4.4，配体效率为0.25-0.32。这些值完全在利平斯基的五分法则之内。(4)由于先导化合物对西尼罗河病毒和所有四种血清型DENV蛋白水解酶均有抑制作用，因此对其中一种或多种化合物进行优化可以得到广谱抑制相关病毒的药物(S)。我们提出了以下具体目标。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射线结晶学，将HTS中确定的两个HIT优化为有效的WNV和DENV2蛋白酶抑制剂。这两个先导化合物的衍生物在体外对病毒蛋白水解酶的抑制能力(IC50)将被测定，并将建立构效关系(SAR)。在IM 1.2中，我们建议优化WNV和DENV2蛋白酶在大肠杆菌中的表达系统，以生产适合结晶的纯化的蛋白酶并确定配体结合的复合体的结构。在目标2中，我们建议用WNV和DENV2肾型荧光素酶(Rluc)报告复制子表达的猴肾(Vero)和BHK-21细胞分别测定这些化合物对WNV和DENV2复制抑制50%的EC50值和有效性。此外，我们建议在BSL-3设施中与Ted Pierson博士(NIAID)合作，使用WNV Reporter复制子病毒样颗粒(RVP)和传染性WNV验证两种化合物的EC50值。将测定显示最大效力(IC50和EC50值)的先导化合物的细胞毒性(CC50)。在R21阶段，我们计划实现以下里程碑：我们将鉴定1-3个化合物，其IC50值和EC50s值在~500 nM范围内，在低细胞毒性(E 200 MM)的Vero和BHK-21细胞中抑制WNV和DENV2病毒的复制。我们将在第一年对两个选定的先导化合物进行P450抑制、小鼠代谢稳定性和Caco-2细胞通透性研究，并在第二年对五个优化化合物进行P450抑制、小鼠代谢稳定性和Caco-2细胞通透性研究。此外，我们将对五个化合物进行血浆蛋白结合，并对最有希望的两个化合物进行小鼠PK研究。作为未来的目标(在R21阶段之后)，将通过与NIAID/NIH分子免疫实验室首席医学博士Phil Murphy博士的合作，以及与加利福尼亚州拉霍亚过敏和免疫学研究所的Sujan Shresta博士的合作，在WNV小鼠模型中测试两种具有理想药物样特性的优化化合物，并在AG129小鼠模型中测试登革热病毒。