Development of West Nile Virus/Broad Spectrum Flavivirus Protease Inhibitors

描述（由申请人提供）：西尼罗河病毒（WNV），黄病毒科的蚊媒成员，于1999年首次引入流行病，并迅速在美国蔓延。在西尼罗河病毒感染短暂下降后，2012年突然激增，这是美国最大的一次，在48个州有5，387例病例，243例死亡（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，西尼罗河病毒感染导致严重的神经系统后遗症，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发病机制。然而，目前还没有疫苗或抗病毒药物可供人类使用。这一多学科R21提案解决了这一关键需求。使用高通量筛选（HTS），我们已经确定了五个先导化合物的IC 50值在低至亚微摩尔范围内抑制WNV和登革病毒蛋白酶。我们的建议的优点是：（1）我们的实验室是第一个建立一个敏感的基于荧光的体外检测登革病毒（DENV）蛋白酶，现在采用在各种HTS活动的几个团体。(2)我们在哈佛医学院的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，其代表在10 mM下表现出对DENV 2和WNV蛋白酶的51-90%抑制的五种不同的化学支架。（3）从该列表中选择的五种化合物都具有范围在338-392 Da之间的分子量，cLogP：1.8-4.4，以及配体效率：0.25-0.32.这些值完全符合Lipinski的五点法则。(4)由于发现先导化合物抑制WNV和所有四种血清型的DENV蛋白酶，因此这些化合物中的一种或多种的优化可以产生用于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，生物化学病毒学，分子建模和X射线晶体学将HTS中鉴定的两种命中物优化为有效的WNV和DENV 2蛋白酶抑制剂。将测定两种先导化合物衍生物体外抑制病毒蛋白酶的效价（IC 50），并建立构效关系（SAR）。在im 1.2中，我们对WNV和DENV 2蛋白酶在大肠杆菌中的表达系统进行了优化。大肠杆菌，以产生mg量的纯化的蛋白酶，适合于结晶和确定配体结合的复合物的结构。在目的2中，我们提出分别使用表达WNV和DENV 2海肾荧光素酶（Rluc）报告复制子的猴肾（Vero）和BHK-21细胞来确定EC 50值，即这些化合物抑制WNV和DENV 2复制50%的功效。此外，我们建议在BSL-3设施中与Ted Pierson博士（NIAID）合作，使用WNV报告复制子病毒样颗粒（RVP）和感染性WNV验证两种化合物的EC 50值。将测定显示最大效力（IC 50和EC 50值）的先导化合物的细胞毒性（CC 50）。 在R21阶段，我们计划实现以下里程碑：我们将鉴定1-3种化合物，其在Vero和BHK-21细胞中抑制WNV和DENV 2病毒复制的IC 50和EC 50值在约500 nM范围内，具有低细胞毒性（e 200 mM）。我们将在第1年对两种选择的先导化合物进行P450抑制、小鼠代谢稳定性和Caco-2细胞渗透性研究，并在第2年对五种优化化合物进行研究。此外，我们将对5种化合物进行血浆蛋白结合，并对其中两种最有前途的化合物进行小鼠PK研究。 作为未来的目标（超越R21阶段），两种具有理想药物样特征的优化化合物将通过与Phil Murphy博士合作在WNV小鼠模型中进行测试，NIAID/NIH分子免疫学实验室主任，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AG 129小鼠模型。

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.