Discovery of Enterovirus D68 2A protease inhibitors for antiviral therapy

发现用于抗病毒治疗的肠道病毒 D68 2A 蛋白酶抑制剂

• 批准号: 9907158
• 负责人: Gai Liu
• 金额: $ 30万
• 依托单位: MICROBIOTIX, INC
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-09 至 2021-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9907158
• 关键词: Address; Affect; Affinity; Animals; Antiviral Agents; Antiviral Therapy; Arizona; Binding; Biochemical; Biological Assay; Canada; Cell Culture Techniques; Cell Line; Cells; Cellular Assay; Cellular biology; Chemicals; Chemistry; Cleaved cell; Conserved Sequence; Coughing; Country; Data; Development; Disease Outbreaks; Dose; Drug Kinetics; Drug Targeting; Enterovirus; Enterovirus 68; Enzymes; Europe; Evaluation; FDA approved; Fluorescence Resonance Energy Transfer; Goals; Hand, Foot and Mouth Disease; Hepatitis C virus; Hospitalization; In Vitro; Infection; Institutes; Knowledge; Measures; Medical; Modeling; Myalgia; National Institute of Allergy and Infectious Disease; Peptide Hydrolases; Pharmaceutical Preparations; Phase; Property; Protease Inhibitor; Respiratory Signs and Symptoms; Scanning; Series; Serine Proteinase Inhibitors; Serotyping; Site; Specificity; Structure; Structure-Activity Relationship; Symptoms; Therapeutic; Time; Triad Acrylic Resin; United States National Institutes of Health; Vertebral column; Viral; Viral Cytopathogenic Effect; Virus; Virus Diseases; acute flaccid myelitis; base; cytotoxicity; experience; global health; high throughput screening; in vivo; inhibitor/antagonist; lead series; medical schools; novel; polypeptide; prevent; priority pathogen; prophylactic; small molecule; small molecule inhibitor; small molecule libraries; therapeutic vaccine

Abstract Enterovirus D68 (EV-D68), a highly contagious non-polio enterovirus, caused the 2014 outbreak affecting thousands of people in multiple countries, including the US, Canada and Europe. Historically, symptoms of EV- D68 infections include rhinorrhea, muscle aches and cough. However, during the 2014 outbreak, there were increasing cases involving more severe respiratory symptoms and hospitalization. Also, because the EV-D68 infections coincided with an increase of acute flaccid myelitis (AFM) cases, EV-D68 is suspected to be associated with AFM even though more data are needed to understand the relationship between them. EV-D68 is currently listed as one of NIAID’s priority pathogens. Unfortunately, there are no FDA-approved drugs or vaccines for the treatments of the EV-D68 infection. Therefore, there is an urgent medical need for more potent therapeutics tailored for EV-D68. The overall goal of this project is to identify and develop small molecule protease inhibitors of EV-D68 2Apro as prophylactics and/or therapeutics for EV-D68 infections and preferably also for infections of the highly related virus EV-A71, which is responsible for hand-foot-and-mouth disease and whose 2Apro shares a conserved sequence and catalytic triad with 2Apro of EV-D68. Our strategy is to address the unmet medical need by identifying small molecule inhibitors targeting the enzymatic activity of a newly characterized EV-D68 protease, 2Apro. The approach is to leverage our experience with 2Apro and homogeneous biochemical screens utilizing FRET to identify small molecules that inhibit the enzymatic activity of 2Apro. In Preliminary Studies, we developed a FRET assay for EV-D68 2Apro and have applied the assay in a low/medium-throughput screen of ~2000 compounds with Z’-factors ≥0.7. The pilot screen identified telaprevir as an inhibitor of EV-D68 2Apro with confirmed antiviral activity against a panel of contemporary EV-D68 strains. In Phase I, for Aim 1, an FRET HTS will be optimized and applied to diverse chemical libraries for the identification of small molecules that inhibit the enzymatic activity of 2Apro. In Aim 2, FRET secondary assays, a thermal shift assay and a cell-based CPE assay will be optimized and used to validate confirmed hits, to determine the binding affinity, and to measure in vitro antiviral activity against strains of serotypes D68 and A71. In Aim 3, validated hits prioritized based on their drug- likeness and antiviral spectrum will be evaluated for their ADME properties. We will also explore preliminary SAR of prioritized validated hits. In Phase II, we will chemically optimize priority inhibitors for potency and selectivity, in vitro and in vivo pharmacokinetic properties, and evaluate them in animal infection models.

抽象的 肠病毒D68（EV-D68）是一种高度传染性的非Polio肠病毒，引起了2014年爆发的影响 包括美国，加拿大和欧洲在内的多个国家中的成千上万人。从历史上看，ev-症状 D68感染包括鼻漏，肌肉酸痛和咳嗽。但是，在2014年爆发期间，有 增加病例涉及更严重的呼吸道症状和住院。另外，因为EV-D68 感染随着急性松弛性脊髓炎（AFM）病例的增加，怀疑EV-D68是相关的 即使需要更多数据来了解它们之间的关系，也有更多数据。 EV-D68目前 被列为NIAID的优先病原体之一。不幸的是，没有FDA批准的药物或疫苗 EV-D68感染的治疗方法。因此，紧急医疗需要更多潜在的治疗 为EV-D68量身定制。该项目的总体目标是识别和开发小分子蛋白酶抑制剂 EV-D68 2APRO作为预防药物和/或治疗EV-D68感染，最好也用于感染 高度相关的病毒EV-A71，该病毒负责手脚疾病，其2APRO共享 具有EV-D68的2APRO的保守序列和催化三合会。我们的策略是解决未满足的医疗 通过鉴定针对新表征的EV-D68的酶促活性的小分子抑制剂的需求 蛋白酶，2apro。方法是利用我们在2APRO和均质生化屏幕上的经验 使用FRET鉴定抑制2APRO酶促活性的小分子。在初步研究中，我们 开发了用于EV-D68 2APRO的FRET分析 〜2000化合物，Z'Factors≥0.7。试验屏幕将Telaprevir确定为EV-D68 2APRO的抑制剂 确认对当代EV-D68菌株小组的抗病毒活性。在第一阶段，对于AIM 1，fret hts 将优化并应用于潜水化学文库，以鉴定抑制的小分子 2apro的酶活性。在AIM 2中，FRET次要测定，热移测定和基于细胞的CPE测定 将被优化并用于验证已确认的命中，以确定结合亲和力并在体外测量 针对血清型D68和A71菌株的抗病毒活性。在AIM 3中，经过验证的命中根据其药物优先级 - 将评估相似性和抗病毒光谱的ADME特性。我们还将探索初步 优先级验证命中的SAR。在第二阶段，我们将化学优化优先级抑制剂的效力和 选择性，体外和体内药代动力学特性，并在动物感染模型中评估它们。