Targeting Coronavirus through Nucleocapsid Phosphorylation

通过核衣壳磷酸化靶向冠状病毒

• 批准号: 10239590
• 负责人: PETER S KLEIN
• 金额: $ 44.69万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-14 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10239590
• 关键词: 2019-nCoV; Antiviral Agents; Arginine; Attenuated; Binding; Biology; COVID-19; COVID-19 testing; COVID-19 treatment; Cell Culture Techniques; Cells; Clinical; Clinical Trials; Collaborations; Coronavirus; Coronavirus Infections; Coronavirus nucleocapsid protein; Data; Disease; Disease Outbreaks; Drug Targeting; Epidemic; Epithelial Cells; Future; Genetic Transcription; Glycogen Synthase Kinase 3; Glycogen Synthase Kinases; Human; Impairment; Infection; Institutes; Knock-out; Lead; Lithium; Lung; Modeling; Mutation; Nucleocapsid; Nucleocapsid Proteins; Pathogenicity; Patients; Pharmaceutical Preparations; Phosphorylation; Phosphorylation Site; Phosphotransferases; Protein Inhibition; Protein Kinase; Proteins; RNA replication; Reporting; Research; Risk; SARS coronavirus; Serine; Severe Acute Respiratory Syndrome; Signal Transduction; Site; Testing; Therapeutic; Viral; Virion; Virus Replication; alveolar epithelium; clinical efficacy; efficacy testing; experimental study; genomic RNA; inhibitor/antagonist; kinase inhibitor; knock-down; loss of function; protective effect

Summary Coronaviruses express a nucleocapsid protein (N) that is essential for viral replication, transcription, and virion assembly. Phosphorylation of N from SARS-CoV (responsible for SARS) by glycogen synthase kinase 3 (GSK- 3) is required for its function. GSK-3 inhibition attenuates infectivity of SARS-CoV and other coronaviruses and is therefore an intriguing therapeutic strategy for coronavirus infections. Lithium, a widely used medication, directly inhibits GSK-3 and impairs N phosphorylation, viral transcription, replication, and infectivity of diverse coronaviruses. However, GSK-3 phosphorylation of N protein from SARS-CoV-2 (the cause of COVID19) has not been reported. This proposal leverages our long-standing expertise with GSK-3 to block N function and SARS-CoV-2 replication. In a preliminary review of 70,000 subjects undergoing PCR testing for SARS- CoV-2, we found that patients on lithium had reduced risk of COVID19 (odds ration = 0.5 [0.36 - 0.80], p = 0.0002). We show that phosphorylation of N from SARS-CoV-2 is inhibited by lithium and that other GSK-3 inhibitors block N phosphorylation with IC50s in the low micromolar range. GSK3 loss of function supports that GSK-3 is required for SARS-CoV-2 N protein phosphorlyation. We identified clinically-tolerated drugs that unexpectedly inhibit GSK-3 and impair N phosphorylation at clinically-tolerated levels. Aim 1 of this proposal describes approaches to enhance inhibition of N phosphorylation by lithium and by selective GSK-3 inhibitors, with a focus on those shown to be safe in humans through clinical trials for other diseases. As GSK-3 phosphorylation of N protein requires pre-phosphorylation at a distinct site by another, unkown protein kinase, Aim 2 will identify and target the priming kinase as an alternative strategy to block SARS-CoV-2 transcription and replication. This aim will also test whether inhibitors of either GSK-3 or the priming kinase interfere with replication of other pathogenic coronaviruses. If successful, the project will identify clinically safe medications that can be repurposed to treat COVID19 as well as future coronavirus outbreaks.

总结 冠状病毒表达一种对病毒复制、转录和病毒粒子至关重要的核衣壳蛋白（N 组装件.糖原合成酶激酶3（GSK-3）对SARS冠状病毒（SARS病毒）N的磷酸化作用 3)是其功能所必需的。GSK-3抑制减弱SARS-CoV和其他冠状病毒的感染性， 因此是一种有趣的冠状病毒感染治疗策略。锂，一种广泛使用的药物， 直接抑制GSK-3并损害N磷酸化、病毒转录、复制和多种病毒的感染性。 冠状病毒然而，来自SARS-CoV-2的N蛋白的GSK-3磷酸化（COVID 19的原因）具有以下作用： 没有被报道。该提案利用了我们在GSK-3方面的长期专业知识来阻断N功能 和SARS-CoV-2复制。在对7万名接受SARS PCR检测的受试者进行的初步审查中， CoV-2，我们发现服用锂盐的患者患COVID 19的风险降低（比值比= 0.5 [0.36 - 0.80]，p = 0.001）。 0.0002）。我们发现，锂抑制了SARS-CoV-2的N磷酸化，而其他GSK-3 抑制剂以低微摩尔范围的IC 50阻断N磷酸化。GSK 3功能丧失支持， GSK-3是SARS-CoV-2 N蛋白磷酸化所必需的。我们发现了临床耐受的药物， 出乎意料地抑制GSK-3并以临床耐受水平损害N磷酸化。本提案的目标1 描述了增强锂和选择性GSK-3抑制剂对N磷酸化的抑制的方法， 重点是那些通过对其他疾病的临床试验证明对人类安全的药物。作为GSK-3 N蛋白的磷酸化需要在不同位点被另一种未知的蛋白激酶预磷酸化， 目的2将鉴定并靶向引发激酶作为阻断SARS-CoV-2转录的替代策略 和复制。该目的还将测试GSK-3或引发激酶的抑制剂是否会干扰 其他致病性冠状病毒的复制。如果成功，该项目将确定临床安全 这些药物可以重新用于治疗COVID 19以及未来的冠状病毒爆发。