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.

概括 冠状病毒表达核蛋白蛋白（N），这对于病毒复制，转录和病毒粒子至关重要 集会。通过糖原合酶激酶3的SARS-COV（负责SARS）N的N磷酸化（GSK- 3）其功能是必需的。 GSK-3抑制作用减轻了SARS-COV和其他冠状病毒的感染力， 因此，是冠状病毒感染的有趣的治疗策略。锂，一种广泛使用的药物， 直接抑制GSK-3并损害不同的N磷酸化，病毒转录，复制和感染力 冠状病毒。但是，SARS-COV-2的N蛋白的GSK-3磷酸化（COVID19的原因）已有 没有报道。该提案利用了我们在GSK-3的长期专业知识来阻止N功能 和SARS-COV-2复制。在对正在接受SARS PCR测试的70,000名受试者的初步审查中 COV -2，我们发现锂的患者降低了COVID19的风险（赔率= 0.5 [0.36-0.80]，p = 0.0002）。我们表明，锂抑制了来自SARS-COV-2的N的磷酸化，而其他GSK-3则抑制 抑制剂在低微摩尔范围内用IC50阻断N磷酸化。 GSK3功能丧失支持 SARS-COV-2 N蛋白磷光磷酸化是GSK-3。我们确定了临床上耐受性的药物 出乎意料地抑制GSK-3并在临床耐受水平下损害N磷酸化。目的1本提案 描述了通过锂和选择性GSK-3抑制剂增强N磷酸化抑制的方法， 通过针对其他疾病的临床试验，专注于在人类中显示安全的。作为GSK-3 N蛋白的磷酸化需要在不同的位点预磷酸化，而另一种未造成的蛋白激酶， AIM 2将识别并靶向启动激酶作为阻断SARS-COV-2转录的替代策略 和复制。该目标还将测试GSK-3的抑制剂或启动激酶是否干扰 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 可以重新使用以治疗Covid19以及未来的冠状病毒爆发的药物。