Targeting Coronavirus through Nucleocapsid Phosphorylation

通过核衣壳磷酸化靶向冠状病毒

基本信息

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
项目类别：
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

项目摘要

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衣壳蛋白 (N)，该蛋白对于病毒复制、转录和病毒粒子至关重要集会。糖原合成酶激酶 3 (GSK- 3) 是其功能所必需的。 GSK-3 抑制可减弱 SARS-CoV 和其他冠状病毒的感染性因此，这是一种有趣的冠状病毒感染治疗策略。锂是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（COVID19 的病因）的 N 蛋白的 GSK-3 磷酸化已没有被报道。该提案利用了我们在 GSK-3 方面的长期专业知识来阻止 N 功能和 SARS-CoV-2 复制。在对 70,000 名接受 SARS PCR 检测的受试者进行的初步审查中， CoV-2，我们发现服用锂剂的患者感染 COVID19 的风险降低（比值比 = 0.5 [0.36 - 0.80]，p = 0.0002）。我们发现，锂可以抑制 SARS-CoV-2 中 N 的磷酸化，而其他 GSK-3 抑制剂阻断 N 磷酸化，IC50 在低微摩尔范围内。 GSK3 功能丧失支持这一点 SARS-CoV-2 N 蛋白磷酸化需要 GSK-3。我们确定了临床耐受的药物出乎意料地抑制 GSK-3 并损害临床耐受水平的 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，重点关注通过其他疾病的临床试验证明对人类安全的药物。作为GSK-3 N 蛋白的磷酸化需要通过另一种未知的蛋白激酶在不同位点进行预磷酸化，目标 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 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可重新用于治疗新冠肺炎以及未来冠状病毒爆发的药物。