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Discovery and Optimization of Inhibitors of SARS-CoV-2 Polymerase and Exonuclease

SARS-CoV-2聚合酶和核酸外切酶抑制剂的发现和优化

基本信息

批准号：
10513924
负责人：
JINGYUE JU
金额：
$ 815.25万
依托单位：
HACKENSACK UNIVERSITY MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-05-16 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10513924
关键词：
2019-nCoV Address Affinity Alphavirus Animal Model Animals Antiviral Agents Biochemical Biological Assay COVID-19 COVID-19 test COVID-19 testing COVID-19 therapeutics COVID-19 treatment Cell Culture Techniques Cells Chikungunya virus Clinical Trials Collaborations Complex Coronavirus Cryoelectron Microscopy DNA-Directed RNA Polymerase Dependence Development Dose Drug Combinations Drug Targeting Enzyme Inhibitor Drugs Enzymes Excision Exonuclease Flavivirus Fluorescence Generations Genetic Transcription Genomics Hepatitis C virus High-Throughput Nucleotide Sequencing Human In Vitro Lead Length Libraries Masks Mass Spectrum Analysis Metabolic Methods Modeling Modification Molecular Nucleosides Nucleotides Oral Pharmaceutical Chemistry Pharmaceutical Preparations Pharmacology Play Polymerase Prodrugs Property RNA RNA Polymerase Inhibitor RNA replication RNA-Directed RNA Polymerase Reporting Resolution Roentgen Rays Role SARS-CoV-2 inhibitor Solubility Structure Structure-Activity Relationship Testing Therapeutic Toxic effect Toxicology Viral Viral Genome Viral Physiology Virus Virus Inhibitors Virus Replication Zika Virus base biosafety level 3 facility design drug candidate drug development drug efficacy efficacy study high throughput screening improved in vivo inhibitor metropolitan molnupiravir rational design remdesivir scaffold screening structural biology viral RNA virology

项目摘要

Summary: Discovery and Optimization of Inhibitors of SARS-CoV-2 Polymerase and Exonuclease SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) (Nsp12/7/8) and exonuclease (ExoN) (Nsp14/10) play critical roles in viral genome replication and transcription. These enzymes are highly conserved among the coronaviruses and have no counterparts in human host cells, serving as unique inhibitor targets. We discovered that SARS-CoV-2 ExoN, acting as a proofreader, removes nucleotide inhibitors, such as Remdesivir, that are incorporated by RdRp into viral RNA during its synthesis. This mechanism might be responsible for the relatively low efficacy of these drugs for treating COVID-19. We reasoned that combinations of inhibitors of the viral RdRp and ExoN could overcome this deficiency. We showed that inhibitors of the RdRp and ExoN act synergistically to overcome deficiencies of using RdRp inhibitors alone to block SARS-CoV-2 replication in vitro. We have developed high resolution molecular assays to assess SARS-CoV-2 RdRp and ExoN activities to screen inhibitors of these enzymes. We identified and validated a set of bona fide inhibitors for both enzymes. We demonstrated that the oral drug Pibrentasvir, an HCV NS5A inhibitor, also inhibits SARS-CoV-2 ExoN, and in the presence of Pibrentasvir, RNAs terminated with the RdRp inhibitors Remdesivir, Favipiravir, Molnupiravir, and AT-527 were largely protected from ExoN excision. These results indicate that all the nucleoside/nucleotide- based oral drug candidates currently in COVID-19 clinical trials will potentially benefit from this RdRp/ExoN inhibitor combination approach. In this project, we will focus on the discovery and optimization of new inhibitors of these two enzymes. Their ability to stop SARS-CoV-2 replication will be systematically tested. We will perform molecular, structural, and in vitro/in vivo studies to optimize this synergistic strategy to develop new molecules as effective COVID-19 drugs. In close collaboration with all the Cores of the MAVDA Center, first, we’ll screen and characterize SARS-CoV-2 RdRp and ExoN inhibitors using optimized molecular assays. Second, we’ll perform structural characterization of SARS-CoV-2 RdRp and ExoN complexed with the identified inhibitors. Third, we’ll design and synthesize new SARS-CoV-2 RdRp and ExoN inhibitors based on the molecular scaffolds of the above compounds that have demonstrated viral inhibitor activity. Finally, we’ll test SARS-CoV-2 RdRp and ExoN inhibitors for their potential synergistic effects using in vitro and in vivo assays. This four-pronged approach will be performed iteratively for the development of oral COVID-19 therapeutics. We expect the newly developed SARS-CoV-2 RdRp inhibitors will inhibit RNA polymerases in flaviviruses (ZIKV, DENV) and alphaviruses (CHIKV, EEEV), and we will assess their ability to inhibit these viruses as well.

SARS-CoV-2聚合酶和核酸外切酶抑制剂的发现与优化 SARS-CoV-2依赖RNA聚合酶(RdRp)(Nsp12/7/8)和外显子(Nsp14/10)的作用在病毒基因组复制和转录中的关键作用。这些酶在冠状病毒，并且在人类宿主细胞中没有对应的基因，作为独特的抑制物靶标。我们发现 SARS-CoV-2外显子起校对作用，移除核苷酸抑制物，如Remdesivir，这些抑制物是 RdRp在合成过程中被RdRp掺入病毒RNA。这一机制可能负责相对这些药物治疗新冠肺炎的疗效较低。我们推断，病毒RdRp的抑制剂组合而外显子可以克服这一缺陷。我们发现RdRp和外显子的抑制剂具有协同作用。克服单独使用RdRp抑制剂在体外阻断SARS-CoV-2复制的不足。我们有建立高分辨分子检测SARS-CoV-2 RdRp和外显子活性的方法这些酶的抑制剂。我们鉴定并验证了一组针对这两种酶的真正的抑制剂。我们证明了口服药物匹布列韦，一种丙型肝炎病毒NS5A抑制剂，也抑制SARS-CoV-2外显子，并且在 Pibrentasvir的存在，以RdRp抑制剂Remdesivir，Favipiravir，Molnupiravir，和AT-527在很大程度上没有被外显子切除。这些结果表明，所有的核苷/核苷酸- 目前正在进行新冠肺炎临床试验的口服候选药物可能会从这个RdRp/外显子中受益抑制剂组合方法。在这个项目中，我们将专注于发现和优化这两种酶的新抑制剂。他们的将对阻止SARS-CoV-2复制的能力进行系统测试。我们将执行分子、结构和体外和体内研究旨在优化这一协同策略，以开发新的分子作为有效的新冠肺炎药物。在与MAVDA中心所有核心的密切合作下，首先，我们将对SARS-CoV-2进行筛查和特征分析 RdRp和外显子抑制剂使用优化的分子分析。第二，我们将执行结构表征 SARS-CoV-2的RdRp和外显子与已鉴定的抑制物形成络合。第三，我们将设计和合成新的基于上述化合物的分子支架的SARS-CoV-2 RdRp和外显子抑制剂显示出病毒抑制活性。最后，我们将测试SARS-CoV-2 RdRp和外显子抑制剂的潜力采用体外和体内试验的协同效应。这个四管齐下的方法将迭代执行，用于口腔新冠肺炎疗法的发展。我们期待新开发的SARS-CoV-2 RdRp抑制剂将抑制黄病毒(ZIKV，DENV)和甲病毒(CHIKV，EEEV)中的RNA聚合酶，我们将评估它们抑制这些病毒的能力也是如此。