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.

摘要：SARS-CoV-2 聚合酶和核酸外切酶抑制剂的发现和优化 SARS-CoV-2 RNA 依赖性 RNA 聚合酶 (RdRp) (Nsp12/7/8) 和核酸外切酶 (ExoN) (Nsp14/10) 发挥作用这些酶在病毒基因组复制和转录中高度保守。我们发现，冠状病毒在人类宿主细胞中没有分子，可以作为独特的抑制剂靶点。 SARS-CoV-2 ExoN 作为校对器，去除了核苷酸抑制剂，例如瑞德西韦 (Remdesivir)，在病毒RNA合成过程中，RdRp将其整合到病毒RNA中，这种机制可能是造成这种现象的原因。这些药物治疗 COVID-19 的疗效较低，我们推断病毒 RdRp 抑制剂的组合。 ExoN 可以克服这一缺陷，我们发现 RdRp 和 ExoN 的抑制剂具有协同作用。为了克服单独使用 RdRp 抑制剂在体外阻断 SARS-CoV-2 复制的缺陷。开发了高分辨率分子测定法来评估 SARS-CoV-2 RdRp 和 ExoN 活性以进行筛选我们鉴定并验证了这两种酶的一组真正的抑制剂。证明口服药物 Pibrentasvir（一种 HCV NS5A 抑制剂）也能抑制 SARS-CoV-2 ExoN，并且在 Pibrentasvir 的存在，用 RdRp 抑制剂 Remdesivir、Favipiravir、Molnupiravir 终止的 RNA，和 AT-527 在很大程度上免受 ExoN 切除。这些结果表明所有核苷/核苷酸- 目前正在进行 COVID-19 临床试验的口服候选药物将可能受益于该 RdRp/ExoN 抑制剂组合方法。在这个项目中，我们将重点关注这两种酶的新型抑制剂的发现和优化。我们将系统地测试阻止 SARS-CoV-2 复制的能力，包括分子、结构和功能。体外/体内研究旨在优化这种协同策略，以开发新分子作为有效的 COVID-19 药物。首先，我们将与 MAVDA 中心的所有核心人员密切合作，筛选并表征 SARS-CoV-2 其次，我们将使用优化的分子测定来进行 RdRp 和 ExoN 抑制剂的结构表征。第三，我们将设计和合成新的抑制剂。基于上述化合物的分子支架的 SARS-CoV-2 RdRp 和 ExoN 抑制剂最后，我们将测试已证实的 SARS-CoV-2 RdRp 和 ExoN 抑制剂的潜力。使用体外和体内测定的协同效应将反复进行。我们期待新开发的 SARS-CoV-2 RdRp 抑制剂的口服治疗。将抑制黄病毒（ZIKV、DENV）和甲病毒（CHIKV、EEEV）中的 RNA 聚合酶，我们将评估他们也有抑制这些病毒的能力。