EAGER: Collaborative Research: Design of Inhibitors for ORF7a and ORF7b Oligomerization in COVID-19

EAGER：合作研究：COVID-19 中 ORF7a 和 ORF7b 寡聚化抑制剂的设计

• 批准号: 2029895
• 负责人: Bryan Berger
• 金额: $ 15万
• 依托单位: University of Virginia Main Campus
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-15 至 2022-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2029895&HistoricalAwards=false
• 关键词: EAGER; Collaborative; Research; Design; Inhibitors

With this award, the Chemistry of Life Processes Program in the Chemistry Division and the Chemical and Biochemical Engineering Program in the Chemical, Bioengineering, Environmental and Transport Systems Division are funding Dr. Bryan Berger (University of Virginia) and Dr. Jeffery Klauda (University of Maryland) to investigate two proteins named ORF7a and OR7b from the COVID19 virus that have been implicated in how harmful the virus is to its host, e.g. the human cells. The research will focus on how these two proteins form larger protein complexes that in turn affect the interactions between the virus and the infected cells and influence the immune response of the host. The research informs the development of peptides that could be used to probe the viral propagation. The research is based on the use of a combination of computational and experimental methods. Dr. Berger and Dr. Klauda distribute to the scientific community free of charge through Addgene the plasmids and associated protocols developed for this project, thus enabling the global scientific community that works on finding a solution to the current pandemic and to minimizing the possibility of future outbreaks to quickly use the outcomes of their research. This work will provide training for post-doctoral fellows working on critical challenges using state-of-the-art experimental and computational methods. The results of the research will be disseminated by the team to the greater community through conferences and workshops at University of Virginia and University of Maryland and through publications. The researchers also plan to inform and educate students on possible mechanisms of virus transmission and prevention by participation in existing outreach programs at their Institutions.This research project seeks to understand the basis of specificity for transmembrane and juxtamembrane oligomerization of ORF7a with BST-2 and for homooligomerization of ORF7b. Using bacterial transcriptional assays for membrane protein dimerization based on the E. coli AraC protein (AraTM and DN-AraTM assays), the researches determine specific amino acid residues and structural motifs responsible for the protein oligomerization in bacterial membranes. This knowledge informs computational models for formation of BST-2/ORF7a heterooligomers and ORF7b homooligomers. In turn, the computational models are used to make critical new predictions of sequences for transmembrane peptides that could influence protein-protein interactions involving ORF7a and ORF7b. These predictions and the properties of the peptides are tested by synthesizing peptide libraries and using AraTM, DN-AraTM, and mammalian, cell-based fluorescence resonance energy transfer assays. Validation of candidate sequences are achieved using mammalian cell-based assays for BST-2 function and apoptosis. The results of these studies could provide high-resolution, experimentally validated models for OR7a and ORF7b homo and heterooligomerization, as well as peptide sequences that can be used to probe the roles of ORF7a and ORF7b in viral propagation in vivo.This grant is being awarded using funds made available by the Coronavirus Aid, Relief, and Economic Security (CARES) Act supplement allocated to MPS and ENG.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

有了这个奖项，化学生命过程的化学过程和化学，生物工程，环境和运输系统部的化学和生物化学工程计划资助了Bryan Berger博士（弗吉尼亚大学）（弗吉尼亚大学）和Jeffery Klauda（马里兰州大学），马里兰大学（University of Maryland）将两种名为ORF7A和ORF7A和OR7B的蛋白质与COVID19 VIRUS的危害相关的危害，该蛋白质与该蛋白质有关，该蛋白质曾是曾经的危害率，该蛋白质与曾经的构成了该病毒us的危害，该蛋白质曾经是该病毒us的危害。例如人类细胞。 该研究将集中于这两种蛋白质如何形成较大的蛋白质复合物，进而影响病毒与感染细胞之间的相互作用并影响宿主的免疫反应。 该研究为可用于探测病毒传播的肽的发展提供了信息。 该研究基于计算方法和实验方法的结合。 Berger博士和Klauda博士通过为该项目开发的质粒和相关协议免费分发给科学界，从而使全球科学界致力于找到解决当前大流行的解决方案，并最大程度地减少未来爆发的可能性，以快速利用其研究成果。 这项工作将为博士后研究员提供培训，该研究员使用最先进的实验和计算方法来应对关键挑战。该研究的结果将由团队通​​过弗吉尼亚大学和马里兰大学的会议和研讨会以及通过出版物将其传播给更大的社区。 研究人员还计划通过参与其机构的现有外展计划来告知和教育学生有关病毒传播和预防的可能机制。该研究项目旨在了解跨膜和ORF7A对ORF7A的特异性的基础，而ORF7A则使用BST-2和ORF7B的Homooligomeratization。使用细菌转录测定基于大肠杆菌ARAC蛋白（ARATM和DN-ARATM分析）进行膜蛋白二聚化，研究确定了负责细菌膜中蛋白质低聚的特定氨基酸残基和结构基序。这些知识为计算模型提供了形成BST-2/ORF7A异性恋者和ORF7B家族的模型。 反过来，计算模型用于对跨膜肽的序列进行关键的新预测，这些预测可能影响涉及ORF7A和ORF7B的蛋白质 - 蛋白质相互作用。这些预测和肽的特性通过合成肽库，使用ARATM，DN-ARATM和哺乳动物，基于细胞的基于细胞的荧光共振能量传递测定测试。使用基于哺乳动物细胞的BST-2功能和凋亡的基于哺乳动物的细胞测定法实现了候选序列的验证。这些研究的结果可以为OR7A和ORF7B HOMO和异质化的高分辨率，经过实验验证的模型，以及可用于探测ORF7A和ORF7B在病毒式传播中的作用的肽序列。 Eng。该奖项反映了NSF的法定任务，并通过使用基金会的知识分子优点和更广泛的影响审查标准来评估值得支持。