Mechanism of Inhibition of Entry Inhibitors against SARS-CoVs

SARS-CoV 进入抑制剂的抑制机制

• 批准号: 10702782
• 负责人: di s xia
• 金额: $ 11.2万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10702782
• 关键词: 2019-nCoV; ACE2; Address; Antiviral Agents; Antiviral Therapy; Avian Influenza; Binding; Biological Assay; Cell membrane; Cells; Chicago; Chimera organism; China; Collection; Complex; Computer software; Coronavirus; Cryoelectron Microscopy; Crystallization; Data; Development; Disease Outbreaks; Drug Design; Drug Interactions; Drug Kinetics; Drug resistance; Ebola virus; Ensure; Environment; Escherichia coli; Evaluation; FDA Emergency Use Authorization; Freezing; Funding; Future; Grant; HIV; Human; Illinois; In Vitro; Incubated; Individual; Infection; Influenza A Virus, H5N1 Subtype; Influenza A virus; Laboratories; Lead; Length; Luciferases; Marburgvirus; Measures; Mediating; Membrane; Membrane Fusion; Membrane Glycoproteins; Membrane Proteins; Microscope; Microscopy; Middle East Respiratory Syndrome Coronavirus; Modeling; Molecular; Molecular Conformation; National Institute of Allergy and Infectious Disease; Negative Staining; Peptide Hydrolases; Peptides; Photons; Pichia; Polysaccharides; Precipitation; Preparation; Process; Protein Fragment; Proteins; Public Health; Receptor Cell; Recombinant Proteins; Recombinants; Reporter; Resolution; Respiratory Disease; Robotics; Roentgen Rays; SARS coronavirus; Sampling; Series; Source; Structure; Structure-Activity Relationship; Surface; TMPRSS2 gene; Testing; Therapeutic; Titan; United States National Institutes of Health; Universities; Vaccine Design; Vaccines; Viral; Viral Proteins; Virion; Virus; Virus Diseases; Work; X-Ray Crystallography; Zoonoses; animal coronavirus; base; beamline; betacoronavirus; cellular engineering; design; detector; experience; experimental study; flexibility; human coronavirus; inhibitor; lead candidate; mortality; neutralizing antibody; novel therapeutics; pandemic disease; pathogenic virus; prevent; protein expression; receptor binding; restraint; small molecule; symposium; virucide; zoonotic coronavirus

Lead identification. Based on the well-established principle that the S proteins carry out all the cell entry functions of CoVs, the CoV S spike were incorporated onto a replication defective HIV core, resulting pseudovirions with SARS-CoV-2 S protein expressed on the surface of viral envelop. Transduction will introduce the pseudovirions into target cells engineered with surface expression of ACE-2 and host protease TMPRSS2, with subsequent expression of viral-encoded luciferase (Luc) reporter providing readouts for high-throughput evaluation of CoV S-directed cell entry functions. Dr. Rong and others have routinely and successfully used this approach to identify and develop entry inhibitors for highly pathogenic viruses, such as Ebola and Marburg viruses, H5N1 bird flu, and SARS-CoV. The SARS-CoV and SARS-CoV-2 pseudovirus infection assays in a 384-well format have been well optimized. The luciferase activities were measured after 48 h of incubation. Using the robust viral entry assay, we have identified several compounds as well as viral derived peptides that are able to inhibit S protein mediated entry. My group studies membrane proteins using both X-ray crystallography and cryo-EM. For the proposed work, we have obtained purified recombinant ectodomain of the S protein, the S1 subunit, the RBD domain, the S2 subunit, and the human ACE-2 with the amount sufficient for cryo-EM studies. Currently, we have obtained small amounts of purified recombinant proteins sufficient for cryo-EM studies. Specifically, we have obtained ectodomain of the S protein, S1, S2, and the RBD fragments of the S protein. While the amounts for the large full-length ectodomain trimer and the S2 fusion trimer are sufficient for cryo-EM studies, some fragments are too small to do EM studies. Instead, they will be studied by X-ray crystallography, such as the S1 protein and the RBD. These small fragments will have to be produced in large quantities for X-ray crystallographic studies. My group has extensive experience in recombinant protein expression both in E. coli and in Pichia. In the case where the lead compound interferes with proteolytic priming of the S protein, we plan also to purify the human TMPRSS2. My group has access to Titan Krios microscopes equipped with Gatan K2 Summit direct detectors at both CMM (Center for Molecular Microscopy) of NCI and NICE (NIH Intramural cryo-EM facility). We also have assured access to the SERCAT X-ray beam line at Advance Photon Source, Argonne National Lab in Chicago. Lead compounds provided by Dr. Rong's laboratory will be incubated with our protein samples and any precipitations will be removed prior to EM grid preparation. We will first test our samples for quality in screen microscopes by negative stain and by cryo-EM. When the samples are deemed suitable for high resolution EM, data will be collected on the Krios at 300 kV. EM micrographs will be processed using either Relion, CryoSPARC and/or CisTem. Modeling will be performed using Coot or Chimera and structure refinement will be carried with Refmac or Phoenix. Crystallization of smaller S protein fragments will be carried out in house robotically with various commercial kits. Crystallization hit conditions will be refined and diffraction experiments will be performed at SERCAT beamline. Structure determination will be done using CCP4 or Phenix software. We will conduct mechanistic studies of these lead inhibitory compounds based on structural information. Conceivably, potential mechanisms include (1) destabilizing the prefusion complex or other direct disabling of S proteins on virus particles ("direct" virucidal activity), (2) preventing interaction of the virus with host cell receptor ACE-2, (3) interfering host protease priming, or (4) blocking the fusion process of the viral membrane to the host cell membrane. These possibilities will be systematically evaluated both in our structural analyses and in a series of established reductionist assays. Lead candidate optimization and structure-activity relationship development based on established structure-based drug design principles will be started as soon as the complex structures are obtained. Binding environments of lead compounds at atomic resolution will help to determine (1) the limits of steric, electronic and configurational factors in the activity and selectivity within the chemotypes. (2) When pharmacokinetically undesirable features are present in a lead molecule, we will address structural changes of the compound based on structural information to eliminate these features for improvement of the molecule. For instance, if the molecule is too flexible because of a high number of rotatable bonds, we will impose conformational restraints that will reduce the degree of flexibility and also freeze a conformation that might reproduce the conformation required for binding of the inhibitor. (3) we will be able to design new derivatives of hit compounds to maximize druglike features of the new compounds.

线索识别。基于S蛋白执行冠状病毒所有细胞进入功能的既定原理，将冠状病毒S刺突整合到复制缺陷的HIV核心上，产生在病毒表面表达SARS-CoV-2 S蛋白的假病毒粒子信封。转导将把假病毒粒子引入表面表达 ACE-2 和宿主蛋白酶 TMPRSS2 的靶细胞中，随后表达病毒编码的荧光素酶 (Luc) 报告基因，为 CoV S 引导的细胞进入功能的高通量评估提供读数。荣博士和其他人经常成功地使用这种方法来识别和开发高致病性病毒的进入抑制剂，例如埃博拉病毒和马尔堡病毒、H5N1禽流感和SARS-CoV。 384 孔格式的 SARS-CoV 和 SARS-CoV-2 假病毒感染测定已得到很好的优化。孵育48小时后测量荧光素酶活性。使用强大的病毒进入测定，我们已经鉴定了几种能够抑制 S 蛋白介导的进入的化合物以及病毒衍生肽。我的小组使用 X 射线晶体学和冷冻电镜研究膜蛋白。对于拟议的工作，我们获得了 S 蛋白、S1 亚基、RBD 结构域、S2 亚基和人 ACE-2 的纯化重组胞外域，其数量足以用于冷冻电镜研究。目前，我们已经获得了少量纯化的重组蛋白，足以用于冷冻电镜研究。具体来说，我们获得了S蛋白的胞外域、S1、S2以及S蛋白的RBD片段。虽然大的全长胞外域三聚体和 S2 融合三聚体的量足以进行冷冻电镜研究，但某些片段太小而无法进行电镜研究。相反，它们将通过 X 射线晶体学进行研究，例如 S1 蛋白和 RBD。这些小碎片必须大量生产才能用于 X 射线晶体学研究。我的团队在大肠杆菌和毕赤酵母中的重组蛋白表达方面拥有丰富的经验。在先导化合物干扰 S 蛋白的蛋白水解引发的情况下，我们还计划纯化人 TMPRSS2。我的团队可以在 NCI 的 CMM（分子显微镜中心）和 NICE（NIH 校内冷冻电镜设施）使用配备 Gatan K2 Summit 直接探测器的 Titan Krios 显微镜。我们还可以保证使用芝加哥阿贡国家实验室 Advance Photon Source 的 SERCAT X 射线束线。荣博士实验室提供的先导化合物将与我们的蛋白质样品一起孵育，并且在准备 EM 网格之前去除所有沉淀。我们将首先通过负染色和冷冻电镜在屏幕显微镜中测试样品的质量。当样品被认为适合高分辨率 EM 时，将在 Krios 上以 300 kV 收集数据。 EM 显微照片将使用 Relion、CryoSPARC 和/或 CisTem 进行处理。建模将使用 Coot 或 Chimera 进行，结构细化将使用 Refmac 或 Phoenix 进行。较小的 S 蛋白片段的结晶将使用各种商业试剂盒在内部机器人进行。 结晶命中条件将得到改进，衍射实验将在 SERCAT 光束线上进行。结构测定将使用CCP4或Phenix软件完成。我们将根据结构信息对这些先导抑制化合物进行机理研究。可以想象，潜在的机制包括（1）破坏预融合复合物的稳定性或以其他方式直接使病毒颗粒上的 S 蛋白失活（“直接”杀病毒活性），（2）防止病毒与宿主细胞受体 ACE-2 相互作用，（3）干扰宿主蛋白酶引发，或(4)阻断病毒膜与宿主细胞膜的融合过程。这些可能性将在我们的结构分析和一系列已建立的还原分析中进行系统评估。一旦获得复杂结构，将立即开始基于已建立的基于结构的药物设计原则的先导候选物优化和构效关系开发。先导化合物在原子分辨率下的结合环境将有助于确定（1）化学型内活性和选择性的空间、电子和构型因素的限制。 (2) 当先导分子中存在药代动力学不良特征时，我们将根据结构信息解决化合物的结构变化，以消除这些特征，从而改进分子。例如，如果分子由于大量可旋转键而过于柔性，我们将施加构象限制，这将降低柔性程度，并冻结可能重现抑制剂结合所需构象的构象。 (3)我们将能够设计热门化合物的新衍生物，以最大限度地提高新化合物的药物特性。