Mechanism of Inhibition of Entry Inhibitors against SARS-CoVs

SARS-CoV 进入抑制剂的抑制机制

• 批准号: 10262581
• 负责人: di s xia
• 金额: $ 37.05万
• 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/10262581
• 关键词: 2019-nCoV; Address; Animals; Antiviral Agents; Antiviral Therapy; Avian Influenza; Binding; Biological; 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; Electron Transport Complex III; Ensure; Environment; Escherichia coli; Evaluation; Frankfurt-Marburg Syndrome Virus; Freezing; Future; HIV; Human; Illinois; In Vitro; Incubated; Individual; Infection; Influenza A Virus, H5N1 Subtype; Laboratories; Lead; Length; Luciferases; Lung diseases; Measures; Mediating; Membrane; Membrane Fusion; Membrane Glycoproteins; Membrane Proteins; Microscope; Microscopy; Middle East Respiratory Syndrome Coronavirus; Mitochondria; Modeling; Molecular; Molecular Conformation; Negative Staining; Peptide Hydrolases; Peptides; Peptidyl-Dipeptidase A; Photons; Pichia; Polysaccharides; Precipitation; Preparation; Process; Protein Fragment; Proteins; Public Health; Receptor Cell; Recombinant Proteins; Recombinants; Reporter; Resolution; 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; base; beamline; cellular engineering; design; detector; experience; experimental study; flexibility; human coronavirus; inhibitor/antagonist; lead candidate; mortality; neutralizing antibody; novel therapeutics; pandemic disease; pathogenic virus; prevent; protein expression; protein folding; protein purification; receptor binding; respiratory; restraint; small molecule inhibitor; symposium; translocase; virucide

Progress (1) Lead identification. Based on the well-established principle that the S proteins carry out all of 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. (2) My group studies membrane proteins using both X-ray crystallography and cryo-EM. Our most recent work was the structure determination of bcs1, an ATP-driven protein translocase that transport folded proteins across biological membrane, using cryo-EM. We also determined structures of mitochondrial and bacterial Complex III alone or in complex with various respiratory small molecule inhibitors by 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. Specific Aims Aim 1: Large scale protein expression and purification in Pichia. 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. Aim 2: Structure determination for SARS-CoV-2 S protein or its fragments in the presence of lead compounds by cyro-EM or X-ray crystallography and elucidation of the mechanism of inhibition by these compounds. 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 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. Aim 3: Structural analysis to aid optimization of the hits to develop lead inhibitors effective against different coronaviruses, especially SARS-CoV and SARS-CoV-2. 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.

进度（1）铅识别。基于S蛋白具有COV的所有细胞输入功能的完善原理，将CoV的尖峰纳入了复制有缺陷的HIV核心，从而在病毒包裹表面表达的SARS-COV-2 S蛋白产生了伪病毒。转导将将假病变引入通过ACE-2和宿主蛋白酶TMPRSS2的表面表达设计的靶细胞中，随后表达病毒 - 编码的荧光素酶（LUC）报告基因，可为COV S指导的细胞进入功能进行高通量评估的读数。 Rong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Marburg病毒，H5N1鸟流感和SARS-COV。 SARS-COV和SARS-COV-2伪病毒感染的384孔格式已得到很好的优化。孵育48小时后测量荧光素酶活性。使用健壮的病毒进入测定法，我们已经确定了几种化合物以及能够抑制S蛋白介导的进入的病毒衍生肽。 （2）我的小组使用X射线晶体学和冷冻EM研究膜蛋白。我们最近的工作是使用Cryo-EM跨生物膜折叠蛋白的ATP驱动蛋白易位酶BCS1的结构测定。我们还单独确定了线粒体和细菌复合物III的结构，或通过X射线晶体学和冷冻EM与各种呼吸系统小分子抑制剂进行复合物。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的纯化重组外生域，并且足以进行冷冻EM研究。特定目的目标1：大规模蛋白质表达和纯化的pichia。目前，我们获得了足以用于冷冻EM研究的少量纯化的重组蛋白。具体而言，我们获得了S蛋白，S1，S2和S蛋白的RBD片段的e骨架。尽管大型全长外域三聚体和S2融合三聚体的量足以进行冷冻EM研究，但有些片段太小而无法进行EM研究。取而代之的是，将通过X射线晶体学研究，例如S1蛋白和RBD。这些小片段必须大量生产X射线晶体学研究。我的小组在大肠杆菌和肉饼中的重组蛋白表达方面都有丰富的经验。如果铅化合物会干扰S蛋白的蛋白水解启动，我们还计划纯化人类TMPRSS2。 AIM 2：通过Cyro-EM或X射线晶体学在铅化合物存在下SARS-COV-2 S蛋白或其片段的结构测定，并通过这些化合物阐明抑制机制。我的小组可以使用配备Gatan K2 Summit直接检测器的Titan Krios显微镜，这两个CMM（NCI分子显微镜中心）和NIC（NIH壁内冷冻EM设施）。我们还确保在芝加哥Argonne National Lab的Advance Photon Source上访问Sercat X射线梁系列。 Rong博士实验室提供的铅化合物将与我们的蛋白质样品一起孵育，并在EM电网制备之前去除任何沉淀。我们将首先通过负污渍和低温EM测试样品在屏幕显微镜中的质量。当样品被认为适用于高分辨率EM时，将在300 kV的Krios上收集数据。 EM显微照片将使用与/或Cistem进行处理。将使用COOT或CHIMERA进行建模，并将使用RefMac或Phoenix进行结构改进。较小的S蛋白片段的结晶将使用各种商业套件在房屋中进行。结晶命中条件将进行完善，并将在Sercat梁线上进行衍射实验。结构确定将使用CCP4或Phenix软件完成。我们将根据结构信息对这些铅抑制化合物进行机械研究。可以想象，潜在的机制包括（1）破坏病毒颗粒上S蛋白的预融合复合物或其他直接禁用（“直接”的病毒活性），（2）防止病毒与宿主细胞受体ACE-2，（3）阻止宿主宿主的融合量的宿主型宿主量的相互作用。这些可能性将在我们的结构分析和一系列既定的还原主义测定中进行系统评估。 AIM 3：结构分析以帮助优化命中以开发针对不同冠状病毒，尤其是SARS-COV和SARS-COV-2的铅抑制剂。一旦获得复杂的结构，将开始基于既定的基于结构的药物设计原理的铅候选优化和结构活性关系发展。原子分辨率下铅化合物的结合环境将有助于确定（1）在化学型中活性和选择性中空间，电子和构型因子的限制。 （2）当铅分子中存在药代动力学的不良特征时，我们将根据结构信息解决化合物的结构变化，以消除这些特征以改善分子。例如，如果由于大量可旋转键，该分子太灵活了，我们将施加构象约束，以降低柔韧性程度，并冻结构象，以重现抑制剂结合所需的构象。 （3）我们将能够设计新化合物的新衍生物，以最大化新化合物的吸毒特征。