Mechanism of Inhibition of Entry Inhibitors against SARS-CoVs

线索识别。根据S蛋白执行冠状病毒的所有细胞进入功能这一公认的原理，将冠状病毒S刺突插入到复制缺陷的HIV核心上，产生假病毒粒子，并在病毒囊膜表面表达SARS-CoV-2 S蛋白。转导将假病毒粒子引入表面表达血管紧张素转换酶-2和宿主蛋白酶TMPRSS2的靶细胞，随后表达病毒编码的荧光素酶(Luc)报告，为高通量评估冠状病毒S定向的细胞进入功能提供读数。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，如埃博拉和马尔堡病毒、H5N1禽流感和SARS-CoV。384孔格式的SARS-CoV和SARS-CoV-2伪病毒感染检测得到了很好的优化。孵育48h后检测荧光素酶活性。利用强大的病毒侵入实验，我们已经鉴定了几种化合物以及病毒衍生的多肽，它们能够抑制S蛋白介导的侵入。我的团队同时使用X射线结晶学和低温电子显微镜研究膜蛋白。在所提出的工作中，我们已经获得了纯化的S蛋白、S1亚基、RBD结构域、S2亚基和人血管紧张素转换酶-2的重组胞外域，其量足以用于冷冻-EM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-EM研究。具体来说，我们已经获得了S蛋白的胞外结构域、S1、S2和S蛋白的RBD片段。虽然大的全长胞外结构域三聚体和S2融合三聚体的数量足以进行冷冻-EM研究，但有些片段太小，无法进行EM研究。相反，它们将通过X射线结晶学进行研究，例如S1蛋白和RBD。为了进行X射线结晶学研究，必须大量生产这些小碎片。我的团队在重组蛋白在大肠杆菌和毕赤酵母中的表达方面都有丰富的经验。在先导化合物干扰S蛋白的蛋白水解性启动的情况下，我们也计划纯化人TMPRSS2。我的团队可以在NCI的CMM(分子显微镜中心)和NICE(NIH壁内冷冻-EM设施)使用配备Gatan K2 Summit直接探测器的Titan Krios显微镜。我们还确保可以使用位于芝加哥阿贡国家实验室的高级光子源的SERCAT X射线束线。荣博士的实验室提供的铅化合物将与我们的蛋白质样本一起孵化，任何沉淀物将在EM网格准备之前被去除。我们将首先用底片染色和低温电子显微镜在屏幕显微镜上测试样品的质量。当样品被认为适合于高分辨率EM时，将在300千伏的Krios上收集数据。EM显微照片将使用Relion、CryoSPARC和/或CisTem进行处理。将使用Coot或Chimera进行建模，并使用Refmac或Phoenix进行结构优化。较小的S蛋白质片段的结晶将在室内使用各种商业试剂盒进行机器人操作。将改进结晶命中条件，并在SERCAT光束线上进行衍射实验。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铅抑制化合物进行机理研究。可以想见，潜在的机制包括(1)破坏病毒颗粒上S蛋白的预融合复合体或其他直接失效的稳定性(“直接”杀毒活性)，(2)阻止病毒与宿主细胞受体ACE2的相互作用，(3)干扰宿主蛋白酶的启动，或(4)阻止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既定的简化论分析中进行系统评估。一旦获得复杂的结构，将立即开始基于既定的基于结构的药物设计原则的主要候选优化和构效关系开发。原子分辨的铅化合物的结合环境将有助于确定(1)空间、电子和构型因素在化学类型中的活性和选择性的限制。(2)当铅分子中存在药动学上不受欢迎的特征时，我们将根据结构信息处理化合物的结构变化，以消除这些特征，以改进分子。例如，如果分子由于大量的可旋转键而过于灵活，我们将施加构象限制，这将降低灵活性程度，并冻结可能复制结合抑制剂所需的构象的构象。(3)我们将能够设计新的HIT化合物的衍生物，以最大限度地发挥新化合物的药物特性。

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.