Mechanism of Inhibition of Entry Inhibitors against SARS-CoVs

进展(1)引线识别。基于S蛋白执行冠状病毒的所有细胞进入功能的既定原理，将冠状病毒S刺突结合到复制缺陷的HIV核心上，从而在病毒包膜表面表达带有SARS-CoV-2 S蛋白的假病毒粒子。转导将假病毒粒子引入靶细胞，表面表达ACE-2和宿主蛋白酶TMPRSS2，随后表达病毒编码的荧光素酶（Luc）报告基因，为高通量评估冠状病毒s定向细胞进入功能提供数据。荣博士和其他人经常并成功地使用这种方法来识别和开发高致病性病毒（如埃博拉病毒和马尔堡病毒、H5N1禽流感和sars冠状病毒）的进入抑制剂。对384孔格式的SARS-CoV和SARS-CoV-2假病毒感染检测方法进行了优化。孵育48 h后测定荧光素酶活性。使用强大的病毒进入实验，我们已经确定了几种化合物以及病毒衍生的肽，它们能够抑制S蛋白介导的进入。(2)我的小组使用x射线晶体学和低温电镜研究膜蛋白。我们最近的工作是利用低温电镜技术测定bcs1的结构，bcs1是一种atp驱动的蛋白质转位酶，可在生物膜上运输折叠蛋白。我们还通过x射线晶体学和冷冻电镜测定了线粒体和细菌复合物III单独或与各种呼吸小分子抑制剂的复合物的结构。在本研究中，我们获得了S蛋白的重组外结构域、S1亚基、RBD结构域、S2亚基和人ACE-2，其数量足以用于冷冻电镜研究。目的1：毕赤酵母蛋白的大规模表达和纯化。目前，我们已经获得了少量的纯化重组蛋白，足以进行低温电镜研究。具体来说，我们获得了S蛋白的外结构域、S1、S2和S蛋白的RBD片段。虽然大长度外三聚体和S2融合三聚体的数量足以进行低温电镜研究，但有些片段太小而无法进行电镜研究。相反，它们将被x射线晶体学研究，比如S1蛋白和RBD。为了进行x射线晶体学研究，必须大量生产这些小碎片。我的团队在大肠杆菌和毕赤酵母的重组蛋白表达方面有丰富的经验。如果先导化合物干扰S蛋白的蛋白水解启动，我们也计划纯化人TMPRSS2。目的2：利用电子显微镜或x射线晶体学测定先导化合物存在下sars - cov - 2s蛋白或其片段的结构，并阐明这些化合物的抑制机制。我的团队可以在NCI的CMM（分子显微镜中心）和NICE（美国国立卫生研究院校内冷冻电镜设备）使用配备Gatan K2 Summit直接探测器的Titan Krios显微镜。我们还确保了芝加哥阿贡国家实验室先进光子源的SERCAT x射线束流线。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，在制备电磁栅格之前，将清除任何沉淀物。我们将首先在屏幕显微镜下通过阴性染色和低温电镜检测样品的质量。当样品被认为适合高分辨率EM时，将在300千伏的Krios上收集数据。EM显微照片将使用Relion和/或CisTem进行处理。建模将使用Coot或Chimera进行，结构改进将使用Refmac或Phoenix进行。较小的S蛋白片段的结晶将用各种商用试剂盒在室内机器人进行。细化结晶命中条件，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。我们将基于结构信息对这些铅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其他直接使病毒颗粒上的S蛋白失能（“直接”杀病毒活性），(2)阻止病毒与宿主细胞受体ACE-2的相互作用，(3)干扰宿主蛋白酶启动，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论分析中系统地进行评估。目的3：通过结构分析来优化hit，开发针对不同冠状病毒，特别是SARS-CoV和SARS-CoV-2有效的先导抑制剂。基于已建立的基于结构的药物设计原则的先导候选药物优化和构效关系开发将在获得复杂结构后立即启动。在原子分辨率下先导化合物的结合环境将有助于确定(1)化学型中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，以消除这些特征以改善分子。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，这将降低灵活性的程度，并冻结可能再现结合抑制剂所需构象的构象。(3)我们将能够设计hit化合物的新衍生物，以最大限度地发挥新化合物的药物特性。

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.