Mechanism of Inhibition of Entry Inhibitors against SARS-CoVs

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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.