Document text
Principal Investigator: di s xia
Organization: DIVISION OF BASIC SCIENCES - NCI
Fiscal Year: 2021
Award: $23,607
Funding agency: National Cancer Institute
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 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.
Terms: <2019 novel corona virus><2019 novel coronavirus><2019-nCoV><ACE2><AIDS Virus><Acquired Immune Deficiency Syndrome Virus><Acquired Immunodeficiency Syndrome Virus><Address><Animals><Antiviral Agents><Antiviral Drugs><Antiviral Therapy><Antivirals><Assay><Avian Influenza><Binding><Bioassay><Biologic Assays><Biological Assay><Bird Flu><COVID-19 virus><COVID19 virus><Cell Body><Cell Surface Glycoproteins><Cell membrane><Cells><Chicago><Chimera><Chimera organism><China><CoV-2><CoV2><Collection><Complex><Computer software><Coronaviridae><Coronavirus><Cryo-electron Microscopy><Cryoelectron Microscopy><Crystallization><Cytoplasmic Membrane><Data><Development><Disease Outbreaks><Drug Design><Drug Interactions><Drug Kinetics><Drug resistance><E coli><E. coli><EBOV><Ebola virus><Ebola-like Viruses><Electron Cryomicroscopy><Ensure><Environment><Epitheliasin Gene><Escherichia coli><Esteroproteases><Evaluation><FDA EUA><FDA Emergency Use Authorization><Food and Drug Administration EUA><Food and Drug Administration Emergency Use Authorization><Fowl Pest><Fowl Plague><Frankfurt-Marburg Syndrome Virus><Freezing><Funding><Future><Glycans><Grant><H5N1><H5N1 virus><HCoV><HIV><Hansenula><Human><Human Immunodeficiency Viruses><Illinois><In Vitro><Incubated><Individual><Infection><Influenza A><Influenza A Virus, H5N1 Subtype><Influenza A virus><Influenza Viruses Type A><Influenzavirus A><LAV-HTLV-III><Laboratories><Lead><Length><Luciferase Immunologic><Luciferases><Lung diseases><Lymphadenopathy-Associated Virus><MERS corona virus><MERS coronavirus><MERS virus><MERS-CoV><Mainland China><Marburg><Marburg virus><Marburg-like Viruses><Marburgvirus><Measures><Mediating><Membrane><Membrane Fusion><Membrane Glycoproteins><Membrane Protein Gene><Membrane Proteins><Membrane-Associated Proteins><Microscope><Microscopy><Middle East Respiratory Syndrome Corona Virus><Middle East Respiratory Syndrome Coronavirus><Middle East Respiratory Syndrome Virus><Middle East Respiratory Syndrome-CoV><Middle Eastern Respiratory Syndrome Corona virus><Middle Eastern Respiratory Syndrome Coronavirus><Middle Eastern Respiratory Syndrome Virus><Middle Eastern Respiratory Syndrome-CoV><Modeling><Modern Man><Molecular><Molecular Configuration><Molecular Conformation><Molecular Interaction><Molecular Stereochemistry><NIAID><NIH><National Institute of Allergy and Infectious Disease><National Institutes of Health><Negative Staining><Orthomyxovirus Type A><Outbreaks><PRSS10><Pb element><Peptidases><Peptide Hydrolases><Peptides><Pharmacokinetics><Photons><Pichia><Plasma Membrane><Polysaccharides><Precipitation><Preparation><Process><Protease Gene><Proteases><Protein Fragment><Proteinases><Proteins><Proteolytic Enzymes><Public Health><Pulmonary Diseases><Pulmonary Disorder><Receptor Cell><Recombinant Proteins><Recombinants><Reporter><Resolution><Respiratory Disease><Respiratory System Disease><Respiratory System Disorder><Robotics><Roentgen Rays><SARS Virus><SARS corona virus><SARS corona virus 2><SARS coronavirus><SARS-Associated Coronavirus><SARS-CoV><SARS-CoV-2><SARS-CoV2><SARS-Related Coronavirus><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><Sampling><Series><Severe Acute Respiratory Distress Syndrome CoV 2><Severe Acute Respiratory Distress Syndrome Corona Virus 2><Severe Acute Respiratory Distress Syndrome Coronavirus 2><Severe Acute Respiratory Syndrome CoV 2><Severe Acute Respiratory Syndrome Virus><Severe Acute Respiratory Syndrome corona virus><Severe Acute Respiratory Syndrome coronavirus><Severe Acute Respiratory Syndrome-associated coronavirus 2><Severe Acute Respiratory Syndrome-related coronavirus 2><Severe acute respiratory syndrome associated corona virus 2><Severe acute respiratory syndrome corona virus 2><Severe acute respiratory syndrome coronavirus 2><Severe acute respiratory syndrome related corona virus 2><Single Crystal Diffraction><Software><Source><Structure><Structure-Activity Relationship><Surface><Surface Glycoproteins><Surface Proteins><TMPRSS2><TMPRSS2 gene><Testing><Therapeutic><Titan><Type A Influenza><United States National Institutes of Health><Universities><Vaccine Design><Vaccines><Viral><Viral Diseases><Viral Gene Products><Viral Gene Proteins><Viral Proteins><Virion><Virus><Virus Diseases><Virus Particle><Virus-HIV><Work><Wuhan coronavirus><X Ray Crystallographies><X-Radiation><X-Ray Crystallography><X-Ray Diffraction Crystallography><X-Ray Radiation><X-Ray/Neutron Crystallography><X-ray><Xray><Xray Crystallography><Zoonoses><Zoonotic><Zoonotic Infection><angiotensin converting enzyme 2><angiotensin converting enzyme II><anti-viral agents><anti-viral drugs><anti-viral therapy><anti-virals><avian flu><avian influenza H5N1><avian influenza H5N1 virus><base><beamline><beta CoV><beta coronavirus><betaCoV><betacoronavirus><cell engineering><cellular engineering><chemical structure function><conference><conformation><conformational state><convention><corona virus><coronavirus disease 2019 virus><cryo-EM><cryoEM><design><designing><detector><developmental><disease of the lung><disorder of the lung><drug resistant><ebolavirus><emergency use authorization><experience><experiment><experimental research><experimental study><flexibility><flexible><hCoV19><heavy metal Pb><heavy metal lead><human CoV><human corona virus><human coronavirus><influenza in birds><inhibitor><inhibitor/antagonist><lead candidate><lung disorder><membrane structure><mortality><nCoV2><neutralizing antibody><new drug treatments><new drugs><new therapeutics><new therapy><next generation therapeutics><novel drug treatments><novel drugs><novel therapeutics><novel therapy><pandemic><pandemic disease><pathogenic virus><plasmalemma><prevent><preventing><protein expression><receptor binding><receptor bound><resistance to Drug><resistant to Drug><restraint><severe acute respiratory syndrome-CoV><structure function relationship><summit><symposia><symposium><viral infection><viral infectious disease treatment><viral pathogen><viricidal><virucidal><virucide><virus infection><virus pathogen><virus protein><virus-induced disease><zoonotic CoV><zoonotic coronavirus><β CoV><β coronavirus><βCoV>