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Principal Investigator: MING LUO
Organization: UNIVERSITY OF MINNESOTA
Fiscal Year: 2022
Award: $8,177,132
Funding agency: National Institute of Allergy and Infectious Diseases
Project 1 – Small Molecule Entry Inhibitors of Pandemic Viruses
ABSTRACT
Coronaviruses (CoVs), arenaviruses (Arv), flaviviruses (FLAVs) and filoviruses (FiVs) are enveloped
viruses. During virus entry, receptor binding and refolding of the fusion protein, followed by lipid mixing, are three
essential steps to release the viral genome. Inhibitors of any one of the three steps may be developed as effective
antiviral drugs. Aim 1. Screen. (A) DEC-Tec screen using the purified ectodomain of the SARS2 S protein,
glycoprotein of Machupo virus (MACV) and envelope (E) protein of Zika virus (ZIKV). (B) HTS will be carried out
using fluorescence assays by targeting the six-helix bundle (6HB) of SARS2 and by competition with compound
ALD-1.2 that binds ZIKV E protein. (C) Virtual screens by AutoDock or other computation methods will also be
conducted since 3D structures of all target proteins are available. Aim 2. Optimization. Mechanism of action by
hit compounds. (SARS2) Hit compounds from aim 1A will be validated by time of addition inhibition assays based
on cell culture infection of SARS2 pseudotype. Validated entry inhibitors will be screened for inhibition of receptor
binding by the S protein, and formation of 6HB. The receptor binding assay is by ELISA using an ACE2-Fc
protein. The 6HB assay is set up by binding a fluorescently labeled HR2 peptide to 5HB. Entry inhibitors of other
viruses will follow the same study approach. Inhibitors of lipid mixing. In preliminary efforts, we have identified
three inhibitors that have EC50 values as low as 190 nM for SARS2 infection of cell culture. Mechanistic studies
confirm that these inhibitors interact with the transmembrane domain of the fusion protein and block membrane
fusion during virus entry. The experimental design for optimization is presented. Ebola virus entry inhibitors.
These inhibitors are at an advanced stage and serve as a proof-of-concept example for our strategy. Med Chem
Optimization. Structure and QSAR-based optimization of the inhibitor compounds will be carried out in
collaboration with Cores C and D. Candidates that meet the criteria for further evaluation will be advanced to
DMPK/toxicity studies (Core C). Escape mutants. The state-of-the-art approach is developed to evaluate
mutants that escape the antiviral activities of the inhibitors, to aid inhibitor optimization. Aim 3. In vivo efficacy.
For SARS2, lead candidates, especially prodrugs, will be evaluated for broad antiviral activities against multiple
SARS2 and SARS isolates. Potent candidates will be evaluated in hamster and mouse models. For Arv, Stat1-/-
and Ifnar1/Ifnar2 double knockout mice will be used as infection models. For Ebola virus, we have identified a
series of small molecule inhibitors targeting the Ebola GP with a novel mechanism. We will evaluate and adapt
these for the broad-spectrum activity against other significant filoviruses and evaluate their in vivo efficacy in the
animal model operating at ABSL4. Top ZIKV inhibitors will be tested in animal models against multiple ZIKV
infections. All technical work is carried out in Core E.
Terms: <2019 novel corona virus><2019 novel coronavirus><2019-nCoV><2019-nCoV S protein><2019-nCoV spike glycoprotein><2019-nCoV spike protein><3-D structure><3-dimensional structure><3D structure><ACE2><Animal Model><Animal Models and Related Studies><Antiviral Agents><Antiviral Drugs><Antivirals><Arenaviridae><Arenavirus><Arenavirus group><Assay><Binding><Binding Proteins><Bioassay><Bioavailability><Biologic Assays><Biologic Availability><Biological Assay><Biological Availability><Bolivian Hemorrhagic Fever Virus><COVID-19 S protein><COVID-19 infection><COVID-19 spike glycoprotein><COVID-19 spike protein><COVID-19 therapy><COVID-19 treatment><COVID-19 virus><COVID19 S protein><COVID19 infection><COVID19 spike glycoprotein><COVID19 spike protein><COVID19 therapy><COVID19 treatment><COVID19 virus><Cell Body><Cell Culture Techniques><Cell Surface Glycoproteins><Cell membrane><Cells><Chimera Protein><Chimeric Proteins><CoV-2><CoV2><Collaborations><Computing Methodologies><Coronaviridae><Coronavirus><Cricetinae><Cytoplasmic Membrane><Data Set><Dataset><Drug Precursors><EBOV><ELISA><Ebola><Ebola virus><Ebola-like Viruses><Endocytosis><Envelope Protein><Enzyme-Linked Immunosorbent Assay><Escape Mutant><Evaluation><Experimental Designs><Filoviridae><Filovirus><Flavivirus><Fluorescence><Fusion Protein><Genome><Glycoproteins><Group B Arbovirus><Hamsters><Hamsters Mammals><In Vitro><Infection><KO mice><Knock-out Mice><Knockout Mice><Label><Libraries><Ligand Binding Protein><Ligand Binding Protein Gene><Lipids><Lung><Lung Respiratory System><Lung infections><Machupo><Machupo virus><Mediating><Membrane><Membrane Fusion><Membrane Glycoproteins><Methods><Mice><Mice Mammals><Midwest><Midwest U.S.><Midwest US><Midwestern United States><Modeling><Molecular Configuration><Molecular Conformation><Molecular Interaction><Molecular Stereochemistry><Murine><Mus><Null Mouse><Oral><Outcome><Peptides><Physiologic Availability><Plasma Membrane><Pro-Drugs><Prodrugs><Protein Binding><Proteins><QSAR><Quantitative Structure-Activity Relationship><Quantitiative Structure Activity Relationship><Receptor Cell><Receptor Inhibition><Receptor Protein><SARS><SARS corona virus 2><SARS coronavirus disease><SARS-CO-V2><SARS-COVID-2><SARS-CoV disease><SARS-CoV-2><SARS-CoV-2 S protein><SARS-CoV-2 infection><SARS-CoV-2 spike glycoprotein><SARS-CoV-2 spike protein><SARS-CoV-2 therapy><SARS-CoV-2 treatment><SARS-CoV2><SARS-CoV2 S protein><SARS-CoV2 infection><SARS-CoV2 spike glycoprotein><SARS-CoV2 spike protein><SARS-associated corona virus 2><SARS-associated coronavirus 2><SARS-coronavirus-2><SARS-related corona virus 2><SARS-related coronavirus 2><SARSCoV2><Series><Severe Acute Respiratory Coronavirus 2><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><Severe Acute Respiratory Syndrome CoV 2><Severe Acute Respiratory Syndrome CoV disease><Severe Acute Respiratory Syndrome coronavirus disease><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 coronavirus 2 S protein><Severe acute respiratory syndrome coronavirus 2 infection><Severe acute respiratory syndrome coronavirus 2 spike glycoprotein><Severe acute respiratory syndrome coronavirus 2 spike protein><Severe acute respiratory syndrome related corona virus 2><Structure><Surface Glycoproteins><TM Domain><Testing><Time><Toxic effect><Toxicities><Transmembrane Domain><Transmembrane Region><Update><Variant><Variation><Viral><Viral Gene Products><Viral Gene Proteins><Viral Genome><Viral Proteins><Virus><Virus Inhibitors><Virus Replication><Work><Wuhan coronavirus><ZIKV><ZIKV infection><ZIKV positive><Zika Virus><Zika virus infection><analog><angiotensin converting enzyme 2><angiotensin converting enzyme II><anti-viral agents><anti-viral compound><anti-viral drugs><anti-viral medication><anti-viral therapeutic><anti-virals><antiviral compound><antiviral medication><antiviral therapeutic><base><bound protein><cell culture><cell cultures><computational methodology><computational methods><computer based method><computer methods><computing method><conformation><conformational state><corona virus><coronavirus disease 2019 S protein><coronavirus disease 2019 infection><coronavirus disease 2019 spike glycoprotein><coronavirus disease 2019 spike protein><coronavirus disease 2019 therapy><coronavirus disease 2019 treatment><coronavirus disease 2019 virus><coronavirus disease-19 virus><design><designing><dimer><ebolavirus><env Antigens><env Gene Products><env Polyproteins><env Protein><enzyme linked immunoassay><hCoV19><in vivo><in vivo Model><indexing><infected with COVID-19><infected with COVID19><infected with SARS-CoV-2><infected with SARS-CoV2><infected with coronavirus disease 2019><infected with severe acute respiratory syndrome coronavirus 2><inhibitor><lead candidate><lead series><membrane structure><model of animal><model organism><mouse model><murine model><nCoV2><novel><pandemic><pandemic disease><plasmalemma><pulmonary><pulmonary infections><receptor><receptor binding><receptor bound><respiratory><severe acute respiratory syndrome coronavirus 2 therapy><severe acute respiratory syndrome coronavirus 2 treatment><small molecule><small molecule inhibitor><stem><three dimensional structure><treat COVID-19><treat COVID19><treat SARS-CoV-2><treat coronavirus disease 2019><treat severe acute respiratory syndrome coronavirus 2><viral inhibitor><viral multiplication><viral replication><virtual><virus envelope><virus genome><virus multiplication><virus protein><zika infection><zika viral infection><zikav>