Journal: bioRxiv

Article Title: Antibody-mediated cell entry of SARS-CoV-2

doi: 10.1101/2023.02.20.529249

Figure Lengend Snippet: ( A ) Binding site locations of selected monoclonal antibodies and ACE2 binding site. Surface regions of the SARS-CoV-2 spike trimer in a top view targeted by eight selected antibodies on the RBD and NTD are highlighted by ellipses. Various domains of one protomer are colored (RBM in magenta, the rest of RBD in red and NTD in blue); the other two protomers in white and gray, respectively. The ACE2 binding site is marked with a yellow dashed line. ( B ) Schematic representation of antibody IgG (heavy and light chains) and FcγRI (α and γ chains) constructs, as well as a diagram for how FcγRI captures an IgG antibody on the surface of membrane based on the crystal structure PDB ID: 4W4O . ( C ) HEK293T cells with or without expressing FcγRI were decorated with eight selected monoclonal antibodies and tested for membrane fusion with the full-length G614 S protein expressing cells in our standard cell-cell fusion assay. Cell-cell fusion led to reconstitution of α and μ fragments of β-galactosidase yielding an active enzyme and thus the fusion activity was quantified by a chemiluminescent assay. Cells only and cells expressing FcγRI with no antibody added were negative controls. ( D ) The cell-cell fusion assay was used to analyze seven polyclonal IgG antibodies purified from serum samples of vaccinated convalescent individuals reported previously .

Article Snippet: The SARS-CoV-2 G614 seed virus (the clinical isolate New York-PV09158/2020; ATCC #NR-53516) was obtained through BEI Resources (Manassas, VA) and amplified in TMPRSS2-E6 cells.

Techniques: Binding Assay, Bioprocessing, Construct, Membrane, Expressing, Cell-Cell Fusion Assay, Activity Assay, Purification

Journal: bioRxiv

Article Title: Antibody-mediated cell entry of SARS-CoV-2

doi: 10.1101/2023.02.20.529249

Figure Lengend Snippet: ( A ) Schematic representation of the full-length human ACE2 and design of antibody-based expression constructs. Various segments for ACE2 include: catalytic peptidase domain, neck domain; TM, transmembrane anchor; and CT, cytoplasmic tail. Expression constructs of antibody-ACE2 chimera, the Fab heavy chain of an antibody is fused with the neck domain, TM and CT of ACE2, coexpressed with the Fab light chain. A diagram showing how Fab is presented on the cell surfaces by the ACE2 TM anchor. ( B ) HEK293T cells transfected with eight different antibody-ACE2 chimeric constructs were tested for membrane fusion with the full-length S protein (G614 or Omicron subvariant BA.2) expressing cells in the β-galactosidase-based cell-cell fusion assay. The wildtype ACE2 was a positive control; no receptor/no S a negative control. ( C ) Infection of HEK293T cells transfected with either ACE2 or various antibody-ACE2 chimeric constructs by HIV-based pseudotyped viruses using the full-length G614 and BA.1 S constructs in a single cycle. Empty vector was used as a negative control. ( D ) Virus foci in MDCK cells transfected with either ACE2 or various antibody-ACE2 chimeric constructs followed by infection of the authentic SARS-CoV-2 G614 isolate. Empty vector was used as a negative control.

Article Snippet: The SARS-CoV-2 G614 seed virus (the clinical isolate New York-PV09158/2020; ATCC #NR-53516) was obtained through BEI Resources (Manassas, VA) and amplified in TMPRSS2-E6 cells.

Techniques: Expressing, Construct, Transfection, Membrane, Cell-Cell Fusion Assay, Positive Control, Negative Control, Infection, Plasmid Preparation, Virus

Journal: bioRxiv

Article Title: Antibody-mediated cell entry of SARS-CoV-2

doi: 10.1101/2023.02.20.529249

Figure Lengend Snippet: ( A ) Schematic representation of the membrane-bound B cell receptor (BCR) expression constructs and a diagram showing how Fab is presented on the cell surfaces by the BCR TM anchor. ( B ) HEK293T cells transfected with eight different membrane-bound BCR constructs were tested for membrane fusion with the full-length S protein (G614 or Omicron subvariant BA.2) expressing cells in the β-galactosidase-based cell-cell fusion assay. The wildtype ACE2 was a positive control and no receptor/no S a negative control. ( C ) Infection of HEK293T cells transfected with either ACE2 or various BCR constructs by HIV-based pseudotyped viruses using the full-length G614 and Omicron BA.1 S constructs in a single cycle. Empty vector was used as a negative control. ( D ) Virus foci in MDCK cells transfected with either ACE2 or various antibody-ACE2 chimeric constructs followed by infection of the authentic SARS-CoV-2 G614 isolate. Empty vector was used as a negative control.

Article Snippet: The SARS-CoV-2 G614 seed virus (the clinical isolate New York-PV09158/2020; ATCC #NR-53516) was obtained through BEI Resources (Manassas, VA) and amplified in TMPRSS2-E6 cells.

Techniques: Membrane, Expressing, Construct, Transfection, Cell-Cell Fusion Assay, Positive Control, Negative Control, Infection, Plasmid Preparation, Virus

Journal: bioRxiv

Article Title: Antibody-mediated cell entry of SARS-CoV-2

doi: 10.1101/2023.02.20.529249

Figure Lengend Snippet: ( A ) and ( B ) Inhibition of viral infectivity by protease inhibitors. Pseudovirus (G614 S) infection of HEK293T cells transfected with ACE2 or antibody constructs with/without TMPRSS2 were treated with either E-64d (a cathepsin L inhibitor) or Camostat (TMPRSS2 inhibitor). DMSO, organic solvent used to dissolve E-64d. ( C - E ) Antibody neutralization of pseudoviruses containing the G614 S protein was determined using IgG antibodies, C63C8, SP1-77, S2H97 and C63C7 in red; G32B6 and C12A2 in magenta; and C12C9 in blue.

Article Snippet: The SARS-CoV-2 G614 seed virus (the clinical isolate New York-PV09158/2020; ATCC #NR-53516) was obtained through BEI Resources (Manassas, VA) and amplified in TMPRSS2-E6 cells.

Techniques: Inhibition, Infection, Transfection, Construct, Solvent, Neutralization

Journal: Frontiers in Molecular Biosciences

Article Title: Heparan Sulfate Facilitates Spike Protein-Mediated SARS-CoV-2 Host Cell Invasion and Contributes to Increased Infection of SARS-CoV-2 G614 Mutant and in Lung Cancer

doi: 10.3389/fmolb.2021.649575

Figure Lengend Snippet: Cell surface SV2-S binding depends on and correlates with expression levels of HS. (A,B) Cell surface expressions of ACE2 and HS were assessed by flow cytometry after incubation with anti-ACE2 and anti-HS antibody 10E4, respectively, and sequential Alexa Fluor 488-tagged secondary antibody. A portion of the cells was treated with heparinases I-III (HSase) before the antibody staining. (C) The binding of SV2-S on cell surface was assessed by flow cytometry after sequential incubation with his-tagged SV2-S and Alexa Fluor 488-tagged anti-his antibody. A portion of the cells was treated with HSase before SV2-S binding. Median fluorescence intensity (MFI) was calculated by subtracting MFI of BSA binding or isotype-match naive control antibody binding. The experiments were repeated 3–6 times and an unpaired t -test was performed for comparison of the same cells without vs. with HSase treatment. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (D–G) The Pearson’s correlation coefficient between SV2-S binding and cell surface expression level of HS or ACE2 was determined by analyzing the data collected in (A–C) using Prism 8. The cells analyzed in (F,G) were pre-treated with HSase.

Article Snippet: For direct binding analysis, SV2-S D614 (Sino Biological, 40591-V08H) and SV2-S G614 (Sino Biological, 40591-V08H3) were diluted in a running HBS-EP buffer containing 0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20, at pH 7.4.

Techniques: Binding Assay, Expressing, Flow Cytometry, Incubation, Staining, Fluorescence

Journal: Frontiers in Molecular Biosciences

Article Title: Heparan Sulfate Facilitates Spike Protein-Mediated SARS-CoV-2 Host Cell Invasion and Contributes to Increased Infection of SARS-CoV-2 G614 Mutant and in Lung Cancer

doi: 10.3389/fmolb.2021.649575

Figure Lengend Snippet: Heparan sulfate (HS) plays a major role in mediating cell surface SV2-S protein binding only when ACE2 expresses at a low level. 293T cells were transfected with ACE2-GFP or scramble-GFP plasmid. In SV2-S binding analysis, the transient 293T-ACE2-GFP-expressing cells were gated into two populations: the ACE2-GFP-low and -high expression cells based on GFP expression. (A) Cell surface ACE2 expression was assessed by flow cytometry after staining with anti-ACE2 antibody and Alexa Fluor 647-conjugated secondary antibody. A portion of the cells was treated with HSase before the specific antibody staining. (B) The binding of SV2-S on cell surface was assessed by flow cytometry after sequential incubation with his-tagged SV2-S and Alexa Fluor 647-tagged anti-his antibody. A portion of the cells was treated with HSase before SV2-S binding. (C) Cell surface HS expression was assessed by flow cytometry after staining with anti-HS antibody and Alexa Fluor 647-conjugated secondary antibody. A portion of the cells was treated with HSase before the specific antibody staining. MFI was calculated by subtracting MFI of BSA binding or isotype-match naive control antibody staining. The experiments were repeated 6- to 12- times and an unpaired t -test was performed for comparison of the same cells without vs. with HSase treatment. Data are presented as mean ± SD. ns, not significant; * p < 0.05, *** p < 0.001, **** p < 0.0001.

Article Snippet: For direct binding analysis, SV2-S D614 (Sino Biological, 40591-V08H) and SV2-S G614 (Sino Biological, 40591-V08H3) were diluted in a running HBS-EP buffer containing 0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20, at pH 7.4.

Techniques: Protein Binding, Transfection, Plasmid Preparation, Binding Assay, Expressing, Flow Cytometry, Staining, Incubation

Journal: Frontiers in Molecular Biosciences

Article Title: Heparan Sulfate Facilitates Spike Protein-Mediated SARS-CoV-2 Host Cell Invasion and Contributes to Increased Infection of SARS-CoV-2 G614 Mutant and in Lung Cancer

doi: 10.3389/fmolb.2021.649575

Figure Lengend Snippet: The modification of heparin and cell surface HS involved in SV2-S binding. (A,B) Inhibition on the binding of SV2-S to cultured A549 cells. After incubation in the absence or presence of different concentrations of heparin (HP), NDS-HP; 2DS-HP, 6DS-HP, or OS-HP, SV2-S that bound to A549 cells was quantified by cell-based ELISA and normalized to the cell density. The SV2-S binding was further normalized to the wells without inhibitors. The experiments were repeated at least three times. Data are presented as mean ± SEM. (C) SV2-S binding on MLEC surface. The tested HS mutant MLEC lines include wildtype (WT) control, Hs2st1 − /− (lack of 2S), Hs6st1 − /− (reduced 6S), Hs6st2 − /− (normal 6S), Hs6st1 − /− ;2 − /− (no 6S), Sulf1 − /− ;2 − /− (increased 6S), Hs3st1 − /− (reduced 3S), Hs3st4 − /− (reduced 3S), and Hs3st1 − /− ;4 − /− (greatly reduced 3S). Cell surface SV2-S protein binding was assessed by cell-based ELISA after incubation of fixed cells with his-tagged SV2-S and normalization to the cell density. The experiments were repeated 6- to 12- times and the unpaired t-test was performed for comparison between the wildtype and the mutants. Data are presented as mean ± SD. Ns, not significant; * p < 0.05, ** p < 0.01.

Article Snippet: For direct binding analysis, SV2-S D614 (Sino Biological, 40591-V08H) and SV2-S G614 (Sino Biological, 40591-V08H3) were diluted in a running HBS-EP buffer containing 0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20, at pH 7.4.

Techniques: Modification, Binding Assay, Inhibition, Cell Culture, Incubation, In-Cell ELISA, Mutagenesis, Protein Binding

Journal: Frontiers in Molecular Biosciences

Article Title: Heparan Sulfate Facilitates Spike Protein-Mediated SARS-CoV-2 Host Cell Invasion and Contributes to Increased Infection of SARS-CoV-2 G614 Mutant and in Lung Cancer

doi: 10.3389/fmolb.2021.649575

Figure Lengend Snippet: Heparinases, surfen, heparin, and chemically modified non-anticoagulant heparins block SV2-S pseudotyped virus infection of 293T-ACE2 cells. The 293T-ACE2 cells were infected with SV2-S pseudotyped virus expressing luciferase after the cells were treated with HSase (5 mU/ml) (A) or in the presence of surfen (20 μM) (B) , heparin (HP, C ), HS or chemically-modified heparins at 2- and 20 μM (D,E) . The luciferase activity was measured 48 h after the virus infection. VSV pseudotyped virus was used as a control to make sure each well has comparable cell numbers (data are not shown). The experiments were repeated at least 3 times and an unpaired t -test was performed for the two-group comparison. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. RLU, relative light unit.

Article Snippet: For direct binding analysis, SV2-S D614 (Sino Biological, 40591-V08H) and SV2-S G614 (Sino Biological, 40591-V08H3) were diluted in a running HBS-EP buffer containing 0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20, at pH 7.4.

Techniques: Modification, Blocking Assay, Infection, Expressing, Luciferase, Activity Assay

Journal: Frontiers in Molecular Biosciences

Article Title: Heparan Sulfate Facilitates Spike Protein-Mediated SARS-CoV-2 Host Cell Invasion and Contributes to Increased Infection of SARS-CoV-2 G614 Mutant and in Lung Cancer

doi: 10.3389/fmolb.2021.649575

Figure Lengend Snippet: Summary of kinetic data of wild type spike and mutant spike protein-heparin interactions*.

Article Snippet: For direct binding analysis, SV2-S D614 (Sino Biological, 40591-V08H) and SV2-S G614 (Sino Biological, 40591-V08H3) were diluted in a running HBS-EP buffer containing 0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20, at pH 7.4.

Techniques: Mutagenesis

Journal: Frontiers in Molecular Biosciences

Article Title: Heparan Sulfate Facilitates Spike Protein-Mediated SARS-CoV-2 Host Cell Invasion and Contributes to Increased Infection of SARS-CoV-2 G614 Mutant and in Lung Cancer

doi: 10.3389/fmolb.2021.649575

Figure Lengend Snippet: The SV2-S variant G614 protein has a higher binding affinity to heparin than the wildtype SV2-S D614 protein, and the entry of SAS-Cov-2 G614 pseudotyped virus into host cells depends on cell surface HS and can be inhibited by heparin. (A,B) SPR binding kinetics sensorgrams between heparin and SV2-S D614 or SV2-S G614. Concentrations of D614 were (from top to bottom): 1,000, 800, 600, 400, and 200 nM, respectively (A) , and G614 were (from top to bottom): 1,000, 600, 400, 200, and 100 nM, respectively (B) . The black curves are the fitting curves using a 1:1 Langmuir model from BIAevaluate 4.0.1. (C) Infection of 293T-ACE2 cells by the same titer of SARS-Cov2-D614 and SARS-Cov2-G614 pseudovirus. (D) SARS-Cov2-G614 pseudovirus depends on HS to infect host cells. The 293T-ACE2 cells were infected with SARS-Cov2-G614 pseudovirus expressing luciferase after the cells were treated with heparinases I-III (5 mU/ml) or in the presence of heparin (2 μM). The luciferase activity was measured 48 h after the virus infection. VSV pseudotyped virus was used as a control to make sure each well has comparable cell numbers (data are not shown). The experiments were repeated at least 3 times and an unpaired t-test was performed for two-group comparison. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01.

Article Snippet: For direct binding analysis, SV2-S D614 (Sino Biological, 40591-V08H) and SV2-S G614 (Sino Biological, 40591-V08H3) were diluted in a running HBS-EP buffer containing 0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20, at pH 7.4.

Techniques: Variant Assay, Binding Assay, Infection, Expressing, Luciferase, Activity Assay

Journal: bioRxiv

Article Title: Identification of novel antiviral drug candidates using an optimized SARS-CoV-2 phenotypic screening platform

doi: 10.1101/2022.07.17.500346

Figure Lengend Snippet: A) Percentage of SARS-CoV-2-infected cells detected in Caco-2 cell lines from different sources infected with different SARS-CoV-2 isolates at a multiplicity of infection (MOI) 0.01 as determined by immunostaining for the viral spike (S protein) 48h post infection. B) Cytopathogenic effect (CPE) formation in SARS-CoV-2 (MOI 0.01)-infected Caco-2 cell lines from different sources as determined 48h post infection. C) Susceptibility of Caco-2-F03 cells to a broad range of SARS-CoV-2 isolates after different times of cultivation. Cells had been frozen at passage 14 and were now resuscitated and cultivated for a further 30 passages. SARS-CoV-2 susceptibility was determined by immunostaining for S 48h after SARS-CoV-2 (MOI 0.01) infection 3 and 30 passages post resuscitation. D) ACE2 and TMPRSS2 levels in Caco-2-F03, Caco-2A, and single-cell derived clones from Caco-2A. E) Susceptibility of Caco-2A clones to selected SARS-CoV-2 isolates as indicated by immunostaining for S and CPE formation in SARS-CoV-2 (MOI 0.01)-infected cells 48h post-infection. F) Correlation of S staining and CPE formation with cellular ACE2 levels.

Article Snippet: To establish a drug-resistant strain, SARS-CoV-2 strain FFM3 (G614) was passaged in the presence of increasing remdesivir concentrations starting with 0.5 µM (the IC50 concentration) until it could be cultivated in the presence of remdesivir 2µM (FFM3 r REM).

Techniques: Infection, Immunostaining, Derivative Assay, Clone Assay, Staining

Journal: bioRxiv

Article Title: Identification of novel antiviral drug candidates using an optimized SARS-CoV-2 phenotypic screening platform

doi: 10.1101/2022.07.17.500346

Figure Lengend Snippet: A) Caspase 3/7, caspase 8, and caspase 9 activity in Caco-2-F03 cells infected with a range of different SARS-CoV-2 isolates (MOI 0.01), as determined by Caspase-Glo assay assay (Promega) 48h post infection. Higher signal-to-basal (S/B) ratios and Z’ scores indicate higher assay robustness. B) Caspase 3/7 activity as determined by Caspase-Glo assay, C) SARS-CoV-2 Spike (S) protein staining, and D) virus titers as indicated by genomic RNA copy numbers determined by qPCR in Caco-2-F03 cells infected with a wide range of uncharacterized SARS-CoV-2 isolates (MOI 0.01) 48h post infection. E) Representative images indicating CPE formation in G614 (MOI 0.01)-infected Caco-2-F03 and Vero cells 48h post infection as indicated by phase contrast microscopy and immunofluoresce staining for the viral S protein in combination with DAPI-stained nuclei. F) Quantification of cellular S protein levels in Caco-2-F03 cells infected with G614 (MOI 0.01) 48h post infection by immunostaining. G) Only caspase 3/7 activity but not viability assays (MTT, CellTiter-Glo measuring ATP production) reflects G614 (MOI 0.01) replication 48h post infection in Vero cells, which do not display a virus-induced CPE. G614 (MOI 0.01)-infected Caco-2-F03 cells served as a control that displays a CPE. P values were calculated by one-way ANOVA. H) Caspase 3/7 activity in Caco-2-F03 cells infected with MERS-CoV, SARS-CoV, and HCoV-229E (MOI 0.01) as determined 48h post infection including S/B ratios and Z’ scores.

Article Snippet: To establish a drug-resistant strain, SARS-CoV-2 strain FFM3 (G614) was passaged in the presence of increasing remdesivir concentrations starting with 0.5 µM (the IC50 concentration) until it could be cultivated in the presence of remdesivir 2µM (FFM3 r REM).

Techniques: Activity Assay, Infection, Caspase-Glo Assay, Staining, Microscopy, Immunostaining

Journal: bioRxiv

Article Title: Identification of novel antiviral drug candidates using an optimized SARS-CoV-2 phenotypic screening platform

doi: 10.1101/2022.07.17.500346

Figure Lengend Snippet: A) Dose-response curves and concentrations that inhibit virus infection by 50% (IC50) of antiviral agents as determined by caspase 3/7 activity and immunostaining for the coronavirus S protein in G614 (MOI 0.01)-infected Caco-2-F03 cells 24h post infection. B) Effects of the approved anti-SARS-CoV-2 drug remdesivir on cellular levels of the viral NP protein in G614 (MOI 1)-infected air liquid interface (ALI) cultures of primary human bronchial epithelial (HBE) cells 120h post infection. C) Effects of remdesivir on caspase 3/7 activity and virus titers (genomic RNA copy numbers determined by PCR) in G614 (MOI 1)-infected ALI HBE cultures 120h post infection. D) Effects of remdesivir on caspase 3/7 activity and virus titers in G614 (MOI 1)-infected primary human cardiomyocytes (CMS) 48h post infection. E) Correlation of the neutralization capacity of sera derived from seven donors two weeks after their second dose of the mRNA-1273 vaccine determined by caspase 3/7 activity or cytopathogenic effect (CPE) scoring in D614, Alpha and Delta-infected Caco-2-F03 cells 48h post infection. F) Determination of neutralization titers by caspase 3/7 activity or CPE scoring using sera derived from seven donors two weeks after their second dose of the mRNA-1273 vaccine in Caco-2-F03 cells infected with D614, Alpha, and Delta isolates 72h post infection. P values were calculated using paired t-test.

Article Snippet: To establish a drug-resistant strain, SARS-CoV-2 strain FFM3 (G614) was passaged in the presence of increasing remdesivir concentrations starting with 0.5 µM (the IC50 concentration) until it could be cultivated in the presence of remdesivir 2µM (FFM3 r REM).

Techniques: Infection, Activity Assay, Immunostaining, Neutralization, Derivative Assay

Journal: bioRxiv

Article Title: Identification of novel antiviral drug candidates using an optimized SARS-CoV-2 phenotypic screening platform

doi: 10.1101/2022.07.17.500346

Figure Lengend Snippet: A) Drug dose response curves and concentrations that reduce cellular levels of the SARS-CoV-2 S protein by 50% (IC50) in Caco-2-F03 and Calu-3 cells as determined by immunostaining 24h (Caco-2-F03) or 48h (Calu-3) post infection with the SARS-CoV-2 strain FFM3 or its remdesivir-adapted substrain FFM3rREM at MOI 0.01. B) Drug concentrations that reduce caspase 3/7 activity in FFM3 and FFM3rREM (MOI 0.01)-infected Caco-2-F03 cells 48h post infection. C) Sequence variants in FFM3rREM compared to FFM3. D) The polymerase complex with nsp7 and nsp8 and a template-primer RNA (cyan and deep teal) and remdesivir (magenta) bound. Gly671Ser is shown in red (as serine). E) The SARS-CoV-2 polymerase Gly671Ser sequence variant. Residue 671 is shown in red as serine, which would be able to form a hydrogen bond with Thr402 which would not be present as Gly671. All p values were calculated by two-way ANOVA.

Article Snippet: To establish a drug-resistant strain, SARS-CoV-2 strain FFM3 (G614) was passaged in the presence of increasing remdesivir concentrations starting with 0.5 µM (the IC50 concentration) until it could be cultivated in the presence of remdesivir 2µM (FFM3 r REM).

Techniques: Immunostaining, Infection, Activity Assay, Sequencing, Variant Assay

Journal: bioRxiv

Article Title: Identification of novel antiviral drug candidates using an optimized SARS-CoV-2 phenotypic screening platform

doi: 10.1101/2022.07.17.500346

Figure Lengend Snippet: A) Overview of the proof-of-concept screen for anti-SARS-CoV-2 compounds using the Kinase inhibitor library L-1200 (Selleckchem, Germany) containing 1796 compounds (Selleckchem, Germany) in Delta (MOI 0.01)-infected Caco-2-F03 cells using caspase 3/7 activity as read-out 48h post infection. For the screen, every compound was tested at a concentration of 10 and 1 µM. 21 selected hits were then confirmed by determining drug-response curves. B) Quality controls, Z’scores served as quality controls (left). Only plates with a Źscore ≥ 0.5 were further analyzed. Remdesivir (10 µM) was used as positive control on each plate and produced consistent results (right). C) Number of hits at different inhibition cut-offs. D) Visualization of the distribution of hits according to their targets. Targets for which inhibitors were selected for confirmation are indicated. E) Heatmaps of the anti-SARS-CoV-2 activity of 21 hits by the determination of dose-response in Delta and Omicron (MOI 0.01)-infected Caco-2-F03 cells using immunostaining for the viral S protein as read-out 24h post infection.

Article Snippet: To establish a drug-resistant strain, SARS-CoV-2 strain FFM3 (G614) was passaged in the presence of increasing remdesivir concentrations starting with 0.5 µM (the IC50 concentration) until it could be cultivated in the presence of remdesivir 2µM (FFM3 r REM).

Techniques: Infection, Activity Assay, Concentration Assay, Positive Control, Produced, Inhibition, Immunostaining

Journal: bioRxiv

Article Title: Identification of novel antiviral drug candidates using an optimized SARS-CoV-2 phenotypic screening platform

doi: 10.1101/2022.07.17.500346

Figure Lengend Snippet: A) Scheme of the testing of NCT-503 for anti-SARS-CoV-2 activity in air liquid interface (ALI) cultures of primary human bronchial epithelial (HBE) cells. Effect of NCT-503 on (B) caspase 3/7 activity, (C) virus titers (determined as genomic RNA copy numbers by qPCR), (D) transepithelial electrical resistance (TEER), and (E) LDH release in ALI HBE cultures infected with Delta (MOI 1) 120h post infection. F) Anti-SARS-CoV-2 effects of NCT-503 in combination with 2-Deoxy-D-glucose (2DG). Illustration of how NCT-503 and 2DG can exert combined effects on a common metabolic pathway. G) Representative fluorescence images indicating the number of Delta and Omicron (MOI 0.01)-infected cells in NCT503 and/ or 2DG-treated Caco-2-F03 cultures 24h post infection. H) and I) Weighted combination indices (CIwt) determined by the method of Chou and Talalay indicating a strong synergism of NCT-503 and 2DG.

Article Snippet: To establish a drug-resistant strain, SARS-CoV-2 strain FFM3 (G614) was passaged in the presence of increasing remdesivir concentrations starting with 0.5 µM (the IC50 concentration) until it could be cultivated in the presence of remdesivir 2µM (FFM3 r REM).

Techniques: Activity Assay, Infection, Fluorescence