To determinate the optimal remdesivir concentration for human dermal fibroblasts, upon confluency of the respective primary fibroblast cell culture from each participant, cells were incubated with 0.00, 0.05, 0.50 and 1.00 µM remdesivir (MedChemExpress, Germany) for 24 h. Cell viability was measured using the Trypan Blue Exclusion Test (Strober 2015 (link)).
Remdesivir
Manufactured by MedChemExpress
Sourced in United States, Germany
Remdesivir is a broad-spectrum antiviral medication developed by Gilead Sciences. It is a nucleotide analog that inhibits viral RNA-dependent RNA polymerase, an enzyme essential for viral replication. The product is used for research purposes only and its intended use may vary.
Automatically generated - may contain errors
Lab products found in correlation
Nirmatrelvir, by MedChemExpress (4 mentions)
Gs 441524, by MedChemExpress (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Chloroquine, by Merck Group (2 mentions)
Camostat, by Merck Group (2 mentions)
μclear black 96 well plate, by Greiner (2 mentions)
Sb412515, by Cayman Chemical (2 mentions)
Molnupinavir, by MedChemExpress (2 mentions)
Molnupiravir, by MedChemExpress (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Anti nucleocapsid antibody, by Sino Biological (1 mentions)
Doxycycline, by Merck Group (1 mentions)
Ivermectin, by Merck Group (1 mentions)
Cp100356, by Merck Group (1 mentions)
Ruxolitinib, by Cayman Chemical (1 mentions)
Innuprep rna mini kit 2, by Analytik Jena (1 mentions)
Human ifn β 1a, by PBL Assay Science (1 mentions)
Torin 1, by Thermo Fisher Scientific (1 mentions)
Cell proliferation kit, by Sartorius (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Nacalai Tesque (1 mentions)
36 protocols using remdesivir
1
Optimizing Remdesivir Concentration for Fibroblasts
2
SARS-CoV-2 Antiviral Assay in Tonsil Organoids
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Antiviral assays were performed according to our previous report with some modifications [60 ]. Briefly, 7-day–cultured tonsil organoids were infected with SARS-CoV-2 at an MOI of 0.1 and embedded in 20 μL of fresh TeM plus Matrigel (6 × 104 cells per well in 48-well plates). Remdesivir (purity 99.84%; MedChemExpress, Monmouth Junction, NJ, USA) was 10-fold serially diluted in TeM from 10 to 0.1 μM and used to treat SARS-CoV-2-infected organoids. For determining EC50 values, which are chemical concentrations to reduce viral RNA titers by 50%, three-fold serial dilutions of remdisivir (from 100 μM to 1.7 nM) were treated to the SARS-CoV-2-infected organoids from three different donors in triplicate. On day 2, the culture supernatants were harvested to measure the amount of viral RNA by quantitative RT-PCR. In parallel, cell lysates were collected from four wells using RIPA buffer (LPS solution, Daejeon, Korea), and viral protein expression levels were compared by Western blot analysis. The immuno-transferred membrane was probed with anti-spike primary antibody (GeneTex, Irvine, CA, USA) or anti-nucleocapsid antibody (Sino Biological, Beijing, China) followed by HRP–conjugated anti-mouse goat IgG as the secondary antibody (Invitrogen).
3
Antiviral Drug Preparation Protocol
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Remdesivir (HY-104077; MedChemExpress, Monmouth Junction, NJ, USA) was prepared in 100 mM and 10 mM stock solutions in sterile water. Chloroquine (C6628; Sigma-Aldrich, St. Louis, MO, USA) and ivermectin (CAS 70288-86-7; Merck, Burlington, MA, USA) were prepared in 10 mM stock solutions in dimethyl sulfoxide (DMSO). Doxycycline (CAS 24390-14-5; Merck, Burlington, MA, USA) was prepared in 10 mM stock solution in sterile water. For treatment experiments, all drug dilutions were prepared in 0.5% DMSO with the respective cell culture medium.
4
Synthesis and Characterization of Antiviral Compounds
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Ensitrelvir (Shionogi & Co. Ltd., Osaka, Japan) was synthesized as previously described.14 (link) Nirmatrelvir was also synthesized at Shionogi & Co., Ltd. Remdesivir and NHC, the active ingredients of the prodrug molnupiravir, were obtained from MedChemExpress (Monmouth Junction, NJ, USA). The permeability glycoprotein (P-gp) inhibitor CP-100356 was purchased from Sigma–Aldrich Co. Ltd.
5
SARS-CoV-2 Inhibition by IAV DIPs
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Confluent Calu-3 cells in 96-well plates (~6 × 104 cells/well) were infected with SARS-CoV-2 (2000 pfu per well). At 1 or 24 hpi, we added active or inactive IAV DIPs (DI244 or OP7) at indicated fractions (% v/v) with respect to the cell culture volume of 100 µL. Whenever indicated, we additionally added 0.8 µM ruxolitinib (Cayman Chemical (Ann Arbor, MI, USA), #Cay11609-1) to these wells. Alternatively, remdesivir (MedChem Express (Monmouth Junction, NJ, USAnited States), #HY-104077) or human IFN-β-1A (PBL assay science (Piscataway, NJ, USA), #11415-1) (instead of IAV DIPs) were added at indicated concentrations at 1 hpi. Supernatants were collected at indicated time points for quantification of SARS-CoV-2 titers (plaque assay) and for protein quantification of secreted IFNs using commercially available ELISA kits (see below). In addition, infected cells were lysed using solution RL for subsequent total RNA extraction using the innuPREP RNA Mini Kit 2.0 (Analytik Jena (Jena, Germany), #845-KS-2040050), according to the manufacturer’s instructions, for gene expression analysis via real-time RT-qPCR.
6
SARS-CoV-2 Cytopathic Effect Inhibition
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SARS-CoV-2 (GISAID accession EPI_ISL_406862) was kindly provided by Bundeswehr Institute of Microbiology, Munich, Germany. Virus stocks were propagated (four passages) and titered on Vero E6 cells (Vero E6, ATCC® CRL-1586™). Handling and working with SARS-CoV-2 virus were conducted in a BSL3 facility in accordance with the biosafety guidelines of the Israel Institute for Biological Research (IIBR). Vero E6 cells were seeded at a density of 3 × 104 cells per well in 96-well plates. After overnight incubation, cells were treated in three replicates with Torin-1 (Fisher Scientific) or Remdesivir (Medchemexpress) as indicated. Cells were infected 1 h later with SARS-CoV-2 (MOI, 0.01–0.015). Cell viability was determined 72 h after infection by using the Cell Proliferation Kit (XTT based, Biological Industries, Israel) according to manufacturer's protocol. For positive control, cells were treated with Remdesivir, for negative control cells were not treated prior to infection.
7
Cell Viability Assay for SARS-CoV-2 Inhibitors
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The MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) (Nacalai Tesque) assay was performed to evaluate cell viability following viral infection according to methods previously described [46 (link)]. Camostat, E-64d, nafamostat (FUJIFILM Wako Pure chemical) and remdesivir (MedChemExpress) were serially diluted 2-fold increments in duplicates and plated on 96-well microplates in MEM containing 2% FBS. Vero-TMPRSS2 were infected with either WT or S mutants of SARS-CoV-2 at 4–10 TCID50 and added to the plates. Plates were incubated at for 3 days, and CPE was determined for calculation of 50% endpoints using MTT assay. The concentration achieving 50% inhibition of cell viability (effective concentration; EC50) was calculated.
8
Bergamottin, Clofazimine, and Remdesivir Analysis
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Bergamottin (HY-N2194) was purchased from MedChemExpress and Weikeqi Biotech (wkq21050802), and it was dissolved in DMSO. The purity of bergamottin obtained from both companies was identified by HPLC as greater than 98%.
Clofazimine (HY-B1046) and Remdesivir (HY-104077) were purchased from MedChemExpress.
Clofazimine (HY-B1046) and Remdesivir (HY-104077) were purchased from MedChemExpress.
9
Evaluating antiviral compounds against SARS-CoV-2
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Cells were seeded in a μClear black 96-well plate (Greiner Bio-One) and pretreated for 2 hours with Camostat (Sigma, cat# E8640), E-64d (Sigma, cat# SML0057), SB412515 (Cayman Chemical, cat# 23249), Nirmatrelvir (MedChemExpress; cat# HY-138687), Remdesivir (MedChemExpress; cat# HY-104077), or Molnupinavir (MedChemExpress; cat# HY-135853) at concentrations as described in the figure legends. Cells were infected with the indicated SARS-CoV-2 strains. After 24h, infection was revealed as described above. The percentage of inhibition of infection was calculated using the area of N-positive cells as a value with the following formula: 100 × (1 − (value with drugs − value in ’non-infected’)/(value in ’no drugs’ − value in ’non-infected’)).
The monoclonal antibodies used in this study were previously described 36 ,69 . Neutralizing activity and ED50 were measured as described in the “S-Fuse neutralization assay ” section.
The monoclonal antibodies used in this study were previously described 36 ,69 . Neutralizing activity and ED50 were measured as described in the “
10
Neutralizing Antibody and Antiviral Efficacy against EBOV
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Neutralizing antibody c2G4 (provided by Yi Shi, Institute of Microbiology, Chinese Academy of Sciences) was serially diluted to the indicated concentrations and incubated with EBOV-trVLP for 1 h at 37°C and used to infect Huh7-4PX cells. The cells were fixed at 48 h postinfection, and VP40-positive foci were analyzed by immunofluorescence. Remdesivir (MedChemExpress) was serially diluted to the indicated concentrations and mixed with EBOV-trVLP to infect Huh7-4PX cells. The cell lysates were harvested at 72 h posttreatment for measuring RNA levels. IFN-α (Roche), IFN-γ (R&D Systems), and IFN-λ3 (R&D system) were serially diluted to the indicated concentrations and added to Huh7-4PX cells 12 h before EBOV-trVLP infection. The cell lysates were harvested at 72 h posttreatment for measuring RNA levels.
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