Guava leaf, green tea leaf, and rose petal extracts were purchased from Daejong Biotech Co., Ltd. (Seoul, Korea). We then prepared BEN815 by mixing these extracts in a 65:30:5 ratio, respectively. The active compounds were quercetin, EGCG, ellagic acid, gallic acid, kaempferol, and myricetin, the content and efficacy of which were analyzed in a previous study [15 (link)]. The mixture was dissolved in 0.9% saline and stored at -20°C. Nirmatrelvir was obtained from MedChemExpress (Monmouth Junction, NJ, USA); a stock solution was prepared by dissolving Nirmatrelvir in 0.9% saline containing 1% dimethyl sulfoxide and 20% sulfobutylether-β-cyclodextrin (MedChemExpress), which was stored at -20°C.
Nirmatrelvir
Manufactured by MedChemExpress
Sourced in United States
Nirmatrelvir is a synthetic organic compound used as a laboratory tool for research purposes. It is a protease inhibitor that targets the main protease (Mpro) of SARS-CoV-2, the virus that causes COVID-19 disease. Nirmatrelvir is utilized in scientific research to study its potential therapeutic applications.
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Lab products found in correlation
Remdesivir, by MedChemExpress (4 mentions)
Molnupiravir, by MedChemExpress (3 mentions)
Sulfobutylether β cyclodextrin, by MedChemExpress (2 mentions)
μclear black 96 well plate, by Greiner (2 mentions)
Camostat, by Merck Group (2 mentions)
Sb412515, by Cayman Chemical (2 mentions)
Molnupinavir, by MedChemExpress (2 mentions)
Tween 80, by Merck Group (1 mentions)
Methylcellulose, by Merck Group (1 mentions)
Ritonavir, by Merck Group (1 mentions)
Eidd 1931, by MedChemExpress (1 mentions)
Ultima gold xr scintillation fluid, by PerkinElmer (1 mentions)
Eidd2801, by MedChemExpress (1 mentions)
3h uridine, by Moravek Biochemicals (1 mentions)
3h adenosine, by Moravek Biochemicals (1 mentions)
Nafamostat, by MedChemExpress (1 mentions)
Molnupiravir, by Merck Group (1 mentions)
Ritonavir, by MedChemExpress (1 mentions)
Gs 441524, by MedChemExpress (1 mentions)
Ruxolitinib, by InvivoGen (1 mentions)
12 protocols using nirmatrelvir
1
Antiviral Extract Preparation and Characterization
2
Nirmatrelvir and Ritonavir Treatment Protocol
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Experimental animals were administered vehicle alone [1% (w/v) Soluplus (BASF), 1% (w/v) Tween 80 (Sigma Aldrich), 0.5% (w/v) methylcellulose (Sigma Aldrich) in purified water], high dose nirmatrelvir alone (300mg/kg; MedChem Express), or an animal equivalent dose of nirmatrelvir boosted with ritonavir (1.7 mg nirmatrelvir/dose [MedChem Express], 0.6 mg ritonavir/dose [Sigma Aldrich]). Animal equivalent doses were calculated as described (66 (link)) by converting the standard human dose of nirmatrelvir and ritonavir (68 ) to a body-surface-area equivalent for mice (66 (link)) using a standardized body surface area for mice of 0.007 mg/m2, which is recommended for conversion of animal doses to human equivalent doses (87 ), along with an assumed mass of 30 g for all calculations so that pregnant and nonpregnant animals receive the same amount per dose. Mice were administered treatment via oral gavage twice daily for 5 days or until tissue collection, starting 4 hours after infection as described in the original published preclinical study of nirmatrelvir (64 (link)).
3
Antiviral Drug Formulation and Storage
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Nirmatrelvir and molnupiravir were purchased from MedChemExpress (Monmouth Junction, USA). Remdesivir was purchased from Ambeed (Illinois, USA). Stock solutions of all these antiviral drugs were prepared in 2% DMSO and 20% sulfobutylether-β-cyclodextrin (MedChemExpress) in 0.9% saline and stored at −20°C.
4
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 “
5
Formulation and Characterization of Antiviral Compounds
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GS-5245 was synthesized at Gilead Sciences, Inc., and their chemical composition and purity was quality controlled by nuclear magnetic resonance, high resolution mass spectrometry, and high-performance liquid chromatography. Molnupiravir was purchased from MedChemExpress (Monmouth Junction, NJ). Nirmatrelvir (PF-07321332) was purchased from MedChemExpress (Monmouth Junction, NJ) or WuXi AppTec (Shanghai, China). GS-5245 was solubilized in 2.5% dimethyl sulfoxide, 10% Kolliphor HS-15; 10% Labrasol; 2.5% propylene glycol; 75% water; pH 2-3 or 10% ethanol, 90% propylene glycol for mouse in vivo studies. Molnupiravir was solubilized in 2.5% Kolliphor RH 40, 10% polyethylene glycol, 87.5% water for mouse in vivo studies. PF-07321332 was formulated in 10% ethanol, 90% propylene glycol. Oral antiviral drugs were made available to UNC Chapel Hill under an existing material transfer agreement with Gilead Sciences, Inc.
6
Antiviral Compound Evaluation Protocol
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Ensitrelvir (S-217622) was kindly provided by Shionogi Co., Ltd. Active components of remdesivir (GS-441524), molnupiravir (EIDD-1931), and nirmatrelvir (PF-07321332) were purchased from MedChemExpress. Compounds were dissolved in dimethyl sulfoxide for in vitro experiments or 0.5% methyl cellulose for in vivo experiments prior to use.
7
Compound Screening and Radiolabeling Protocol
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EIDD-1931, EIDD2801, and Nirmatrelvir (PF-073213332) were purchased from MedChemExpress (Shanghai, China). Stock solutions of the compounds were freshly prepared in dimethyl sulfoxide (DMSO). Non-radiolabeled chemicals were obtained from Merck/Sigma-Aldrich (St. Louis, MO, USA). All chemicals were of analytical grade. 3H-Uridine ([5-3H], 20.9 Ci/mmol), and 3H-Adenosine ([2,8-3H], 29.3 Ci/mmol) were purchased from Moravek Biochemicals Inc. (Brea, CA, USA). Ultima Gold XR scintillation fluid was purchased from PerkinElmer (Waltham, MA, USA).
8
Antivirals against SARS-CoV-2 in A549-AT cells
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A549-AT cells (3.5 × 104/well, 96-well plate) were seeded in 96-well plates one day prior to infection. On the following day, cells were treated with Remdesivir, Nafamostat mesylate, Nirmatrelvir (PF-07321332), and Molnupiravir (EIDD-2801) (MedChem Express, Monmouth Junction, NJ, USA) with a starting concentration of 3000 ng/mL and 1:3 dilutions to a final concentration of 0.3 ng/mL and subsequently infected with indicated viruses at an MOI of 0.1 for 48 h. The cells were fixed in a 3% PFA-PBS and analyzed by confluency measurement using Spark Cyto 400 multimode plate reader as described in the previous section. Infected and non-infected wells containing no antiviral drug were used as positive and negative control to determine relative confluency. The 50% maximal inhibitory concentration (IC50) was normalized to control infections and used to determine the inhibitory potency of the employed drug.
9
Antiviral Drug Preparation and Storage
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The antiviral drugs GS-441524, teriflunomide, ritonavir, and nirmatrelvir utilized in this study were purchased from MedChemExpress (Princeton, NJ, USA). Ruxolitinib was obtained from InvivoGen (San Diego, CA, USA), while molnupiravir was sourced from Sigma-Aldrich (St. Louis, MO, USA). The antiviral compounds were dissolved or resuspended in dimethyl sulfoxide to prepare stock solutions (5 mM) of all six drugs. The stock solutions of the drugs were stored at −20 °C for Ruxolitinib and −80 °C for GS-441524, molnupiravir, nirmatrelvir, ritonavir, and teriflunomide. The drugs were further diluted to working concentrations using EMEM medium for experimental use.
10
SARS-CoV-2 Inhibitor Screening Assay
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Each tested inhibitor was diluted in dimethyl sulfoxide (DMSO) to the concentration used for each test condition. To each well 5 µL of inhibitor diluted in DMSO (1.10 g/mL) was added with 1 mL of medium (MEM +10% FBS +50 U/mL Pen/Strep). Therefore, for each condition, DMSO with a final concentration of 5.5 mg/mL was used, as well as the DMSO control for each tested condition. The following compounds were used: Remdesivir (MedChem Express), GC376 (Selleck Chemicals), Nirmatrelvir (PF-07321332; MedChem Express), and Amodiaquine dihydrochloride (Amodiaquine; MedChem Express).
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