Journal: Scientific Reports

Article Title: In vitro enzymatic and cell culture assays for SARS-CoV-2 main protease interaction with ambenonium

doi: 10.1038/s41598-025-94283-9

Figure Lengend Snippet: Chain A of crystal structure of SARS-CoV-2 main protease. ( A ): Cartoon representation of 3CL pro (PDB ID: 7KFI) with domain I (residues 8–101) shown in pink, domain II (residues 102–184) shown in green, and domain III (residues 201–301) shown in gold. ( B ): Active site of 3CL pro. The catalytic dyad, HIS41 in domain I and CYS145 in domain II, are shown in red. The substrate is shown in stick representation in gray. This figure was prepared using PyMol (Schrodinger LLC).

Article Snippet: The steady-state kinetic parameters assay was performed in a 96 - well plate using the 3CL Protease MBP-tag Assay Kit (BPS Bioscience, San Diego, CA) and the manufacturer’s recommendations were followed.

Techniques:

Journal: Scientific Reports

Article Title: In vitro enzymatic and cell culture assays for SARS-CoV-2 main protease interaction with ambenonium

doi: 10.1038/s41598-025-94283-9

Figure Lengend Snippet: Ambenonium in 3CL pro active site . This figure shows the ambenonium in the active site of the main protease, this was done using AutoDock Vina, in an experiment conducted by De Souza Gomes et al., 2022. The ligands (in blue) are well fitted in the 3CL pro active site (in orange). Hydrogen bonds are represented by green dashed lines; attractive electrostatic interactions, as orange dashed lines; orbital π interactions, as pink, yellow and purple dashed lines; unfavorable interactions, as red. Residues involved in hydrophobic interactions are represented by light green circles.

Article Snippet: The steady-state kinetic parameters assay was performed in a 96 - well plate using the 3CL Protease MBP-tag Assay Kit (BPS Bioscience, San Diego, CA) and the manufacturer’s recommendations were followed.

Techniques:

Journal: Scientific Reports

Article Title: In vitro enzymatic and cell culture assays for SARS-CoV-2 main protease interaction with ambenonium

doi: 10.1038/s41598-025-94283-9

Figure Lengend Snippet: 3CL pro enzymatic inhibition assay. Different concentrations of ambenonium were tested to assess their effects on viral replication over different time intervals. The relative fluorescence units (RFUs) at 12 h after reaction initiation were normalized to control and used to indicate the enzymatic activities. Each bar represents the average of experiments performed in triplicate. The fluorescence values are presented as an arbitrary unit. The data were subjected to a Kruskal–Wallis test followed by Dunn’s Test using GraphPad Prism version 5.03. The asterisks mean statistical difference with p < 0.05 in relation to control. Amb: Ambenonium. GC376: positive control. The data are representative STD and mean.

Article Snippet: The steady-state kinetic parameters assay was performed in a 96 - well plate using the 3CL Protease MBP-tag Assay Kit (BPS Bioscience, San Diego, CA) and the manufacturer’s recommendations were followed.

Techniques: Inhibition, Fluorescence, Control, Positive Control

Journal: Scientific Reports

Article Title: In vitro enzymatic and cell culture assays for SARS-CoV-2 main protease interaction with ambenonium

doi: 10.1038/s41598-025-94283-9

Figure Lengend Snippet: Hydrogen bonds after molecular docking between the 3CL pro and ambenonium . The carbon atoms are represented in magenta, oxygen atoms in red, nitrogen atoms in blue, and ambenonium in white. The yellow dashed lines indicate the hydrogen bonds formed between ambenonium and the protein. ( A ) Hydrogen bonds formed between the monomeric form of 3CL pro and ambenonium after docking analysis. ( B ) Hydrogen bonds formed between the chain B of 3CL pro dimeric form and ambenonium after docking analysis. The figures highlight the involvement of the catalytic dyad (HIS41 and CYS145) in the interaction, along with other key residues present as sticks. These visualizations were generated using PyMOL (Schrödinger LLC).

Article Snippet: The steady-state kinetic parameters assay was performed in a 96 - well plate using the 3CL Protease MBP-tag Assay Kit (BPS Bioscience, San Diego, CA) and the manufacturer’s recommendations were followed.

Techniques: Generated

Journal: Scientific Reports

Article Title: In vitro enzymatic and cell culture assays for SARS-CoV-2 main protease interaction with ambenonium

doi: 10.1038/s41598-025-94283-9

Figure Lengend Snippet: Calculated binding free energy (ΔG) values for the 3CL PRO and ambenonium systems obtained using MM-GBSA and MM-PBSA methods.

Article Snippet: The steady-state kinetic parameters assay was performed in a 96 - well plate using the 3CL Protease MBP-tag Assay Kit (BPS Bioscience, San Diego, CA) and the manufacturer’s recommendations were followed.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Identification of SARS-CoV-2 3CL Protease Inhibitors by a Quantitative High-throughput Screening

doi: 10.1101/2020.07.17.207019

Figure Lengend Snippet: Schematic representation of the fluorogenic SARS-CoV-2 protease enzymatic assay. The peptide substrate exhibits low fluorescent because the fluorescence intensity of Edans in the C-terminal is quenched by the Dabcyl in the N-terminal of the substrate. The protease cleaves the substrate which breaks the proximity of the quencher molecule Dabcyl with the fluorophore Edans, resulting in an increase in fluorescence signal. This increase in fluorescence signal is proportional to the protease activity.

Article Snippet: 3CL pro of SARS-CoV-2 with an N-terminal MBP-tag, sensitive internally quenched fluorogenic substrate, and assay buffer were obtained from BPS Bioscience (San Diego, CA, USA).

Techniques: Enzymatic Assay, Fluorescence, Activity Assay

Journal: bioRxiv

Article Title: Identification of SARS-CoV-2 3CL Protease Inhibitors by a Quantitative High-throughput Screening

doi: 10.1101/2020.07.17.207019

Figure Lengend Snippet: SARS-CoV-2 3CL pro enzyme assay optimization. (a) Concentration-response curve of enzyme titration. With a fixed concertation of substrate (20 μM), the fluorescent intensity increased with enzyme concentrations. The linear response was observed at low enzyme concentrations. Measurement was conducted 2 h after initiating the reaction at RT. (b) The signal-to-basal (S/B) ratios of three enzyme concentrations within the linear range, at various incubation times. Dotted line represents the S/B = 1. (c) Enzyme kinetics. Michealis-Menton plot exhibited a Km of 75.41 μM and Vmax of 1392 RFU/min for SARS-CoV-2 3CL pro . (d) The S/B ratios determined at RT and 37 °C. No difference was observed in 1 h incubation between the two temperatures.

Article Snippet: 3CL pro of SARS-CoV-2 with an N-terminal MBP-tag, sensitive internally quenched fluorogenic substrate, and assay buffer were obtained from BPS Bioscience (San Diego, CA, USA).

Techniques: Enzymatic Assay, Concentration Assay, Titration, Incubation

Journal: bioRxiv

Article Title: Identification of SARS-CoV-2 3CL Protease Inhibitors by a Quantitative High-throughput Screening

doi: 10.1101/2020.07.17.207019

Figure Lengend Snippet: (a) Concentration response of the known 3CL pro inhibitor, GC376. An IC 50 of 0. 17 µM was determined for the inhibition of SARS-CoV-2 3CL pro . The substrate concentration was 20 μM and enzyme concentration was 50 nM in this experiment. (b) Scatter plot of the results from a DMSO plate in the 3CL pro enzymatic assay in a 1536-well plate, where columns 1 and 2 in the plate contain substrate only, column 3 includes GC376 titration (1:3 dilution series from 57.5 µM), and columns 5-48 contain DMSO (23 nL DMSO in 4 µL reaction solution).

Article Snippet: 3CL pro of SARS-CoV-2 with an N-terminal MBP-tag, sensitive internally quenched fluorogenic substrate, and assay buffer were obtained from BPS Bioscience (San Diego, CA, USA).

Techniques: Concentration Assay, Inhibition, Enzymatic Assay, Titration

Journal: bioRxiv

Article Title: Identification of SARS-CoV-2 3CL Protease Inhibitors by a Quantitative High-throughput Screening

doi: 10.1101/2020.07.17.207019

Figure Lengend Snippet: Triage strategy used to narrow down the starting 10,755 small molecules from the primary HTS campaign which led to the identification of 23 compounds having IC50s < 30 μM, maximal inhibition > 60% against SARS-CoV-2 3CL pro .

Article Snippet: 3CL pro of SARS-CoV-2 with an N-terminal MBP-tag, sensitive internally quenched fluorogenic substrate, and assay buffer were obtained from BPS Bioscience (San Diego, CA, USA).

Techniques: Inhibition

Journal: bioRxiv

Article Title: Identification of SARS-CoV-2 3CL Protease Inhibitors by a Quantitative High-throughput Screening

doi: 10.1101/2020.07.17.207019

Figure Lengend Snippet: Concentration-response curves of the 6 most potent compounds with IC 50 values < 15 μM and maximal inhibition > 80% determined in the SARS-CoV-2 3CL pro enzyme assay. Enzyme assay (blue) and counter screen (red) curves correspond to left y-axis showing inhibitory results, CPE (black) and cytotoxicity (green) curves correspond to right y-axis showing cell viability. (a) Walrycin B, IC 50 = 0.26 μM. (b) Hydroxocobalamin, IC 50 = 3.29 μM. (c) Z-DEVD-FMK, IC 50 = 6.81 μM. (d) Suramin sodium, IC 50 = 6.5 μM. (e) LLL-12, IC 50 = 9.84 μM. (f) Z-FA-FMK, IC 50 = 11.39 μM. Primary CPE and cytotoxicity screens were conducted in 4 concentrations, only the hits were further confirmed with 8 concentrations with dilution ratio of 1:3.

Article Snippet: 3CL pro of SARS-CoV-2 with an N-terminal MBP-tag, sensitive internally quenched fluorogenic substrate, and assay buffer were obtained from BPS Bioscience (San Diego, CA, USA).

Techniques: Concentration Assay, Inhibition, Enzymatic Assay

Journal: iScience

Article Title: SLC38A9 regulates SARS-CoV-2 viral entry

doi: 10.1016/j.isci.2024.110387

Figure Lengend Snippet:

Article Snippet: SARS-CoV-1 spike protein-conjugated pseudovirus , BPS Bioscience , 78614.

Techniques: Labeling, Recombinant, Lysis, Mutagenesis, Fluorescence, Protease Inhibitor, Screening Assay, Luciferase, shRNA, Control, Software

Journal: Biosensors & Bioelectronics

Article Title: Magnetic beads combined with carbon black-based screen-printed electrodes for COVID-19: A reliable and miniaturized electrochemical immunosensor for SARS-CoV-2 detection in saliva

doi: 10.1016/j.bios.2020.112686

Figure Lengend Snippet: The MBs-based assay for SARS-CoV-2 detection in untreated saliva.

Article Snippet: The use of the high sensitive antibodies is one of the main task to develop high sensitive and selective analytical device, thus for the selection of antibodies, spectrophotometric ELISA was carried out to assess the reactivity of MAb and two different PAb (Sinobiological, Germany and ProSci, USA) towards two different S proteins namely SARS Coronavirus 2019 Spike Recombinant protein (1000–1200 aa) and Recombinant Spike protein SARS-CoV Spike protein, S1 subunit.

Techniques:

Journal: Biosensors & Bioelectronics

Article Title: Magnetic beads combined with carbon black-based screen-printed electrodes for COVID-19: A reliable and miniaturized electrochemical immunosensor for SARS-CoV-2 detection in saliva

doi: 10.1016/j.bios.2020.112686

Figure Lengend Snippet: A) ELISA response for two different PAb anti-SARS-CoV-2 1 μg/mL (Sinobiological and ProSci) towards two different Spike proteins coated at 2 ng/mL. B) Binding curve of colorimetric ELISA for MAb anti-SARS-CoV-2 ranging from 0.12 – 2 μg/mL. Coating of Spike protein: 2 ng/mL. C) Electrochemical response using the MBs-based assay using CB-based modified electrode (blue line) and bare electrode (black line). The mean value (n = 3) with the corresponding standard deviation was reported for each measurement. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The use of the high sensitive antibodies is one of the main task to develop high sensitive and selective analytical device, thus for the selection of antibodies, spectrophotometric ELISA was carried out to assess the reactivity of MAb and two different PAb (Sinobiological, Germany and ProSci, USA) towards two different S proteins namely SARS Coronavirus 2019 Spike Recombinant protein (1000–1200 aa) and Recombinant Spike protein SARS-CoV Spike protein, S1 subunit.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Modification, Standard Deviation

Journal: Biosensors & Bioelectronics

Article Title: Magnetic beads combined with carbon black-based screen-printed electrodes for COVID-19: A reliable and miniaturized electrochemical immunosensor for SARS-CoV-2 detection in saliva

doi: 10.1016/j.bios.2020.112686

Figure Lengend Snippet: A) Study of the signal response for evaluating PAb concentration. 10 μL of MBs, 200 μL of PAb anti SARS-CoV-2 0.5, 1, and 2 μg/mL (in PBS) + 200 μL of PAb-AP anti rabbit IgG 0.5, 1, and 2 μg/mL (in PBS) + 300 μL of tested sample, incubation time 30 min without stirring, 3 washing steps, testing a negative control and 0.16 μg/mL of S protein. B) Study of the signal response for evaluating the effect of incubation time. 10 μL of MBs, 200 μL of PAb anti SARS-CoV-2 1 μg/mL (in PBS) + 200 μL of PAb-AP anti rabbit IgG 1 μg/mL (in PBS) + 300 μL of tested sample, incubation time (15/30 min) without stirring, 3 washing steps, testing a negative control, 0.16 and 2.5 μg/mL of S protein. C) Study of the signal response as function of mixing during the incubation step. 10 μL of MBs, 200 μL of PAb anti SARS-CoV-2 1 μg/mL (in PBS) + 200 μL of PAb-AP anti rabbit IgG 1 μg/mL (in PBS) + 300 μL of tested sample, incubation time 30 min with and without stirring, 3 washing steps, testing a negative control and 0.16 and 2.5 μg/mL of S protein. D) Study of the signal response for evaluating the effect of number of washing steps. 10 μL of MBs, 200 μL of PAb anti SARS-CoV-2 1 μg/mL (in PBS) + 200 μL of PAb-AP anti rabbit IgG 1 μg/mL (in PBS) + 300 μL of tested sample, incubation time 30 min without stirring, 1/2/3 washing steps, testing a negative control and 0.16 μg/mL of S protein. The mean value (n = 3) with the corresponding standard deviation was reported for each measurement.

Article Snippet: The use of the high sensitive antibodies is one of the main task to develop high sensitive and selective analytical device, thus for the selection of antibodies, spectrophotometric ELISA was carried out to assess the reactivity of MAb and two different PAb (Sinobiological, Germany and ProSci, USA) towards two different S proteins namely SARS Coronavirus 2019 Spike Recombinant protein (1000–1200 aa) and Recombinant Spike protein SARS-CoV Spike protein, S1 subunit.

Techniques: Concentration Assay, Incubation, Negative Control, Standard Deviation

Journal: Biosensors & Bioelectronics

Article Title: Magnetic beads combined with carbon black-based screen-printed electrodes for COVID-19: A reliable and miniaturized electrochemical immunosensor for SARS-CoV-2 detection in saliva

doi: 10.1016/j.bios.2020.112686

Figure Lengend Snippet: A) Electrochemical calibration curve (inset the voltammograms) using the optimized parameters for S protein detection in buffer (black line) and in untreated saliva (red line) (n = 3). B) Electrochemical calibration curve (inset the voltammograms) for N protein detection in buffer (black line) and untreated saliva (red line) (n = 3). C) Voltammograms obtained without (black line) and with (blue line) cultured SARS-CoV-2 virus at concentration 6.5 PFU/mL using MBs-based immunoassay for S protein. D) Voltammograms obtained without (black line) and with (blue line) cultured SARS-CoV-2 virus at concentration 6.5 x 10 4 PFU/mL using MBs-based immunoassay for N protein. E) Exclusivity test on seasonal influenza virus A (H1N1) (10 2.9 TCID50 mL −1 ) and Influenza 2009 pH1N1 virus (10 4.15 TCID50 mL −1 ) in comparison to SARS-CoV-2 (6.5 PFU/mL) using assay for S protein (n = 3). F) Exclusivity test on seasonal influenza virus A (H1N1) (10 2.9 TCID50 mL −1 ) and 2009 influenza virus pH1N1 (10 4.15 TCID50 mL −1 ) in comparison to SARS-CoV-2 (6.5 x 10 4 PFU/mL) using assay for N protein (n = 3). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The use of the high sensitive antibodies is one of the main task to develop high sensitive and selective analytical device, thus for the selection of antibodies, spectrophotometric ELISA was carried out to assess the reactivity of MAb and two different PAb (Sinobiological, Germany and ProSci, USA) towards two different S proteins namely SARS Coronavirus 2019 Spike Recombinant protein (1000–1200 aa) and Recombinant Spike protein SARS-CoV Spike protein, S1 subunit.

Techniques: Cell Culture, Concentration Assay

Journal: Biosensors & Bioelectronics

Article Title: Magnetic beads combined with carbon black-based screen-printed electrodes for COVID-19: A reliable and miniaturized electrochemical immunosensor for SARS-CoV-2 detection in saliva

doi: 10.1016/j.bios.2020.112686

Figure Lengend Snippet: Overview of biosensors for SARS-CoV-2 detection.

Article Snippet: The use of the high sensitive antibodies is one of the main task to develop high sensitive and selective analytical device, thus for the selection of antibodies, spectrophotometric ELISA was carried out to assess the reactivity of MAb and two different PAb (Sinobiological, Germany and ProSci, USA) towards two different S proteins namely SARS Coronavirus 2019 Spike Recombinant protein (1000–1200 aa) and Recombinant Spike protein SARS-CoV Spike protein, S1 subunit.

Techniques: CRISPR, Amplification, Reporter Assay, SPR Assay, Transduction, Fluorescence, Immunochromatographic Assay