Journal: International Journal of Molecular Sciences

Article Title: Molecular Interactions of Tannic Acid with Proteins Associated with SARS-CoV-2 Infectivity

doi: 10.3390/ijms23052643

Figure Lengend Snippet: Inhibitory effects of TA, TGG, and corilagin on human transmembrane protease serine 2 (TMPRSS2) activity. The effects of different concentrations (0.1 to 100 µM) of ( A ) TA, ( B ) TGG, and ( C ) corilagin are tested on the activity of TMPRSS2. The fluorescence units in control conditions are considered as 100%. Blank values are subtracted from all the readings before the conversion into percentage of activity. Results are expressed as mean ± SD (n = 3). Statistical analysis is performed using one-way ANOVA followed by Tukey post hoc test with *** p < 0.001 compared to positive control wells.

Article Snippet: Recombinant human TMPRSS2 protein (106–492 aa), with a MW of 44.8 kDa, was purchased from Creative Biomart.

Techniques: Activity Assay, Fluorescence, Control, Positive Control

Journal: International Journal of Molecular Sciences

Article Title: Molecular Interactions of Tannic Acid with Proteins Associated with SARS-CoV-2 Infectivity

doi: 10.3390/ijms23052643

Figure Lengend Snippet: Biophysical characterization of the molecular interactions between TA and TMPRSS2. ( A ) The recombinant protein TMPRSS2 is immobilized on a CM5 sensor chip, and increasing concentrations of TA are injected to evaluate binding kinetics by SPR. ( B ) TMPRSS2 is adsorbed to a gold QCMD sensor, and various concentrations of TA are flowed over the surface for 30 min. TA adsorption is expressed by the dimensionless molar ratio of adsorbed TA to adsorbed TMPRSS2.

Article Snippet: Recombinant human TMPRSS2 protein (106–492 aa), with a MW of 44.8 kDa, was purchased from Creative Biomart.

Techniques: Recombinant, Injection, Binding Assay, Adsorption

Journal: International Journal of Molecular Sciences

Article Title: Molecular Interactions of Tannic Acid with Proteins Associated with SARS-CoV-2 Infectivity

doi: 10.3390/ijms23052643

Figure Lengend Snippet: Binding free energy between proteins (RBD, TMPRSS2, 3CLpro) and TA for the best poses found during docking. The MD MMPBSA binding free energy is computed over the interval 750 to 1000 ns using the g\_mmpbsa tools [ 49 ].

Article Snippet: Recombinant human TMPRSS2 protein (106–492 aa), with a MW of 44.8 kDa, was purchased from Creative Biomart.

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Molecular Interactions of Tannic Acid with Proteins Associated with SARS-CoV-2 Infectivity

doi: 10.3390/ijms23052643

Figure Lengend Snippet: Molecular structures (pose 1) of: ( A ) TA/RBD, ( B ) TA/TMPRSS2, and ( C ) TA/3CLpro complexes, before (green) and after (turquoise) 1000-ns MD simulations.

Article Snippet: Recombinant human TMPRSS2 protein (106–492 aa), with a MW of 44.8 kDa, was purchased from Creative Biomart.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Molecular Interactions of Tannic Acid with Proteins Associated with SARS-CoV-2 Infectivity

doi: 10.3390/ijms23052643

Figure Lengend Snippet: Molecular structures after 1000 ns of MD: ( A ) TA/TMPRSS2 complex (pose 1; MMPBSA binding free energy of −68 kcal/mol) and ( B ) ligand interaction map. The interaction map of TA with TMPRSS2 is shown from the center of the biggest cluster computed on the convergence interval using the protein backbone atoms and ligand non-hydrogen atoms. The other contacts, defined by a distance smaller than 0.40 nm between the ligand and the protein, are shown as red arcs. H-bonds and their donor/acceptor distances are shown in green. The interaction map is generated using LigPlot [ , ].

Article Snippet: Recombinant human TMPRSS2 protein (106–492 aa), with a MW of 44.8 kDa, was purchased from Creative Biomart.

Techniques: Binding Assay, Generated

Journal: EBioMedicine

Article Title: Camostat mesylate inhibits SARS-CoV-2 activation by TMPRSS2-related proteases and its metabolite GBPA exerts antiviral activity

doi: 10.1016/j.ebiom.2021.103255

Figure Lengend Snippet: TMPRSS2 protease domain and GBPA interaction. A TMPRSS2 structure model is shown in the left panel, the active site is highlighted in cyan and catalytic triad residues are shown in black. The representative structure of GBPA bound to TMPRSS2 in a reactive complex is shown in the right panel. The GBPA guanidinium head forms a salt bridge with Asp-435 inside the S1 pocket. This transient complex, which is similar for Camostat, is prone to be catalyzed at the ester bond interacting with Ser-441, leading to a covalent complex with TMPRSS2 inhibited.

Article Snippet: Human TMPRSS2 (Recombinant N-terminus 6xHis, aa106-492) (Cat # LS-G57269-20) protein was acquired from LifeSpan Biosciences.

Techniques:

Journal: EBioMedicine

Article Title: Camostat mesylate inhibits SARS-CoV-2 activation by TMPRSS2-related proteases and its metabolite GBPA exerts antiviral activity

doi: 10.1016/j.ebiom.2021.103255

Figure Lengend Snippet: Activation of SARS-2-S by TMPRSS2-related proteases can be suppressed by Camostat mesylate. The experiment was performed as described for Fig. 1 with the modifications that only TMPRSS2, TMPRSS11D, TMPRSS11E, TMPRSS11F and TMPRSS13 were investigated and target cells were pre-treated with either 50 mM ammonium chloride (red), 100 µM Camostat mesylate (blue) or a combination of both (green). DMSO-treated cells served as controls. At 16 h post inoculation with viral particles bearing SARS-2-S, pseudotype entry was analyzed by measuring virus-encoded luciferase activity in cell lysates. Data were further normalized and entry efficiency into control-treated cells was set as 100%. Shown are the average (mean) data obtained from three biological replicates, each performed with four technical replicates. Error bars indicate the SEM. Statistical significance of differences in entry efficiency in ammonium chloride-, Camostat mesylate- or ammonium chloride + Camostat mesylate-treated cells versus control-treated cells was analyzed by two-way ANOVA with Dunnett's posttest ( P values, from left to right: NH 4 Cl [0.0001; 0.0001; 0.0001; 0.0001; 0.0001; 0.7334]; Camostat [0.9999; 0.8995; 0.9969; 0.9999; 0.9731; 0.9999]; NH 4 Cl/Camostat [0.0001; 0.0001; 0.0001; 0.0001; 0.0001; 0.0001]).

Article Snippet: Human TMPRSS2 (Recombinant N-terminus 6xHis, aa106-492) (Cat # LS-G57269-20) protein was acquired from LifeSpan Biosciences.

Techniques: Activation Assay, Virus, Luciferase, Activity Assay, Control

Journal: EBioMedicine

Article Title: Camostat mesylate inhibits SARS-CoV-2 activation by TMPRSS2-related proteases and its metabolite GBPA exerts antiviral activity

doi: 10.1016/j.ebiom.2021.103255

Figure Lengend Snippet: Camostat mesylate and FOY-251 inhibit the activity of recombinant TMPRSS2. Incubation of recombinant TMPRSS2 with the Boc-Gln-Ala-Arg-MCA peptide substrate leads to the cleavage of the substrate and the release of the AMC(7-Amino-4-methylcoumarin) fluorophore, resulting in a fluorescent signal. Data were normalized against the fluorescence signals obtained in the absence of test compounds (Camostat mesylate, FOY-251, GBA). The concentration-response data for each test compound were plotted and modeled by a four-parameter logistic fit to determine the 50% effective concentration (EC 50 ) value. Inhibitory activity of Camostat mesylate (blue), FOY-251(light blue) and GBA (red) against TMPRSS2 recombinant protein were visualized and curve fitting was performed using GraphPad Prism. The average of two biological replicates, each performed with four (Camostat mesylate and FOY-251) or two technical replicates (GBA) is shown. EC 50 values were 4 nM (Camostat mesylate), 70 nM (FOY-251), >10 µM (GBA).

Article Snippet: Human TMPRSS2 (Recombinant N-terminus 6xHis, aa106-492) (Cat # LS-G57269-20) protein was acquired from LifeSpan Biosciences.

Techniques: Activity Assay, Recombinant, Incubation, Fluorescence, Concentration Assay

Journal: Journal of Medical Virology

Article Title: Native and activated antithrombin inhibits TMPRSS2 activity and SARS‐CoV‐2 infection

doi: 10.1002/jmv.28124

Figure Lengend Snippet: Antithrombin inhibits TMPRSS2 protease activity. (A) Docking analysis of AT (orange, from PDB 3KCG) and TMPRSS2 (homology model, UniProtKB O15393, green). The heparin pentasaccharide is shown as spheres and glycoside residues as sticks. The inset shows the AT‐TMPRSS2 catalytic complex after structural refinement. The AT RCL is depicted in orange, TMPRSS2 residues in cyan; water molecules (sticks) within a radius of 5Å and hydrogen bonds (blue lines) are shown. (B) Recombinant TMPRSS2 (residues 106–492) was incubated with two commercially available formulations of AT (Anbinex, Kybernin) or the small molecule TMPRSS2 inhibitor CM, 1h before the addition of fluorogenic TMPRSS2 substrate BOC‐QAR‐AMC. Data are shown as means ± SD derived from n = 2 experiments performed in triplicates. (C) HEK293T cells expressing TMPRSS2 were incubated with AT or CM 1 h before the addition of fluorogenic TMPRSS2 substrate BOC‐QAR‐AMC. Results were corrected for the signal of nontransfected HEK293T cells. Data are shown as means± SEM derived from n = 3 experiments performed in duplicates. AT, antithrombin; CM, camostat mesylate; RCL, reactive center loop; SD, standard deviation; SEM, standard error of the mean.

Article Snippet: Recombinant TMPRSS2 was obtained from CreativeBiomart (TMPRSS2‐1856H) or LSBio (LS‐G57269).

Techniques: Activity Assay, Recombinant, Incubation, Derivative Assay, Expressing, Standard Deviation

Journal: Journal of Medical Virology

Article Title: Native and activated antithrombin inhibits TMPRSS2 activity and SARS‐CoV‐2 infection

doi: 10.1002/jmv.28124

Figure Lengend Snippet: Antithrombin inhibits activity of cathepsin L, while moderately affecting cathepsin B. Recombinant cathepsin L (A) or isolated cathepsin B (B) were incubated with AT (Anbinex), small molecule TMPRSS2 inhibitor CM or small molecule cathepsin inhibitor E64‐d, 1 h before the addition of fluorogenic substrate Z‐L‐R‐AMC (for cathepsin L) or Z‐R‐R‐AMC (for cathepsin B). Data are shown as means ± SEM derived from n = 3 experiments performed in triplicates. AT, antithrombin; CM, camostat mesylate; SEM, standard error of the mean.

Article Snippet: Recombinant TMPRSS2 was obtained from CreativeBiomart (TMPRSS2‐1856H) or LSBio (LS‐G57269).

Techniques: Activity Assay, Recombinant, Isolation, Incubation, Derivative Assay

Journal: Journal of Medical Virology

Article Title: Native and activated antithrombin inhibits TMPRSS2 activity and SARS‐CoV‐2 infection

doi: 10.1002/jmv.28124

Figure Lengend Snippet: Activation of antithrombin increases anti‐TMPRSS2 and anti‐SARS‐CoV‐2 activity. (A) Heparin (Hep)‐ and Fondaparinux (FPX)‐activated antithrombin (Anbinex, 0.0137 µM) was incubated with recombinant TMPRSS2 enzyme before the addition of fluorogenic TMPRSS2 substrate BOC‐QAR‐AMC. Data are shown as means ± SEM derived from n = 3 experiments performed in triplicates. (B) HEK293T cells expressing TMPRSS2 were incubated with Hep‐ or FPX‐activated Anbinex (0.17 µM) before the addition of fluorogenic TMPRSS2 substrate BOC‐QAR‐AMC. Results were corrected for the signal of nontransfected HEK293T cells. Data are shown as means± SEM derived from n = 3 experiments performed in duplicates. (C) Caco2 cells were treated with Hep‐ or FPX‐activated Anbinex (13.75 µM) for 1 h before infection of cells with SARS‐CoV‐2 isolate Wuhan/Hu‐1 (Spike mutation D614G) at an MOI of 0.0002. Data are shown as means ± SD derived from n = 2 experiments performed in triplicates. (D) Caco2 cells were treated with FPX‐activated Anbinex (13.75 µM) for 1 h before infection of cells with the indicated SARS‐CoV‐2 isolates at an MOI of 0.005. Data are shown as means ± SEM derived from n = 3 experiments. Infection rates of (C) and (D) were assessed by flow cytometric analysis of SARS‐CoV‐2 nucleocapsid (N) protein expression in single cells at 2 dpi (C) or 1 dpi (D). Maximum final concentrations of Hep and FPX on cells were 0.4 mg/ml, corresponding to 22.2 or 232 µM, respectively. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, assessed by two‐way analysis of variance with Dunnett's multiple comparisons test. MOI, multiplicity of infection; SD, standard deviation; SEM, standard error of the mean.

Article Snippet: Recombinant TMPRSS2 was obtained from CreativeBiomart (TMPRSS2‐1856H) or LSBio (LS‐G57269).

Techniques: Activation Assay, Activity Assay, Incubation, Recombinant, Derivative Assay, Expressing, Infection, Mutagenesis, Standard Deviation

Journal: eLife

Article Title: Coagulation factors directly cleave SARS-CoV-2 spike and enhance viral entry

doi: 10.7554/eLife.77444

Figure Lengend Snippet: ( A ) Peptides derived from two known cleavage sites of SARS-CoV-2 spike were designed with C-terminal fluorophore 5-FAM and N-terminal fluorescence resonance energy transfer (FRET) quencher QXL-520. ( B ) FDA-approved and investigational serine protease inhibitors were screened by enzymatic assay to inhibit TMPRSS2 cleavage of SARS-CoV-2 S1/S2 peptide substrate. Relative change in fluorescence with respect to DMSO vehicle is shown. Colors indicate the described target of the drugs screened. All drugs screened at 10 µM final concentration. ( C ) Active form of dabigatran in enzymatic assay for TMPRSS2 inhibition. Relative fluorescence with respect to its corresponding 0.1 N HCl vehicle is shown. ( D ) Schematic of constructs used to generate SARS-CoV-2 spike-pseudotyped/HIV-1-based particles. ( E ) Calu3 cells were treated with 10 µM of the indicated drugs for 24 hr prior to infection with HIV-1 NL /SARS-CoV-2 pseudovirus. Media was changed at 24 hr post infection and pseudoviral entry was measured by nanoluciferase luminescent signal at 40 hr. ( F ) Calu3 cells treated with 10 µM of the indicated drugs were monitored for confluence by Incucyte for 40 hr. ( G ) Pseudoviral entry was measured by nanoluciferase luminescent signal in Calu3 cells treated various concentrations of the indicated drugs for 4 hr prior to infection with SARS-CoV-2 pseudovirus. ( H ) Caco2 cells were infected with lenti-Cas9-blast and U6-sgRNA-EFS-puro-P2A-tRFP and selected. Neutral controls targeting CD4 (not endogenously expressed) or PHGDH intron 1, two sgRNAs each targeting different regions of ACE2 and TMPRSS2 were included. Cells were subsequently infected with HIV-1 NL /SARS-CoV-2 pseudovirus. ( I ) Caco2 cells co-expressing Cas9 and sgRNAs targeting CD4 (not expressed) or TMPRSS2 were treated with 10 µM camostat, nafamostat, or DMSO vehicle. N = 3, *p < 0.05, two-tailed t-test. Data represented as mean ± SEM. Figure 1—source data 1. Data and summary statistics for enzymatic and pseudovirus assays.

Article Snippet: Recombinant TMPRSS2, purified from yeast, was obtained from LSBio (LS-G57269).

Techniques: Derivative Assay, Fluorescence, Förster Resonance Energy Transfer, Enzymatic Assay, Concentration Assay, Inhibition, Construct, Infection, Expressing, Two Tailed Test

Journal: eLife

Article Title: Coagulation factors directly cleave SARS-CoV-2 spike and enhance viral entry

doi: 10.7554/eLife.77444

Figure Lengend Snippet: ( A ) TMPRSS2 enzymatic assay was performed in AB1 (20 mM Tris-HCl, pH 7.3, 100 mM NaCl, 1 mM EDTA, fresh 1 mM DTT) or AB2 (50 mM Tris-HCl, 150 mM NaCl, pH 8) using 10 µM of either S1/S2 or S2’ peptide substrate. ( B ) Titration of enzyme concentration was performed (0–1000 nM) with 10 µM S1/S2 substrate. Initial reaction velocity V 0 (rate of change in fluorescent signal) each enzyme concentration with 10 µM S1/S2 peptide substrate.

Article Snippet: Recombinant TMPRSS2, purified from yeast, was obtained from LSBio (LS-G57269).

Techniques: Enzymatic Assay, Titration, Concentration Assay

Journal: eLife

Article Title: Coagulation factors directly cleave SARS-CoV-2 spike and enhance viral entry

doi: 10.7554/eLife.77444

Figure Lengend Snippet: A549 cells (which do not express ACE2), A549/ACE2 cells (ectopic ACE2 expression from a lentiviral vector), and Caco2 cells (which express endogenous ACE2 and TMPRSS2) infected with HIV-1 NL -based particles pseudotyped with SARS-CoV-2 S or VSV G. N = 3, *p < 0.05, two-tailed t-test. Data represented as mean ± SEM.

Article Snippet: Recombinant TMPRSS2, purified from yeast, was obtained from LSBio (LS-G57269).

Techniques: Expressing, Plasmid Preparation, Infection, Two Tailed Test

Journal: eLife

Article Title: Coagulation factors directly cleave SARS-CoV-2 spike and enhance viral entry

doi: 10.7554/eLife.77444

Figure Lengend Snippet: ( A ) Schematic of constructs used to generate SARS-CoV-2 spike-pseudotyped/VSV-based pseudovirus. ( B ) Nanoluciferase luminescent signal following addition of rVSV∆G pseudovirus complemented with VSV G, SARS-CoV-2 S, SARS-CoV S, or without complementation with any envelope protein to Calu3 cells. Each pseudovirus was titrated by adding the indicated volume of inoculum, supplemented with fresh media up to 200 µl/well in a 96-well plate. ( C–F ) Nanoluciferase luminescent signal following infection of ( C ) Caco2, ( D ) Calu3, ( E ) A549/ACE2, or ( F ) Vero cells with rVSV∆G/SARS-CoV-2 pseudovirus pretreated for 4 hr with 10 µM camostat, nafamostat, dabigatran, or otamixaban, compared with uninfected or infected/untreated cells. Expression status of ACE2 and TMPRSS2 for each cell line is indicated. N = 3. Data represented as mean ± SEM.

Article Snippet: Recombinant TMPRSS2, purified from yeast, was obtained from LSBio (LS-G57269).

Techniques: Construct, Infection, Expressing

Journal: eLife

Article Title: Coagulation factors directly cleave SARS-CoV-2 spike and enhance viral entry

doi: 10.7554/eLife.77444

Figure Lengend Snippet: ( A ) Constructs used for CRISPR experiments. ( B ) Percentage of reads exhibiting wild type, frameshift, or in-frame indels at each locus for the indicated sgRNAs. ( C–F ) Distribution of reads with deletion or insertion by position within amplicon. ( G–J ) Distribution of the size of insertions and deletions in each amplicon. Two sgRNAs targeting ACE2 (g1 and g2) and two sgRNAs targeting TMPRSS2 (g1 and g2) were analyzed.

Article Snippet: Recombinant TMPRSS2, purified from yeast, was obtained from LSBio (LS-G57269).

Techniques: Construct, CRISPR, Amplification

Journal: eLife

Article Title: Coagulation factors directly cleave SARS-CoV-2 spike and enhance viral entry

doi: 10.7554/eLife.77444

Figure Lengend Snippet: Initial velocities for the cleavage of SARS-CoV-2 spike S1/S2 and S2’ peptide substrates by ( A ) TMPRSS2, ( B ) factor Xa, and ( C ) thrombin were measured over a range of 0–160 µM substrate. From initial velocity values, enzyme kinetic constants ( D ) turnover rate K cat (s –1 ), ( E ) affinity constant K m , and ( F ) specificity constant (K cat /K m ) were obtained for the indicated enzymes with S1/S2 and S2’ peptides. ( G–I ) Heatmaps depict the initial velocity V 0 of cleavage of the indicated peptide substrates and concentrations by ( G ) TMPRSS2, ( H ) factor Xa, and ( I ) thrombin. Figure 2—source data 1. Data and summary statistics for enzymatic assays.

Article Snippet: Recombinant TMPRSS2, purified from yeast, was obtained from LSBio (LS-G57269).

Techniques:

Journal: eLife

Article Title: Coagulation factors directly cleave SARS-CoV-2 spike and enhance viral entry

doi: 10.7554/eLife.77444

Figure Lengend Snippet: Kinetics of SARS-CoV-2 spike peptide substrate cleavage. Kinetic constants obtained from initial velocity studies with varying concentrations of SARS-CoV-2 spike S1/S2 and S2’ peptide substrates. Each estimate is based on seven different concentrations of substrate in 1:2 serial dilution (0–160 µM).

Article Snippet: Recombinant TMPRSS2, purified from yeast, was obtained from LSBio (LS-G57269).

Techniques: Serial Dilution

Journal: eLife

Article Title: Coagulation factors directly cleave SARS-CoV-2 spike and enhance viral entry

doi: 10.7554/eLife.77444

Figure Lengend Snippet: ( A–C ) Initial reaction velocity with respect to enzyme concentration for peptide substrates of the SARS-CoV-2 spike S1/S2 site (S1S2), with P1 arginine substituted with alanine (S1S2-P1A), or with substitutions in the P3 and P4 position (RR > SQ) with ( A ) TMPRSS2, ( B ) factor Xa, or ( C ) thrombin. ( D ) List of peptide substrates used in this study. ( E ) Initial reaction velocity of factor Xa cleavage of SARS-CoV-2 S1/S2 or thrombin-R271 peptide substrates in the presence of 0–100 µM phosphatidylcholine/phosphatidylserine (PC/PS) phospholipid vesicles. ( F ) Dilute Russell’s viper venom clotting time (dRVVT) assay of pooled normal human plasma, supplemented with 0–100 µM PC/PS phospholipid vesicles. N = 3, *p < 0.05, two-tailed t-test. Data represented as mean ± SEM.

Article Snippet: Recombinant TMPRSS2, purified from yeast, was obtained from LSBio (LS-G57269).

Techniques: Concentration Assay, Coagulation, Clinical Proteomics, Two Tailed Test

Journal: eLife

Article Title: Coagulation factors directly cleave SARS-CoV-2 spike and enhance viral entry

doi: 10.7554/eLife.77444

Figure Lengend Snippet: ( A ) Calu3 cells were infected with rVSV∆G/SARS-CoV-2 pseudovirus with concomitant treatment with vehicle, 250 nM factor Xa, or 250 nM thrombin. Quantification of the ratio of green fluorescent area to total confluence (4 fields/replicate well, 4 wells/condition). ( B ) Nanoluciferase luminescent signal was measured following infection with rVSV∆G/SARS-CoV-2 pseudovirus and the addition of either vehicle, factor Xa, or thrombin. The effect of factor Xa on rVSV∆G complemented with either ( C ) SARS-CoV spike or ( D ) VSV-G was measured by luminescent signal. Luminescent signal was measured following HIV-1 NL /SARS-CoV-2 pseudovirus infection and concomitant treatment with 125–250 nM factor Xa in ( E ) Calu3 cells, ( F ) A549/ACE2, and ( G ) Vero cells following transduction with lentiviral vectors to express GFP or TMPRSS2. Following selection, cells were infected with HIV-1 NL /SARS-CoV-2 pseudovirus and concomitantly treated with 125–250 nM factor Xa. Subsequently, nanoluciferase luminescent signal was determined and plotted relative to vehicle-treated control. *p < 0.05, two-tailed t-test. Data represented as mean ± SEM. Figure 3—source data 1. Data and summary statistics for pseudovirus assays with exogenous proteases.

Article Snippet: Recombinant TMPRSS2, purified from yeast, was obtained from LSBio (LS-G57269).

Techniques: Infection, Transduction, Selection, Control, Two Tailed Test

Journal: eLife

Article Title: Coagulation factors directly cleave SARS-CoV-2 spike and enhance viral entry

doi: 10.7554/eLife.77444

Figure Lengend Snippet: Initial velocities for the cleavage of 10 µM SARS-CoV-2 spike S1/S2 (top) and S2’ (bottom) peptide substrates by ( A ) TMPRSS2, ( B ) TMPRSS11D/human airway trypsin-like protease ( C ) factor Xa, and ( D ) thrombin were measured in the presence of DMSO vehicle, or 10 µM camostat, nafamostat, otamixaban, or dabigatran. The relative activity of ( E ) factor Xa and ( F ) thrombin were determined over a range of 0–10 µM of the indicated drugs. Calu3 cells were treated with a range of concentrations of nafamostat with or without addition of 250 nM exogenous factor Xa and infected with ( G ) rVSV∆G/SARS-CoV-2 pseudovirus or ( H ) HIV-1 NL /SARS-CoV-2 pseudovirus and infectivity was measured by luminescence. N = 3, data represented as mean ± SEM. Figure 4—source data 1. Data and summary statistics for enzymatic assays to determine the effects of protease inhibitors on host proteases.

Article Snippet: Recombinant TMPRSS2, purified from yeast, was obtained from LSBio (LS-G57269).

Techniques: Activity Assay, Infection

Journal: eLife

Article Title: Coagulation factors directly cleave SARS-CoV-2 spike and enhance viral entry

doi: 10.7554/eLife.77444

Figure Lengend Snippet:

Article Snippet: Recombinant TMPRSS2, purified from yeast, was obtained from LSBio (LS-G57269).

Techniques: Recombinant, Lysis, Luciferase, Clinical Proteomics, Sequencing, Software

Journal: Molecules

Article Title: The Proteolytic Activity of Neutrophil-Derived Serine Proteases Bound to the Cell Surface Arming Lung Epithelial Cells for Viral Defense

doi: 10.3390/molecules29184449

Figure Lengend Snippet: Summary of the proteolytic cleavage sites of proteases at the S2′ site. ( A ) Amino acid alignment of the S2′ region of the SARS-CoV-2 S protein. ( B ) S2′-peptides were incubated with TMPRSS2, furin, NE, CatG, and PR3 for 2 h at 37 °C. The hydrolysis of the peptide bonds is summarized in a digestion map (blue bars denote the fragments, and red arrows indicate the cleavage sites). Three independent experiments, n = 3. ( C ) Peptides were incubated with furin in the presence or absence of additional Ca 2+ ions, with a CaCl 2 final concentration of 1.2 mM (left panel). Quantification (right panel). n = 3.

Article Snippet: Human NE (4 μg/mL, neutrophil-derived human NE, PN: 16-14-051200, Lot No. EH 2020-03, Athens Research and Technology, Athens, GA, USA), human CatG (4 μg/mL, neutrophil-derived CatG, PN: 16-14-030107, Lot No. CG 2017-01, Athens Research and Technology, Athens, GA, USA), 4 μg/mL recombinant human furin (4 μL furin, containing 5 mM CaCl 2 based on the company’s production, was added to 94 μL PBS pH 7.4, with a final concentration of 0.2 mM; furin Cat. No. 450-47, Lot No. 1011516, Peprotech, Cranbury, NJ, USA) with or without the addition of CaCl 2 to a final concentration of 1.2 mM, or recombinant human TMPRSS2 (4 μg/mL, Cat. No. CSB-YP023924HU, Batch No. DA0471a3g001, BIOZOL, CUSABIO Technology, LLC, TX, USA) was incubated with 200 μg/mL peptide in PBS pH 7.4 (or in the case of TMPRSS2, Tris pH 7.8, 7.7 mM Tris/HCl and 150 mM NaCl) for 2 h at 37 °C.

Techniques: Incubation, Concentration Assay