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: 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: ACS Infectious Diseases

Article Title: Suite of TMPRSS2 Assays for Screening Drug Repurposing Candidates as Potential Treatments of COVID-19

doi: 10.1021/acsinfecdis.2c00172

Figure Lengend Snippet: (A) Scheme displaying the enzymatic assay principle for the fluorogenic peptide substrate. The fluorogenic peptide substrate Boc-Gln-Ala-Arg-AMC has low fluorescence compared to the fluorescent 7-amino-4-methylcoumarin (AMC), which is released upon proteolytic cleavage. The scissile bond is indicated in red. (B) Schematic of the truncated yeast-expressed recombinant TMPRSS2 used in the fluorogenic assay, containing the low-density lipoprotein receptor A (LDLRA) domain, scavenger receptor cysteine-rich (SRCR) domain, and protease domain.

Article Snippet: Recombinant Human TMPRSS2 protein expressed from yeast (human TMPRSS2 residues 106–492, N-terminal 6× His-tag) (cat. # CSB-YP023924HU) was acquired from Cusabio.

Techniques: Enzymatic Assay, Fluorescence, Recombinant

Journal: ACS Infectious Diseases

Article Title: Suite of TMPRSS2 Assays for Screening Drug Repurposing Candidates as Potential Treatments of COVID-19

doi: 10.1021/acsinfecdis.2c00172

Figure Lengend Snippet: Assay performance for primary screening of the compound libraries in the TMPRSS2 fluorogenic biochemical activity assay. Z′ scores and signal-to-background values are plotted as data points for each plate. There were 32 positive and 64 negative control wells on each 1536-well plate. (A) NCATS Pharmaceutical Collection (NPC), 2,678 compounds, (B) Mechanism Interrogation Plate (MIPE) library, 2,480 compounds, and (C) protease inhibitor library (PIL), 872 compounds. Black horizontal lines represent the mean values.

Article Snippet: Recombinant Human TMPRSS2 protein expressed from yeast (human TMPRSS2 residues 106–492, N-terminal 6× His-tag) (cat. # CSB-YP023924HU) was acquired from Cusabio.

Techniques: Activity Assay, Negative Control, Protease Inhibitor

Journal: ACS Infectious Diseases

Article Title: Suite of TMPRSS2 Assays for Screening Drug Repurposing Candidates as Potential Treatments of COVID-19

doi: 10.1021/acsinfecdis.2c00172

Figure Lengend Snippet: Label-free mass spectrometry biochemical assay. (A) Peptide derived from the known S2′ cleavage site of SARS-CoV-2 spike (S) protein. (B) Scheme displaying the enzymatic assay principle for the unlabeled peptide substrate. The unlabeled peptide substrate, Cbz-SKPSKRFIED, is cleaved by TMPRSS2 to create two cleavage products, Cbz-SKPSKR and SFIED. The scissile bond is indicated in red. (C) Initial velocity ( V 0 ) was calculated for each substrate concentration by plotting product formation versus time. (D) The V 0 for each concentration were plotted against the various substrate concentrations to obtain the V max and K m . V max : 1.95 μmol/min and K m : estimated 2320 μM. (E) Comparison of TMPRSS2 fluorogenic detection and mass spectrometry detection assays by assessing dose–response inhibition by camostat and gabexate. (F) Mass spectrometry traces of the cleavage product ( m / z : 418.8/702.4) showing that addition of camostat prevents product formation in dose–response. Each peak is labeled with the concentration of camostat (nM) for that condition. (G) Assay performance from the mass spectrometry detection assay when screening the hits identified from the primary screening. Z′ of 0.71 and S/B of 7.37. (H) Dose–response inhibition of 7-hydroxycoumarin against TMPRSS2 in both fluorogenic and mass spectrometry detection assays showing the fluorescent molecule as a false-positive hit in the fluorogenic assay, but not interfering with the mass spectrometry detection assay.

Article Snippet: Recombinant Human TMPRSS2 protein expressed from yeast (human TMPRSS2 residues 106–492, N-terminal 6× His-tag) (cat. # CSB-YP023924HU) was acquired from Cusabio.

Techniques: Mass Spectrometry, Derivative Assay, Enzymatic Assay, Concentration Assay, Comparison, Inhibition, Labeling, Detection Assay

Journal: ACS Infectious Diseases

Article Title: Suite of TMPRSS2 Assays for Screening Drug Repurposing Candidates as Potential Treatments of COVID-19

doi: 10.1021/acsinfecdis.2c00172

Figure Lengend Snippet: Detailed TMPRSS2 Fluorogenic Biochemical Assay Protocol for qHTS

Article Snippet: Recombinant Human TMPRSS2 protein expressed from yeast (human TMPRSS2 residues 106–492, N-terminal 6× His-tag) (cat. # CSB-YP023924HU) was acquired from Cusabio.

Techniques: Control, Incubation, Fluorescence

Journal: ACS Infectious Diseases

Article Title: Suite of TMPRSS2 Assays for Screening Drug Repurposing Candidates as Potential Treatments of COVID-19

doi: 10.1021/acsinfecdis.2c00172

Figure Lengend Snippet: Detailed TMPRSS2 Mass Spectrometry Detection Biochemical Assay

Article Snippet: Recombinant Human TMPRSS2 protein expressed from yeast (human TMPRSS2 residues 106–492, N-terminal 6× His-tag) (cat. # CSB-YP023924HU) was acquired from Cusabio.

Techniques: Mass Spectrometry, Control, Incubation

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