Journal: iScience

Article Title: SARS-CoV-2 infects an in vitro model of the human developing pancreas through endocytosis

doi: 10.1016/j.isci.2022.104594

Figure Lengend Snippet:

Article Snippet: Mouse anti-TMPRSS2 , DSHB , Cat# P5H9-A3; RRID: AB_2205599.

Techniques: Control, Virus, Recombinant, Reverse Transcription, Luminex, Software

Journal: bioRxiv

Article Title: High-throughput amino acid-level characterization of the interactions of plasminogen activator inhibitor-1 with variably divergent proteases

doi: 10.1101/2024.09.16.612699

Figure Lengend Snippet: (A) PAI-1 specificity fingerprints are shown for uPA, FXIIa, and TMPRSS2. The PAI-1 amino acid sequence is shown along the x-axis with potential amino acid substitutions shown along the y-axis. Tolerated mutations are shown in blue (log 2 -fold enrichment score > 0), while loss-of-function mutations are shown in red (log 2 -fold enrichment score ≤ 0). The amino acids of the canonical human PAI-1 sequence are shown in dark grey . Amino acids that were either not present or not present at sufficient levels to quantify (BaseMean ≥ 50 as determined by DESeq2 ) in the phage library are shown in white. (B) Heatmap showing the common amino acid substitutions across PAI-1’s specificity spaces for uPA, FXIIa, and TMPRSS2 with positive ( blue) and negative ( red ) functional enrichment scores. (C) PCA plot comparing the specificity spaces of PAI-1 for uPA ( pink ), FXIIa ( yellow ), TMPRSS2 ( blue ), and the starting I91L PAI-1 DMS library ( black ). Groups are annotated with the minimum enclosing ellipse. (D and E) Venn diagrams depicting the number of significantly (D) enriched and (E) depleted amino acid substitutions shared and unique to each specificity fingerprint.

Article Snippet: Soluble recombinant TMPRSS2 (aa 106-492) with an N-terminal 6xHis tag expressed in yeast was purchased from Creative BioMart (Shirley, NY) and shown to be inhibited by WT PAI-1 ( SI Fig. 1 ).

Techniques: Sequencing, Functional Assay

Journal: bioRxiv

Article Title: High-throughput amino acid-level characterization of the interactions of plasminogen activator inhibitor-1 with variably divergent proteases

doi: 10.1101/2024.09.16.612699

Figure Lengend Snippet: (A) A cartoon of the PAI-1 primary amino acid sequence showing which amino acids were included in each amplicon. Amplicon 12 contains PAI-1’s RCL. (B) PCA plots comparing the specificity spaces of PAI-1 for uPA ( pink ), FXIIa ( yellow ), TMPRSS2 ( blue ), and the I91L PAI-1 variant library ( black ) across PAI-1’s twelve amplicons. Groups are annotated with the minimum enclosing ellipse.

Article Snippet: Soluble recombinant TMPRSS2 (aa 106-492) with an N-terminal 6xHis tag expressed in yeast was purchased from Creative BioMart (Shirley, NY) and shown to be inhibited by WT PAI-1 ( SI Fig. 1 ).

Techniques: Sequencing, Amplification, Variant Assay

Journal: bioRxiv

Article Title: High-throughput amino acid-level characterization of the interactions of plasminogen activator inhibitor-1 with variably divergent proteases

doi: 10.1101/2024.09.16.612699

Figure Lengend Snippet: Conservation scores for each amino acid position in PAI-1 ( grey dots ) are shown as a function of the normalized functional scores determined in our DMS screens of PAI-1 inhibition of (A) uPA (R 2 = 0.14, p = 4.6x10 -14 ), (B) FXIIa (R 2 = 0.06, p = 7.5x10 -7 ), and (C) TMPRSS2 (R 2 = -0.003, p = 0.87). Dashed lines indicated the best fit linear regression with the 95% confidence interval shown in grey shading.

Article Snippet: Soluble recombinant TMPRSS2 (aa 106-492) with an N-terminal 6xHis tag expressed in yeast was purchased from Creative BioMart (Shirley, NY) and shown to be inhibited by WT PAI-1 ( SI Fig. 1 ).

Techniques: Functional Assay, Inhibition

Journal: Scientific Reports

Article Title: Enoxaparin augments alpha-1-antitrypsin inhibition of TMPRSS2, a promising drug combination against COVID-19

doi: 10.1038/s41598-022-09133-9

Figure Lengend Snippet: Overexpression of recombinant TMPRSS2 in HEK293T cells. ( A ) pcDNA3.1 empty vector, pcDNA3.1 eGFP , or pcDNA3.1 TMPRSS2+His plasmids (top panel) used to transfect HEK293T cells. Photomicrograph of pcDNA3.1 eGFP transfected cells (HEK293T eGFP ) (bottom panel). Cell lysates were prepared and immunoblotted with ( B ) anti-His antibody or ( C ) anti-TMPRSS2 antibody. ( D ) Immunocytochemistry staining to detect TMPRSS2 in control (HEK293T pcDNA3.1 ) and TMPRSS2–transfected HEK293T (HEK293T TMPRSS2 ) cells. ( E ) Spontaneous TMPRSS2 activity using the fluorogenic substrate Boc-QAR-AMC as a function of time in the HEK293T pcDNA3.1 and HEK293T TMPRSS2 cells. Data shown are representative or triplicate means ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 compared to HEK293T pcDNA3.1 cells.

Article Snippet: A pcDNA3.1 plasmid containing both the human TMPRSS2 open reading frame and the neomycin resistance gene was obtained from Addgene (Watertown, MA).

Techniques: Over Expression, Recombinant, Plasmid Preparation, Transfection, Immunocytochemistry, Staining, Control, Activity Assay

Journal: Scientific Reports

Article Title: Enoxaparin augments alpha-1-antitrypsin inhibition of TMPRSS2, a promising drug combination against COVID-19

doi: 10.1038/s41598-022-09133-9

Figure Lengend Snippet: Activity of TMPRSS2 in the presence of AAT. ( A ) HEK293T TMPRSS2 cells were left untreated or pre-treated with AAT at the indicated final concentrations or AEBSF (50 µM) for 30 min, followed by addition of the fluorogenic substrate Boc-QAR-AMC. Fluorescence was measured immediately on the fluorescent plate reader and then every 15 min for a total of 90 min. ( B ) TMPRSS2 activity at 90 min. Data shown are the triplicate means ± SEM of three independent experiments. *p < 0.05, **p < 0.01 at 90 min compared to untreated cells. AAT = alpha-1-antitrypsin.

Article Snippet: A pcDNA3.1 plasmid containing both the human TMPRSS2 open reading frame and the neomycin resistance gene was obtained from Addgene (Watertown, MA).

Techniques: Activity Assay, Fluorescence

Journal: Scientific Reports

Article Title: Enoxaparin augments alpha-1-antitrypsin inhibition of TMPRSS2, a promising drug combination against COVID-19

doi: 10.1038/s41598-022-09133-9

Figure Lengend Snippet: Activity of TMPRSS2 in the presence of AAT alone or with UFH, nadroparin, or enoxaparin. HEK293T TMPRSS2 cells were pre-treated with AAT alone at the indicated concentrations or with ( A ) UFH, ( B ) nadroparin (Nadro), or ( C ) enoxaparin (Enox) at the indicated final concentrations for 30 min, followed by addition of the fluorescent substrate Boc-QAR-AMC. Fluorescence was measured immediately on the fluorescent plate reader and then every 15 min for a total of 90 min. ( D ) TMPRSS2 activity at 90 min in untreated cells and cells incubated with AAT ± two concentrations of nadroparin, UFH, and enoxaparin. Data shown are the triplicate means ± SEM of three independent experiments. *p < 0.05, **p < 0.01 at 90 min. Unfractionated heparin = UFH.

Article Snippet: A pcDNA3.1 plasmid containing both the human TMPRSS2 open reading frame and the neomycin resistance gene was obtained from Addgene (Watertown, MA).

Techniques: Activity Assay, Fluorescence, Incubation

Journal: Scientific Reports

Article Title: Enoxaparin augments alpha-1-antitrypsin inhibition of TMPRSS2, a promising drug combination against COVID-19

doi: 10.1038/s41598-022-09133-9

Figure Lengend Snippet: Activity of TMPRSS2 in the presence of UFH, nadroparin, or enoxaparin alone or with AAT. HEK293T TMPRSS2 cells were pre-treated with ( A ) UFH, ( B ) nadroparin, or ( C ) enoxaparin ± AAT at the indicated final concentrations for 30 min, followed by addition of the fluorescent substrate Boc-QAR-AMC. ( D ) HEK293T TMPRSS2 cells were grown in Gibco 293 Serum-free Medium II, pre-treated with enoxaparin at the indicated concentrations for 30 min, followed by addition of Boc-QAR-AMC. Fluorescence was measured immediately on the fluorescent plate reader and then every 15 min for a total of 90 min. Data shown are the mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 at 90 min.

Article Snippet: A pcDNA3.1 plasmid containing both the human TMPRSS2 open reading frame and the neomycin resistance gene was obtained from Addgene (Watertown, MA).

Techniques: Activity Assay, Fluorescence

Journal: Scientific Reports

Article Title: Enoxaparin augments alpha-1-antitrypsin inhibition of TMPRSS2, a promising drug combination against COVID-19

doi: 10.1038/s41598-022-09133-9

Figure Lengend Snippet: Modeling TMPRSS2 and its interaction with AAT and heparin. ( A ) Model of the TMPRSS2–AAT Michaelis complex. TMPRSS2 domain organization is shown in inset (TM = transmembrane region; residue numbers of domain boundaries are indicated). Domains are colored separately: low-density lipoprotein receptor A (LDLR-A, plum), scavenger receptor cysteine-rich repeat (SRCR, green), peptidase S1 (catalytic) domain (cyan). Disulphides are shown as sticks. A conserved disulphide formed between the catalytic domain and SRCR domain (244–365) is indicated. Scissors indicate the cleavage site at R255 at the N-terminus of the catalytic domain that generates the mature form. N- and C-termini are labeled. The catalytic triad (H296, D345 and S441) are shown as red sticks. TM region in lipid bilayer is also indicated as a schematic, indicating proximity to the plasma membrane, resembling the homologue hepsin. Reactive centre loop (RCL of AAT, bound to the active site of TMPRSS2 is labeled. ( B ) Molecular surface of TMPRSS2 showing bound RCL (magenta cartoon). Catalytic residues are labelled. P1 sidechain of AAT occupies the S1 site of TMPRSS2, superimposed on the TMPRSS2 inhibitor nafamostat (wheat). ( C ) Hydrogen-bonding interactions between the RCL of AAT and TMPRSS2. Black broken lines indicate hydrogen bonds. ( D ) Heparin molecules stabilize TMPRSS2–AAT association by acting as electrostatic bridges. Electrostatic potential surfaces of the TMPRSS2–AAT complex (blue = positive, red = negative) showing docked heparin 4mers binding to electropositive patches on the molecular surface. Carbon atoms of each 4mer heparin are coloured differently. ( E ) Location of Lys/Arg residues (sticks: AAT = magenta; TMPRSS2 = cyan) in the vicinity of heparin (grey sticks) binding sites at the TMPRSS2(cyan)–AAT(magenta) interface. Semi-transparent molecular surface is shown, and Lys/Arg residues that contribute to unfavorable electrostatics at the interface are shown and labelled (sidechain nitrogen atoms colored blue). ( F ) Poor electrostatic complementarity at P2′, P1′, P1, P2, P3, and P4 of the RCL (magenta) in the TMPRSS2 active site. P1 residue of the AAT RCL and cognate S1 pocket of TMPRSS2 are labeled. View is orthogonal to ( E ) showing the lysines located at the interface rim that are unfavorably close to positively charged residues of AAT.

Article Snippet: A pcDNA3.1 plasmid containing both the human TMPRSS2 open reading frame and the neomycin resistance gene was obtained from Addgene (Watertown, MA).

Techniques: Residue, Labeling, Clinical Proteomics, Membrane, Binding Assay

Journal: Scientific Reports

Article Title: Enoxaparin augments alpha-1-antitrypsin inhibition of TMPRSS2, a promising drug combination against COVID-19

doi: 10.1038/s41598-022-09133-9

Figure Lengend Snippet: Predicted binding free energies for representative protease-inhibitor complexes.

Article Snippet: A pcDNA3.1 plasmid containing both the human TMPRSS2 open reading frame and the neomycin resistance gene was obtained from Addgene (Watertown, MA).

Techniques: Binding Assay, Protease Inhibitor

Journal: Scientific Reports

Article Title: Enoxaparin augments alpha-1-antitrypsin inhibition of TMPRSS2, a promising drug combination against COVID-19

doi: 10.1038/s41598-022-09133-9

Figure Lengend Snippet: Proposed model for TMPRSS2–AAT–heparin ternary complexation at the cell surface, preventing activation of the S2 domain. Heparin (red) neutralizes the repulsive electrostatic forces at the TMPRSS2 (wheat)–AAT (blue) interaction rim, in accordance with the predicted binding mode shown in Figs. D,E and . AAT inhibition of elastase cleavage at the site created by the D614G mutation of the spike protein (B.1.1.7 variant of SARS-CoV-2) is also shown. The inset shows the possibility that one heparin molecule could bridge multiple TMPRSS2–AAT complexes.

Article Snippet: A pcDNA3.1 plasmid containing both the human TMPRSS2 open reading frame and the neomycin resistance gene was obtained from Addgene (Watertown, MA).

Techniques: Activation Assay, Binding Assay, Inhibition, Mutagenesis, Variant Assay

Journal: Scientific reports

Article Title: Altered plasma levels of the SARS-CoV-2-related proteins ACE2 and TMPRSS2 in patients with Crohn's disease.

doi: 10.1038/s41598-024-81810-3

Figure Lengend Snippet: Fig. 1. Characterization of ACE2 and TMPRSS2 species in human plasma. (A) Schematic representation of ACE2 as a transmembrane type I protein and of the epitopes recognized by the antibodies used for it characterization (not drawn to scale). The carboxypeptidase and the transmembrane (TM) domains are represented. (B) Representative plasma samples from non-disease controls were immunoblotted with the antibodies AF933 (ectodomain) and the ab15348 (C-terminus). The C-terminus antibodies only recognize the full-length ACE2 which retains the C-terminal domain. The 70 and 75 kDa ACE2 immunoreacrive species lacking the C-terminal domain were identified as cleaved fragments, whereas 95, 100, 130 and 150 kDa were assigned as full-length forms. (C) Schematic representation of TMPRSS2 as a transmembrane type II protein and of the epitopes recognized by the antibodies used for it characterization (not drawn to scale). The peptidase and the transmembrane (TM) domains are represented, and the localization of the interdomain disulfide bond that served to link to the membrane-tethered prodomain fragment is indicated. (D) Plasma samples from control individuals (not the same than in B) were immunoblotted with the 14437- 1-AP, an antibody that targets full-length TMPRSS2, recognizing a 55-kDa band and recognizes also the 35 kDa membrane-tethered prodomain fragments (black arrowhead) and the 25 kDa peptidase ectodomain (grey arrowhead). The same samples were also blotted with H00007113 (C-terminus) or the OAAB04388 (N-terminus) antibodies, confirming the identity of peptidase and prodomain fragments. (*) A smaller ~ 22- kDa band was attributed to a nonspecific reactivity, since it was immunoreactive to both the anti-C-terminal antibody and the anti-N-terminal antibody.

Article Snippet: TMPRSS2, a 492 amino acids protein, was resolved with the 14437-1-AP antibody raised against a recombinant protein containing the 108–492 amino acids (Proteintech; rabbit polyclonal; 1:1000 dilution), the anti-C-terminus antibody H00007113 raised against a recombinant protein containing the 383–492 amino acids (Abnova; mouse monoclonal; 1:500 dilution) or the anti-N-terminus antibody OAAB04388 raised against a synthetic peptide between the 1–30 amino acids (Aviva Systems Biology; rabbit polyclonal; 1:1000 dilution).

Techniques: Clinical Proteomics, Membrane, 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: 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: iScience

Article Title: Impact of mutations defining SARS-CoV-2 Omicron subvariants BA.2.12.1 and BA.4/5 on Spike function and neutralization

doi: 10.1016/j.isci.2023.108299

Figure Lengend Snippet: Impact of BA.2.12.1 and BA.4/5 mutations on Spike-mediated infection (A) Infection kinetics of Caco2, A549-ACE2, and A549-ACE2-TMPRSS2 cells infected by VSVpp carrying the indicated mutant S proteins. Infected GFP+ cells were automatically quantified over a period of 22 h. Exemplary kinetic representing one out of four independent experiments (from B), where each dot indicates the mean of technical duplicates. (B) Automatic quantification of infection events by counting GFP positive cells of Caco2, A549-ACE2, and A549-ACE2-TMPRSS2 cells infected by VSVpp carrying SARS-CoV-2 Spike protein of Hu-1 (gray), BA.1 (light green), BA.2 (yellow), BA.2.12.1 (orange), or BA.4/5 (dark red). Bars represent the mean of four independent experiments (±SEM). Statistical significance was tested by one-way ANOVA. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. (C) Automatic quantification of infection events of Caco2 (upper panel), A549-ACE2 (middle panel), and A549-ACE2-TMPRSS2 (lower panel) cells transduced with VSVΔG-GFP pseudotyped with SARS-CoV-2 S of Hu-1 (gray), BA.1 (light green), BA.2 (yellow), BA.2.12.1 (orange), or BA.4/5 (dark red) or indicated mutant S proteins. Graphs in the center show Hu-based S mutants, infection events are normalized on the Hu-1 S (set at 1) and p values indicate difference to the Hu-1 S. Graphs on the right show BA.2-based S mutants and infection events are normalized on the BA.2 S (set at 1) and p values indicate difference to the BA.2 S. Bars represent the mean of four independent experiments (±SEM). Statistical significance was tested by one-way ANOVA. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. (D) Correlation of the pseudotype infection events of the wild-type S or indicated mutant S proteins in A549-ACE2-TMPRSS2/A549-ACE2 (upper panel), A549-ACE2/Caco2 (middle panel), and A549-ACE2-TMPRSS2/Caco2 (lower panel) cells. Each symbol represents the average value derived from four infection experiments (shown in C). Numbers indicate the combined mutations of BA.4/5 S proteins whose infection events do not correlate between A549 and Caco2 cells. Coefficient of correlation (r), coefficient of determination (R 2 -values) and two tailed p values are provided (see also Figure S2 ).

Article Snippet: Primer/Probe for qRT-PCR of Human TMPRSS2: TaqManTM Gene Expression Assay (FAM-MGB) Hs01122322_m1 , Thermo Fisher , Cat#4331182.

Techniques: Infection, Mutagenesis, Transduction, Derivative Assay, Two Tailed Test

Journal: iScience

Article Title: Impact of mutations defining SARS-CoV-2 Omicron subvariants BA.2.12.1 and BA.4/5 on Spike function and neutralization

doi: 10.1016/j.isci.2023.108299

Figure Lengend Snippet: Impact of BA.2.12.1 and BA.4/5 mutations on TMPRSS2 dependency (A) Automatic quantification of infection events HEK293T-ACE2 cells alone or transfected with TMPRSS2, cathepsin L or B, and infected with VSVpp carrying SARS-CoV-2 S of Hu-1 (gray), BA.1 (light green), BA.2 (yellow), BA.2.12.1 (orange), or BA.4/5 (dark red) or indicated mutant S proteins. The left part of the graphs shows Hu-based S mutants, infection events are normalized on the Hu-1 S (set at 1) and p values indicate difference to the Hu-1 S. Graphs on the right show BA.2-based S mutants and infection events are normalized to BA.2 S (set to 1) and p values indicate difference to the BA.2 S. Bars represent the mean of three independent experiments (±SEM). Statistical significance was tested by one-way ANOVA. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. (B) Correlation of VSVpp infection events of the wild-type S or indicated mutant S proteins in HEK293T overexpressing ACE2 alone or together with TMPRSS2 or cathepsins. Each dot represents the average value derived from three infection experiments (shown in A). Coefficient of correlation (r), coefficient of determination (R 2 -values), and two tailed p values are provided. (C) Infection kinetics (left panel) or infection events (right panel) of A549-ACE2 cells infected by VSVpp containing wild type or mutant Hu-based S proteins. The left panel shows an exemplary kinetic representing one out of three independent experiments (from right panel), where each dot indicates the mean of technical duplicates. In the bar diagram, infection events are normalized on the Hu-1 S (set at 1) and p values indicate difference to the Hu-1 S. Bars represent the mean of three independent experiments (±SEM). Statistical significance was tested by one-way ANOVA. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. (D) Infection kinetics (left panel) or infection events (right panel) of A549-ACE2-TMPRSS2 cells infected by VSVpp containing the wild type or the indicated Hu-based S proteins. The left panel shows an exemplary kinetic representing one out of three independent experiments (from right panel), where each dot indicates the mean of technical duplicates. In the bar diagram, infection events are normalized on the Hu-1 S (set at 1) and p values indicate difference to the Hu-1 S. Bars represent the mean of three independent experiments (±SEM). Statistical significance was tested by one-way ANOVA. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. (E) Correlation of the pseudotype infection events of the wild-type S or indicated Hu-based mutant S proteins harboring mutations characteristic of BA.1 and/or BA.2 S in A549 overexpressing ACE2 alone/with TMPRSS2. Each dot represents the average value derived from three infection experiments (shown in C and 3D). Coefficient of correlation (r), coefficient of determination (R 2 -values) and two tailed p values are provided. (F) Automated quantification of GFP fluorescence of A549-ACE2-TMPRSS2 (left panel) or Caco2 (right panel) cells infected with VSVΔG-GFP pseudotyped with the indicated S variants. Cells were pre-treated (1 h, 37°C) with 20 μM of camostat or E64d in the highest concentration and diluted in a 1:5 titration row. Symbols represent the mean of three independent experiments (±SEM). (G) Quantification of viral N RNA levels in the supernatant of A549-ACE2-TMPRSS2 (left panel) and Caco2 (right panel) cells infected with the indicated SARS-CoV-2 variants (MOI = 0.05), collected at 48 h post-infection. Cells were pre-treated (1 h, 37°C) with 20 μM of camostat or E64d. The right graph of each panel shows the reduction of vRNA levels in the supernatants relative to the untreated control (set at 1). Bars represent the mean of three independent experiments (±SEM). Non-infected controls were below the quantification level (<1000). Statistical significance was tested by two-way ANOVA. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. See also Figure S3 .

Article Snippet: Primer/Probe for qRT-PCR of Human TMPRSS2: TaqManTM Gene Expression Assay (FAM-MGB) Hs01122322_m1 , Thermo Fisher , Cat#4331182.

Techniques: Infection, Transfection, Mutagenesis, Derivative Assay, Two Tailed Test, Fluorescence, Concentration Assay, Titration, Control

Journal: iScience

Article Title: Impact of mutations defining SARS-CoV-2 Omicron subvariants BA.2.12.1 and BA.4/5 on Spike function and neutralization

doi: 10.1016/j.isci.2023.108299

Figure Lengend Snippet:

Article Snippet: Primer/Probe for qRT-PCR of Human TMPRSS2: TaqManTM Gene Expression Assay (FAM-MGB) Hs01122322_m1 , Thermo Fisher , Cat#4331182.

Techniques: Virus, Recombinant, Saline, Mutagenesis, Binding Assay, Expressing, Construct, Gene Expression, Plasmid Preparation, Software, Modification, Membrane, Semi Dry Blot, Transfection

Journal: Cells

Article Title: Aprotinin Inhibits SARS-CoV-2 Replication

doi: 10.3390/cells9112377

Figure Lengend Snippet: Antiviral effects of aprotinin in SARS-CoV-2-infected air–liquid interface (ALI) cultures from primary bronchial epithelial cells. ( A ) Abundance of the SARS-CoV-2 proteins N (nucleocapsid) and M (membrane) in primary bronchial epithelial cell ALI cultures infected with SARS-CoV-2/FFM1 (MOI of 1) in the presence or absence of aprotinin (20 µM) as determined 5 days post-infection by multiplexed mass spectrometry analysis using acquisition targeting of previously identified viral peptides modified with TMTpro. The detailed data are presented in . ( B ) Western blots indicating cellular SARS-CoV-2 N and TMPRSS2 levels in primary bronchial epithelial cell ALI cultures infected with SARS-CoV-2/7/Human/2020/Frankfurt (FFM7) (MOI of 1) in the presence or absence of aprotinin as detected 5 days post infection. GAPDH was served as the loading control. Uncropped Western blots are shown in .

Article Snippet: Detection occurred by using specific antibodies against SARS-CoV-2 N (1:1000 dilution, SARS-CoV-2 Nucleocapsid Antibody, Rabbit monoclonal antibody (Mab), #40143-R019, Sino Biological), ACE2 (1:500 dilution, Anti-ACE2 antibody, #ab15348, Abcam, Berlin, Germany), TMPRSS2 (1:1000 dilution, Recombinant Anti-TMPRSS2 antibody [EPR3861], #ab92323, Abcam), and GAPDH (1:1000 dilution, Anti-G3PDH Human Polyclonal Antibody, #2275-PC-100, Trevigen, Wiesbaden, Germany).

Techniques: Infection, Mass Spectrometry, Modification, Western Blot

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