tmprss2 activating protease  (ATCC)

Journal: Journal of Nanobiotechnology

Article Title: A bivalent spike-targeting nanobody with anti-sarbecovirus activity

doi: 10.1186/s12951-025-03243-y

Figure Lengend Snippet: Monovalent and bivalent 7F constructs neutralize authentic SARS-CoV-2 in A549 cells and HAE cell cultures and authentic SARS-CoV in Vero cells. A . Neutralization of SARS-CoV-2 in A549 ACE2+TMPRSS2+ cells. Virus was pre-incubated with serial diluted nanobody, or 10 µM remdesivir, for 30 min before infecting A549 ACE2+TMPRSS2+ cells. Infection was quantified by measuring the virus yield (viral RNA copies/ml, as determined with RT-qPCR) in cell culture supernatants of SARS-CoV-2 infected cells. B . Neutralization of SARS-CoV-2 in HAE cell culture. HAE cultures were incubated with SARS-CoV-2 and 100 nM nanobodies or 10 µM remdesivir on the apical side for 2 h. Nanobody incubation was repeated every 24 h. Graph showing the quantification of viral replication in the cultures, evaluated by RT-qPCR. C . Neutralization of SARS-CoV in Vero cells. Virus was pre-incubated with serial diluted nanobody, or 10 µM remdesivir, for 30 min before infecting Vero cells. Infection was quantified by measuring the virus yield (viral RNA copies/ml, as determined with RT-qPCR) in cell culture supernatants of SARS-CoV infected cells

Article Snippet: A549 cells ( Homo sapiens , lung carcinoma, ATCC CCL-185) expressing human ACE2 receptor protein and TMPRSS2 activating protease (A549 ACE2+TMPRSS2+ ) [ ], Vero cells ( Cercopithecus aethiops , kidney epithelial, ATCC CCL-81) and VeroE6 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% FBS for A549 and Vero cells and 10% FBS for VeroE6 cells, 1 mM sodium pyruvate (Gibco), nonessential amino acids (Lonza), penicillin (100 IU/ml), and streptomycin (100 IU/ml).

Techniques: Construct, Neutralization, Virus, Incubation, Infection, Quantitative RT-PCR, Cell Culture

Journal: mBio

Article Title: Serine Protease Inhibitors Restrict Host Susceptibility to SARS-CoV-2 Infections

doi: 10.1128/mbio.00892-22

Figure Lengend Snippet: Donor differences in infection levels and immune response following SARS-CoV-2 infection of HBEC ALI cultures. HBEC ALI cultures from 8 different donors were infected with SARS-CoV-2 at an MOI of 0.05. The accumulated viral release from the apical side of cultures from (A) female donors, n = 4, and (B) male donors, n = 4, was quantified by qPCR at the indicated time points. (C) Heatmap displaying the mean expression (log 2 ) of ACE2 and TMPRSS2 in HBEC ALI cultures from each donor. (D) Volcano plot showing differentially expressed genes between uninfected (mock) and infected HBEC ALI cultures in group high. The statistical P value (–log 10 ) is plotted against the gene expression difference (log 2 ). Dotted lines highlight the significance cutoff at log fold changes of −1/1 (vertical line) and at a P value of 0.05 (horizontal line). (E) Heatmap displaying the significantly upregulated genes in group high upon infection. Shown are the mean expression difference compared to individual mock samples for each group (log 2 fold difference). (F and G) Cytokine levels in (F) apical and (G) basolateral samples collected at 72 h postinfection were analyzed using Proximity Extension Assay (Olink) and normalized to mock-treated samples for each donor individually. Mean values and standard error of the mean (SEM) are shown; statistical significance was calculated by unpaired t test (*, P < 0.05; **, P < 0.01).

Article Snippet: Active recombinant TMPRSS2 was purchased from Cusabio (CSB-YP023924HU).

Techniques: Infection, Expressing, Gene Expression

Journal: mBio

Article Title: Serine Protease Inhibitors Restrict Host Susceptibility to SARS-CoV-2 Infections

doi: 10.1128/mbio.00892-22

Figure Lengend Snippet: The investigated serpins reduce SARS-CoV-2 infection by inhibition of TMPRSS2-mediated spike protein cleavage. (A) HEK-293T cells transfected with the indicated expression plasmids for 24 h were infected with SARS-CoV-2 (MOI = 0.1) for 6 h, and viral RNA was measured by qPCR. (B and C) Posttransfection (24 h) HEK293T cells were infected for 2 h (MOI = 1) and then trypsinized, washed with PBS, and lysed, and the RNA was extracted. Levels of viral RNA were quantified from cDNA synthesized with (B) random hexamers (C) or only the forward primer selectively quantifying the negative sense RNA. Data are cumulative of three independent experiments performed in triplicate; mean and SEM are shown, and statistical significance was calculated by unpaired t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (D) Surface plasmon resonance analysis of TMPRSS2 binding to individual serpins. A 2-fold dilution series of TMPRSS2 ranging from 125 nM down to 7.8 nM over immobilized SERPINE1 with results shown as response units (RU). Binding kinetics for all serpins are summarized to the right, including the natural target for SERPINE1, tissue plasminogen activator (tPA), as a positive control. (E) TMPRSS2-mediated S-protein cleavage in the presence or absence of individual serpins and the known protease inhibitor nafamostat mesylate. Data from three independent experiments were quantified, and a representative blot is shown. (F) The intensity of bands in panel E corresponding to cleaved S-protein was quantified using ImageJ (Fuji) and normalized to S-protein and TMPRSS2 control. Mean values and SEM are shown; statistical significance was calculated by unpaired t test (*, P < 0.05; **, P < 0.01). (G) HBEC ALI cultures were preincubated apically with recombinant SERPINE1, SERPINA1, or SERPINC1 and infected with SARS-CoV-2 at an MOI of 0.05. The accumulated viral release from the apical side was quantified by qPCR at the indicated time points ( n = 3). Mean and SEM are shown; statistical significance was calculated by unpaired t test (*, P < 0.05; **, P < 0.01). (H) The concentrations of apically released SERPINA1 and SERPINE2 from HBEC ALI cultures from both group high and group low were determined by ELISA. The apical secretions were collected at three time points ( n = 3). Mean and SEM are shown; statistical significance was calculated by unpaired t test (***, P < 0.001).

Article Snippet: Active recombinant TMPRSS2 was purchased from Cusabio (CSB-YP023924HU).

Techniques: Infection, Inhibition, Transfection, Expressing, Synthesized, SPR Assay, Binding Assay, Positive Control, Protease Inhibitor, Control, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: ACS Infectious Diseases

Article Title: Antiviral Potential of the Antimicrobial Drug Atovaquone against SARS-CoV-2 and Emerging Variants of Concern

doi: 10.1021/acsinfecdis.1c00278

Figure Lengend Snippet: Atovaquone restricts wtVSV and VSV-SARS spike infectivity in VeroE6 and VeroE6 hTMPRSS2-hACE2 cells. VeroE6 cells or VeroE6 cells stably transduced with hACE2 and TMPRSS2 were seeded at 2.5 × 10 4 cells/cm 2 in 96-well plates and treated with various concentrations of atovaquone. Fifteen minutes later, cells were infected at an MOI of 1 with wtVSV-spike (Whelan strain) or wtVSV expressing GFP. Eleven hours postinfection, cells were imaged and GFP counts were obtained using the ArrayScan High Content Platform (Thermo Scientific Cellomics). Forty-eight hours postinfection, the viability was determined using resazurin sodium salt (Sigma-Aldrich). (A, D) Graphs show % GFP counts (left axis) normalized to untreated, infected conditions for VSV-spike (green squares, N = 5) and wtVSV (blue triangles, N = 2). The right axis shows % viability normalized to untreated, uninfected conditions for atovaquone alone (black circles, N = 3), atovaquone-treated, VSV-spike-infected cells (green squares, N = 5), or atovaquone-treated, wtVSV-infected cells (blue triangles, N = 2). Symbols boxed in red demonstrate a significant difference over untreated cells ( p < 0.05 and p < 0.005 for all atovaquone-treated, VSV-spike-infected GFP counts using a t -test). Representative fluorescent images are shown for each condition (C, F), and IC 50 values are indicated for VSV-spike and wt-VSV (B,E). Vehicle = medium containing the atovaquone-diluting agent, DMSO. Media = culture medium only.

Article Snippet: The assay was initiated with 2 μL of recombinant active human TMPRSS2 (Cusabio Biotech) at 10 μM, after which the 384-well plate was shaken at 500 rpm for 1 min for complete mixing.

Techniques: Infection, Stable Transfection, Transduction, Expressing

Journal: ACS Infectious Diseases

Article Title: Antiviral Potential of the Antimicrobial Drug Atovaquone against SARS-CoV-2 and Emerging Variants of Concern

doi: 10.1021/acsinfecdis.1c00278

Figure Lengend Snippet: Atovaquone partially requires TMPRSS2 to drive its antiviral action against SARS-CoV-2 and reduces the interaction between the spike protein and its surface receptor ACE2. (A) Schematic of atovaquone administration. (B) VeroE6 hTMPRSS2 cells were treated with atovaquone (10 μM) for 2 h before infection (full time), at the time of infection (entry), or 1 h after infection (postentry), before challenging with original SARS-CoV-2 at an MOI of 0.1. Infection was carried out for 48 h in the presence of the drug and for all conditions. Infection was assessed by immunoblotting of the spike protein within cell lysates. (C) Structure of constructs for a split NanoLuc-based bioreporter. RBD or S1 from either SARS-CoV1 or SARS-CoV2 was linked to Large BiT (LgBiT) on its N-terminus to form LgBiT-RBD or LgBiT-S1; similarly, the Small BiT (SmBiT) peptide was linked to human ACE2 to form SmBiT-ACE2. ACE2 and RBD or S1 constructs were transfected separately or cotransfected into HEK293 cells for 48 h and lysed with a passive lysis buffer. Mixed lysates or lysates from cotransfected cells were incubated with coelenterazine, and the luminescence measured using a plate reader. (D) Following plasmid transfection into HEK293 cells, the cells were lysed in a NanoLuc-compatible passive lysis buffer and lysates were dispensed into a 96-well plate, to which atovaquone was added at a final concentration of 4 μM. The impact of atovaquone on SARS receptor binding was assessed in two ways: (1) with atovaquone added to LgBiT-RBD or LgBiT-S1 for 50 min followed by the addition of an equal quantity of SmBiT-ACE2 for another 10 min (“mixed lysates)” or (2) with atovaquone added to the preformed SmBiT-ACE2 + LgBiT-RBD/S1 complex for 1 h (“cotransfected).” Following incubation, a nanoluciferase substrate was added and the luminescence was measured. HEK293 cells were also transfected with a nanoluciferase control plasmid and lysates were incubated with 4 μM atovaquone. N = 4 per condition. The graph shows % bioreporter luminescence with the highest value in each of the respective untreated conditions taken as 100% ( N = 4 per condition, the p values were determined using a t -test). (E, F). The different cell lines mentioned were subjected to immunoblotting (E) and flow cytometry analysis (F) of TMPRSS2 and ACE2 expression. For ACE2, unstained samples of the matched cell line were used as a control. For TMPRSS2, secondary antibody-stained samples of the matched cell line were used as a control. Controls for the A549 hACE2 cell line are represented in the figure. The same controls were used for all cell lines studied. (G) Vero hTMPRSS2, Calu-3, and A549 hACE2 cells were pretreated with atovaquone (100 μM) for 2 h before infection with the original SARS-CoV-2 (MOI of 0.1). Viral RNA levels were determined 48 h postinfection by qPCR. The data represent the means ± SEM of one experiment performed in biological triplicates. (H) HEK293T cells expressing hACE2 cells were mock-transfected or transfected with a plasmid encoding hTMPRSS2. Twenty-four hours post-transfection, cells were seeded in 96-well plates and preincubated with the different drugs E64d (10 μM), camostat (25 μM), and atovaquone (100 μM) + 5 μg/mL polybrene for 1 h before infection with purified SARS-CoV-2 pseudotypes. LacZ + cells were quantified using the Beta-Glo assay system and luminescence measurement. The data are the means ± SEM of two experiments performed in biological triplicates. Similar results were obtained by X-gal staining. (I) Effector cells (zipV2+) expressing SARS-CoV-2 spike and target cells (zipV1+) expressing ACE2 with or without TMPRSS2 were cocultured for 3 h in the presence of the indicated concentration of drugs or DMSO. Cell–cell fusion was assessed by measuring the fluorescence of the Venus protein complementation (zipV1 + zipV2). Data were normalized to the fusion obtained with target cells expressing ACE2 but not TMPRSS2 (ACE2 + TMPRSS2) and are the means ± SEM of two experiments performed in triplicates. The p values were calculated using a t -test where * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The assay was initiated with 2 μL of recombinant active human TMPRSS2 (Cusabio Biotech) at 10 μM, after which the 384-well plate was shaken at 500 rpm for 1 min for complete mixing.

Techniques: Infection, Western Blot, Construct, Transfection, Lysis, Incubation, Plasmid Preparation, Concentration Assay, Binding Assay, Control, Flow Cytometry, Expressing, Staining, Purification, Glo Assay, Fluorescence