Journal: Molecules

Article Title: Evaluation of the Cytotoxic and Antiviral Effects of Small Molecules Selected by In Silico Studies as Inhibitors of SARS-CoV-2 Cell Entry

doi: 10.3390/molecules28207204

Figure Lengend Snippet: Docking score of the ten compounds selected from our in-house library, and ponatinib, for the ACE2 binding pocket (6M18) and the RBD of the spike protein (6M0J), calculated using Autodock Vina.

Article Snippet: Cell pellets were collected after 48 h incubation, and the following lysis, quantification, and Western Blot procedures were performed as previously described [ ], except for the following points: (i) no phosphatase inhibitor was used; (ii) a total of 15 μg of protein lysates obtained from MDA-MB-231 cells treatment or 50 μg of protein lysates obtained from Vero CCL-81 cells were loaded and separated in 10% SDS-Page gels; (iii) the transference to a nitrocellulose membrane (GE Healthcare Life science, Chalfont St Giles, UK; GE10600002) occurred for 1 h 45 min at 100 V; (iv) the primary antibodies (1:2000) used were: mouse anti-ACE2 monoclonal antibody (CL4035; Thermo Fischer Scientific; Waltham, MA, USA) and mouse anti-actin (sc-47778; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Binding Assay

Journal: Molecules

Article Title: Evaluation of the Cytotoxic and Antiviral Effects of Small Molecules Selected by In Silico Studies as Inhibitors of SARS-CoV-2 Cell Entry

doi: 10.3390/molecules28207204

Figure Lengend Snippet: In silico virtual screening. Top-ranked selected test compounds docked into ( A ) ACE2 and ( B ) the RBD of the spike protein. The compounds selected from the in silico studies are represented with sticks in different colors. Crystal structures were obtained from the Protein Data Bank (ACE2, PDB code 6M18 and the RBD of the spike protein, PDB code 6M0J ) and are represented as surface plots.

Article Snippet: Cell pellets were collected after 48 h incubation, and the following lysis, quantification, and Western Blot procedures were performed as previously described [ ], except for the following points: (i) no phosphatase inhibitor was used; (ii) a total of 15 μg of protein lysates obtained from MDA-MB-231 cells treatment or 50 μg of protein lysates obtained from Vero CCL-81 cells were loaded and separated in 10% SDS-Page gels; (iii) the transference to a nitrocellulose membrane (GE Healthcare Life science, Chalfont St Giles, UK; GE10600002) occurred for 1 h 45 min at 100 V; (iv) the primary antibodies (1:2000) used were: mouse anti-ACE2 monoclonal antibody (CL4035; Thermo Fischer Scientific; Waltham, MA, USA) and mouse anti-actin (sc-47778; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: In Silico

Journal: Molecules

Article Title: Evaluation of the Cytotoxic and Antiviral Effects of Small Molecules Selected by In Silico Studies as Inhibitors of SARS-CoV-2 Cell Entry

doi: 10.3390/molecules28207204

Figure Lengend Snippet: Molecular visualization of sulfonamide xanthenes 1 and 2 in ACE2. ( A ) General view of compounds 1 (green) and 2 (brown). ( B ) Interaction between compound 1 and ACE2. ( C ) Interaction between compound 2 and ACE2. Polar interactions are represented as yellow dashes. Residues involved on those interactions are labeled: Ala—alanine; Asn—asparagine; Asp—aspartic acid; His—histidine; Tyr—tyrosine.

Article Snippet: Cell pellets were collected after 48 h incubation, and the following lysis, quantification, and Western Blot procedures were performed as previously described [ ], except for the following points: (i) no phosphatase inhibitor was used; (ii) a total of 15 μg of protein lysates obtained from MDA-MB-231 cells treatment or 50 μg of protein lysates obtained from Vero CCL-81 cells were loaded and separated in 10% SDS-Page gels; (iii) the transference to a nitrocellulose membrane (GE Healthcare Life science, Chalfont St Giles, UK; GE10600002) occurred for 1 h 45 min at 100 V; (iv) the primary antibodies (1:2000) used were: mouse anti-ACE2 monoclonal antibody (CL4035; Thermo Fischer Scientific; Waltham, MA, USA) and mouse anti-actin (sc-47778; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Labeling

Journal: Molecules

Article Title: Evaluation of the Cytotoxic and Antiviral Effects of Small Molecules Selected by In Silico Studies as Inhibitors of SARS-CoV-2 Cell Entry

doi: 10.3390/molecules28207204

Figure Lengend Snippet: Molecular visualization of bile acid derivative 4 in ( A ) ACE2 and ( B ) the RBD of the spike protein. Polar interactions are represented as yellow dashes. Residues involved on those interactions are labeled: Asn—asparagine; Asp—aspartic acid; His—histidine; Tyr—tyrosine.

Article Snippet: Cell pellets were collected after 48 h incubation, and the following lysis, quantification, and Western Blot procedures were performed as previously described [ ], except for the following points: (i) no phosphatase inhibitor was used; (ii) a total of 15 μg of protein lysates obtained from MDA-MB-231 cells treatment or 50 μg of protein lysates obtained from Vero CCL-81 cells were loaded and separated in 10% SDS-Page gels; (iii) the transference to a nitrocellulose membrane (GE Healthcare Life science, Chalfont St Giles, UK; GE10600002) occurred for 1 h 45 min at 100 V; (iv) the primary antibodies (1:2000) used were: mouse anti-ACE2 monoclonal antibody (CL4035; Thermo Fischer Scientific; Waltham, MA, USA) and mouse anti-actin (sc-47778; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Labeling

Journal: Molecules

Article Title: Evaluation of the Cytotoxic and Antiviral Effects of Small Molecules Selected by In Silico Studies as Inhibitors of SARS-CoV-2 Cell Entry

doi: 10.3390/molecules28207204

Figure Lengend Snippet: Molecular visualization of derivatives glucosulfated xanthone 7 and 9 in ACE2. ( A ) General view of compounds 7 (yellow) and 9 (blue). ( B ) Interaction between compound 7 and residues of ACE2. ( C ) Interaction between compound 9 and residues of ACE2. Polar interactions are represented as yellow dashes. Residues involved on those interactions are labeled: Ala—alanine; Arg—arginine; Asp—aspartic acid; Gln—glutamine; Gly—glycine; NAG—N-acetylglucosamine; Thr—threonine; Tyr—tyrosine.

Article Snippet: Cell pellets were collected after 48 h incubation, and the following lysis, quantification, and Western Blot procedures were performed as previously described [ ], except for the following points: (i) no phosphatase inhibitor was used; (ii) a total of 15 μg of protein lysates obtained from MDA-MB-231 cells treatment or 50 μg of protein lysates obtained from Vero CCL-81 cells were loaded and separated in 10% SDS-Page gels; (iii) the transference to a nitrocellulose membrane (GE Healthcare Life science, Chalfont St Giles, UK; GE10600002) occurred for 1 h 45 min at 100 V; (iv) the primary antibodies (1:2000) used were: mouse anti-ACE2 monoclonal antibody (CL4035; Thermo Fischer Scientific; Waltham, MA, USA) and mouse anti-actin (sc-47778; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Labeling

Journal: Molecules

Article Title: Evaluation of the Cytotoxic and Antiviral Effects of Small Molecules Selected by In Silico Studies as Inhibitors of SARS-CoV-2 Cell Entry

doi: 10.3390/molecules28207204

Figure Lengend Snippet: Levels of ( A ) spike and ( B ) ACE2 proteins in Vero CCL-81 cells treated with the IC 50 concentrations of the tested compounds (or 50 μM for compound 9 ) and infected with SARS-CoV-2 (MOI = 1), analyzed by Western blot. Actin was used as a loading control. Representative blots are shown. Data represents the mean ± SEM of at least three independent experiments. Analysis was performed by GraphPad using the Student t -test. * p < 0.05; *** p < 0.001 relative to the vehicle (DMSO for compounds 1 , 2 and 4 ; H 2 O for compounds 7 and 9 ).

Article Snippet: Cell pellets were collected after 48 h incubation, and the following lysis, quantification, and Western Blot procedures were performed as previously described [ ], except for the following points: (i) no phosphatase inhibitor was used; (ii) a total of 15 μg of protein lysates obtained from MDA-MB-231 cells treatment or 50 μg of protein lysates obtained from Vero CCL-81 cells were loaded and separated in 10% SDS-Page gels; (iii) the transference to a nitrocellulose membrane (GE Healthcare Life science, Chalfont St Giles, UK; GE10600002) occurred for 1 h 45 min at 100 V; (iv) the primary antibodies (1:2000) used were: mouse anti-ACE2 monoclonal antibody (CL4035; Thermo Fischer Scientific; Waltham, MA, USA) and mouse anti-actin (sc-47778; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Infection, Western Blot

Journal: Molecules

Article Title: Evaluation of the Cytotoxic and Antiviral Effects of Small Molecules Selected by In Silico Studies as Inhibitors of SARS-CoV-2 Cell Entry

doi: 10.3390/molecules28207204

Figure Lengend Snippet: Summary of the results obtained for compounds 1 , 2 , 4 , 7 , and 9 .

Article Snippet: Cell pellets were collected after 48 h incubation, and the following lysis, quantification, and Western Blot procedures were performed as previously described [ ], except for the following points: (i) no phosphatase inhibitor was used; (ii) a total of 15 μg of protein lysates obtained from MDA-MB-231 cells treatment or 50 μg of protein lysates obtained from Vero CCL-81 cells were loaded and separated in 10% SDS-Page gels; (iii) the transference to a nitrocellulose membrane (GE Healthcare Life science, Chalfont St Giles, UK; GE10600002) occurred for 1 h 45 min at 100 V; (iv) the primary antibodies (1:2000) used were: mouse anti-ACE2 monoclonal antibody (CL4035; Thermo Fischer Scientific; Waltham, MA, USA) and mouse anti-actin (sc-47778; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques:

Journal: Viruses

Article Title: Cross-Neutralization of SARS-CoV-2-Specific Antibodies in Convalescent and Immunized Human Sera against the Bat and Pangolin Coronaviruses

doi: 10.3390/v14081793

Figure Lengend Snippet: Expression and cell-cell fusion activity of sarbecovirus spikes in VeroE6 cells. ( A ). The sequence alignment of the RBD of SARS-CoV-2 and that of PCoV-GD, RShSTT182, and RaTG13. Black triangles indicate interacting amino acids of the RBD of SARS-CoV-2 with hACE2. Alignment was performed using QIAGEN CLC Genomics Workbench and percent similarity was analyzed using the web-based platform SIAS (Universidad Complutense de Madrid). ( B ). Expression of each spike variant in HEK-293T cells in the absence and presence of trypsin. The unprocessed spike (S0) and the cleaved spike product (S1) are indicated by arrows. Expression of beta-actin served as a loading control. ( C ). Cell–cell fusion in VeroE6 cells transfected with each spike variant in the presence of trypsin. Images were taken under an inverted light microscope 24 h after transfection (100-μm scale bars). Black arrows mark representative areas where cell–cell fusion was observed.

Article Snippet: For ACE2 detection, mouse monoclonal anti-human ACE2 antibodies (Thermo Scientific) were used as primary antibodies.

Techniques: Expressing, Activity Assay, Sequencing, Variant Assay, Transfection, Light Microscopy

Journal: Viruses

Article Title: Cross-Neutralization of SARS-CoV-2-Specific Antibodies in Convalescent and Immunized Human Sera against the Bat and Pangolin Coronaviruses

doi: 10.3390/v14081793

Figure Lengend Snippet: Spike of RaTG13 could trigger cell–cell fusion in cells strongly expressing human ACE2. ( A ). The level of ACE2 expression in cell lines susceptible to SARS-CoV-2 infection. Using antibodies against human ACE2, Western blot analysis of lysates from each cell line was performed. Beta-actin expression served as a loading control. To detect ACE2 expression in VeroE6 cells, a longer exposure time was used and the lane of HEK-293T-ACE2 was excluded. ( B ) . Cell–cell fusion in HEK293T-ACE2 cells transfected with each variant of the spike protein in the presence of trypsin. Images were taken 24 h following transfection using an inverted light microscope (scale bar of 100 µm). The black arrows indicate areas where cell–cell fusion was observed.

Article Snippet: For ACE2 detection, mouse monoclonal anti-human ACE2 antibodies (Thermo Scientific) were used as primary antibodies.

Techniques: Expressing, Infection, Western Blot, Transfection, Variant Assay, Light Microscopy

Journal: Viruses

Article Title: Cross-Neutralization of SARS-CoV-2-Specific Antibodies in Convalescent and Immunized Human Sera against the Bat and Pangolin Coronaviruses

doi: 10.3390/v14081793

Figure Lengend Snippet: Entry of pseudo-viruses carrying each spike into HEK-293T-ACE2 cells. After pseudo-virus entry, the luciferase signal was used to evaluate the entry efficiency. The entry efficiency of SARS-CoV-2 wild-type pseudo-viruses was taken as 100%. The error bars depict the standard deviation ( n = 5) with each dot representing each replication. Normalization of the relative luminescence unit (RLU) of Luc reporter gene expression to the p24 content of the pseudo-viruses. Adjusted p values were calculated using one-way ANOVA: ** p 0.01, **** p 0.0001.

Article Snippet: For ACE2 detection, mouse monoclonal anti-human ACE2 antibodies (Thermo Scientific) were used as primary antibodies.

Techniques: Luciferase, Standard Deviation, Expressing

Journal: Placenta

Article Title: Placental SARS-CoV-2 distribution correlates with level of tissue oxygenation in COVID-19-associated necrotizing histiocytic intervillositis/perivillous fibrin deposition

doi: 10.1016/j.placenta.2021.12.002

Figure Lengend Snippet: Immunohistochemical analysis of CAIX and ACE2 protein expression in the placenta. Representative immunohistochemical analysis of expression of carbonic anhydrase IX (CAIX) (A–B) and angiotensin converting enzyme 2 (ACE2) (C–E) in the placenta. CAIX immunoreactivity, localized to villous trophoblastic and stromal cells, is more intense in fibrin-encased villi than in free-floating villi (A–B), consistent with relative tissue hypoxia. Similarly, protein expression of the SARS-CoV-2 receptor, ACE2, is more intense in fibrin-trapped villi than in free villi (C–E). (A–E: DAB-peroxidase system with hematoxylin counterstain, original magnification ×100 (A,C) and X 400 (B, D-E)).

Article Snippet: Tissue sections were incubated sequentially with polyclonal rabbit anti-SARS-CoV-2 antibody, Alexa Fluor 594-conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), mouse monoclonal anti-ACE2 antibody (ThermoFisher Scientific (Invitrogen), Waltham, MA), biotinylated anti-mouse IgG (Vector Laboratories, Inc., Burlingame, CA) and Alexa Fluor 488-conjugated streptavidin (Jackson ImmunoResearch Laboratories, Inc.).

Techniques: Immunohistochemical staining, Expressing

Journal: Placenta

Article Title: Placental SARS-CoV-2 distribution correlates with level of tissue oxygenation in COVID-19-associated necrotizing histiocytic intervillositis/perivillous fibrin deposition

doi: 10.1016/j.placenta.2021.12.002

Figure Lengend Snippet: Combined immunofluorescence analysis of placental colocalization of SARS-CoV-2 and ACE2. Confocal fluorescence microscopy of the placenta subjected to combined anti-ACE2 (green) and anti-SARS-CoV-2 (red) immunofluorescence, captured at the same settings. Yellow dots represent regions of colocalization of ACE2 and SARS-CoV-2 in trophoblastic cells. In selected fields, colocalization was confirmed quantitatively using Pearson's correlation coefficient analysis whereby a cutoff of r 2 > 0.5 was used to indicate positive colocalization. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Tissue sections were incubated sequentially with polyclonal rabbit anti-SARS-CoV-2 antibody, Alexa Fluor 594-conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), mouse monoclonal anti-ACE2 antibody (ThermoFisher Scientific (Invitrogen), Waltham, MA), biotinylated anti-mouse IgG (Vector Laboratories, Inc., Burlingame, CA) and Alexa Fluor 488-conjugated streptavidin (Jackson ImmunoResearch Laboratories, Inc.).

Techniques: Immunofluorescence, Fluorescence, Microscopy