Journal: Journal of Virology

Article Title: Host cell lectins ASGR1 and DC-SIGN jointly with TMEM106B confer ACE2 independence and imdevimab resistance to SARS-CoV-2 pseudovirus with spike mutation E484D

doi: 10.1128/jvi.01230-24

Figure Lengend Snippet: Huh-7 and NCI-H522 cells allow for ACE2-independent entry of mutant E484D. ( A ) ACE2 dependence of host cell entry. The indicated cell lines were grown in 96-well plates, incubated with ACE2 antibody (10108-MM36, Sino Biological) for 30 min and inoculated with pseudotypes bearing the indicated viral glycoproteins. Luciferase activity in cell lysates was quantified at 16–20 h postinoculation. The average of three independent experiments ± SEM is shown. Data were normalized against the assay background (i.e., particles bearing no glycoprotein, set as 1). Statistical significance was assessed by two-tailed Student’s t -test with Welch’s correction ( P > 0.05, not significant [ns]; P ≤ 0.05, *; P ≤ 0.01, **; P ≤ 0.001, ***; not determined [nd]). ( B ) Mutation E484D does not allow for S protein binding to ACE2 in complex with an entry-inhibiting antibody. The indicated S proteins were transiently expressed in 293T cells and the cells incubated with soluble ACE2 preincubated with the indicated concentrations of anti-ACE2 antibody. ACE2 binding was detected by incubation with a secondary antibody and the cells analyzed by flow cytometry. Soluble ACE2 binding to cells transfected with empty plasmid served as control. The average ±SEM of three biological replicates conducted with unicate samples is shown. Data were normalized against the assay background (i.e., cells incubated with secondary antibody alone). Statistical significance was assessed by two-tailed Student’s t -test with Welch’s correction ( P > 0.05, not significant [ns]; P ≤ 0.05, *; P ≤ 0.01, **).

Article Snippet: Particles bearing EBOV-GP, VSV-G, or no viral surface protein served as controls. (ii) In order to assess ACE2-independent entry into cell lines, Huh-7, Li-7, and NCI-H522 cells were preincubated (30 min, 37°C) with medium containing 7 µg/mL of anti-ACE2 neutralizing mouse monoclonal antibody (10108-MM36, Sino Biological), before addition of equal volumes of pseudotyped particles.

Techniques: Mutagenesis, Incubation, Luciferase, Activity Assay, Two Tailed Test, Protein Binding, Binding Assay, Flow Cytometry, Transfection, Plasmid Preparation, Control

Journal: Journal of Virology

Article Title: Host cell lectins ASGR1 and DC-SIGN jointly with TMEM106B confer ACE2 independence and imdevimab resistance to SARS-CoV-2 pseudovirus with spike mutation E484D

doi: 10.1128/jvi.01230-24

Figure Lengend Snippet: ACE2-independent entry correlates with resistance to imdevimab and bebtelovimab. Pseudotyped particles were preincubated with the indicated antibodies at the indicated concentrations for 1 h before addition to the indicated target cells. Luciferase activity in cell lysates was quantified at 16–20 h postinoculation. The average of three (two in case of NCI-H522 cells) independent experiments ± SEM is shown. Data were normalized against signals measured in the absence of antibody (= 0% inhibition). Curves were calculated using a non-linear regression model (variable slope).

Article Snippet: Particles bearing EBOV-GP, VSV-G, or no viral surface protein served as controls. (ii) In order to assess ACE2-independent entry into cell lines, Huh-7, Li-7, and NCI-H522 cells were preincubated (30 min, 37°C) with medium containing 7 µg/mL of anti-ACE2 neutralizing mouse monoclonal antibody (10108-MM36, Sino Biological), before addition of equal volumes of pseudotyped particles.

Techniques: Luciferase, Activity Assay, Inhibition

Journal: Journal of Virology

Article Title: Host cell lectins ASGR1 and DC-SIGN jointly with TMEM106B confer ACE2 independence and imdevimab resistance to SARS-CoV-2 pseudovirus with spike mutation E484D

doi: 10.1128/jvi.01230-24

Figure Lengend Snippet: Directed expression of ASGR1/DC-SIGN jointly with TMEM106B allows for efficient entry into otherwise non-susceptible cells. ( A ) 293T cells were transfected to express the indicated soluble S proteins (S1 domain protein of B.1 [WT] and B.1 [E484D] S proteins fused to the Fc portion of human immunoglobulin G), and S protein levels in cell lysates and supernatants were analyzed by immunoblot using anti-Fc antibody. The results of a representative experiment are shown and were confirmed in two additional experiments. ( B ) 293T cells transfected to express ASGR1, DC-SIGN, TMEM106B, or ACE2 were analyzed by immunoblot using anti-c-Myc (DC-SIGN, TMEM106B, and ACE2) and anti-AU1 (ASGR1) antibody. The results of a representative experiment are shown and were confirmed in a separate experiment. ( C ) 293T cells transfected to express ASGR1, DC-SIGN, TMEM106B, or ACE2 were incubated with the indicated soluble S proteins, and S protein binding was analyzed by flow cytometry. Presented are the average (mean) data from three (DC-SIGN, TMEM106B) or six (ASGR1, ACE2) biological replicates (each conducted with single samples). Statistical significance was assessed by two-tailed Student’s t -test with Welch’s correction ( P > 0.05, not significant [ns]; P ≤ 0.05, *; P ≤ 0.01, **). ( D ) BHK-21 cells were transfected with plasmids encoding ASGR1, DC-SIGN, TMEM106B, and ACE2 either alone or in combination and transduced with SARS-2-S pp and E484D pp followed by quantification of luciferase activity in cell lysates. Presented are the average (mean) data from three biological replicates (each conducted with four technical replicates), for which transduction was normalized against signals obtained from control-transfected cells inoculated with the respective pseudotyped particles (background, set as 1). Statistical significance was assessed by two-tailed Student’s t -test with Welch’s correction ( P > 0.05, not significant [ns]; P ≤ 0.05, *; P ≤ 0.01, **).

Article Snippet: Particles bearing EBOV-GP, VSV-G, or no viral surface protein served as controls. (ii) In order to assess ACE2-independent entry into cell lines, Huh-7, Li-7, and NCI-H522 cells were preincubated (30 min, 37°C) with medium containing 7 µg/mL of anti-ACE2 neutralizing mouse monoclonal antibody (10108-MM36, Sino Biological), before addition of equal volumes of pseudotyped particles.

Techniques: Expressing, Transfection, Western Blot, Incubation, Protein Binding, Flow Cytometry, Two Tailed Test, Transduction, Luciferase, Activity Assay, Control

Journal: Journal of Virology

Article Title: Host cell lectins ASGR1 and DC-SIGN jointly with TMEM106B confer ACE2 independence and imdevimab resistance to SARS-CoV-2 pseudovirus with spike mutation E484D

doi: 10.1128/jvi.01230-24

Figure Lengend Snippet: Directed expression of DC-SIGN or ASGR1 allows for imdevimab resistance and ACE2 independence of mutant E484D. For analysis of imdevimab resistance (A), 293T cells were transfected with ASGR1 or DC-SIGN encoding plasmids or empty plasmid as control and inoculated with pseudotypes that harbored the indicated S proteins and were pre-incubated with 1 µg/mL of imdevimab. Luciferase activities in cell lysates were quantified at 16–20 h postinoculation. For analysis of ACE2-independent entry (B), the experiment was conducted as described above, but cells were preincubated with 7 µg/mL anti-ACE2 antibody. Presented are the average (mean) data from three biological replicates (each conducted with four technical replicates), for which transduction was normalized against samples that did not contain antibody (= 100% pseudotype entry). Error bars indicate the SEM. Statistical significance was assessed by two-tailed Student’s t -test with Welch’s correction ( P > 0.05, not significant [ns]; P ≤ 0.05, *; P ≤ 0.01, **; P ≤ 0.001, ***).

Article Snippet: Particles bearing EBOV-GP, VSV-G, or no viral surface protein served as controls. (ii) In order to assess ACE2-independent entry into cell lines, Huh-7, Li-7, and NCI-H522 cells were preincubated (30 min, 37°C) with medium containing 7 µg/mL of anti-ACE2 neutralizing mouse monoclonal antibody (10108-MM36, Sino Biological), before addition of equal volumes of pseudotyped particles.

Techniques: Expressing, Mutagenesis, Transfection, Plasmid Preparation, Control, Incubation, Luciferase, Transduction, Two Tailed Test

Journal: Journal of Virology

Article Title: Host cell lectins ASGR1 and DC-SIGN jointly with TMEM106B confer ACE2 independence and imdevimab resistance to SARS-CoV-2 pseudovirus with spike mutation E484D

doi: 10.1128/jvi.01230-24

Figure Lengend Snippet: Endogenous expression of TMEM106B is required for DC-SIGN-dependent imdevimab resistance and ACE2 independence of mutant E484D. For analysis of ACE2-independent entry (A), 293T control (EV) or TMEM106B KO cells were transiently transfected with EV (control), or DC-SIGN plasmid or cotransfected with DC-SIGN and TMEM106B plasmids, incubated with 7 µg/mL of anti-ACE2 antibody and inoculated with pseudotypes bearing the indicated S proteins followed by quantification of luciferase activities in cell lysates at 16–20 h postinoculation. For analysis of imdevimab resistance (B), the experiment was conducted as described above, but pseudotyped particles were preincubated with 1 µg/mL imdevimab. Presented are the results from a representative experiment carried out with four technical replicates; transduction was normalized against samples that did not contain antibody. Error bars indicate SD, similar results were obtained in a separate experiment. Statistical significance was assessed by two-tailed Student’s t -test with Welch’s correction ( P > 0.05, not significant [ns]; P ≤ 0.05, *; P ≤ 0.01, **; P ≤ 0.001, ***).

Article Snippet: Particles bearing EBOV-GP, VSV-G, or no viral surface protein served as controls. (ii) In order to assess ACE2-independent entry into cell lines, Huh-7, Li-7, and NCI-H522 cells were preincubated (30 min, 37°C) with medium containing 7 µg/mL of anti-ACE2 neutralizing mouse monoclonal antibody (10108-MM36, Sino Biological), before addition of equal volumes of pseudotyped particles.

Techniques: Expressing, Mutagenesis, Control, Transfection, Plasmid Preparation, Incubation, Luciferase, Transduction, Two Tailed Test

Journal: Journal of Virology

Article Title: Host cell lectins ASGR1 and DC-SIGN jointly with TMEM106B confer ACE2 independence and imdevimab resistance to SARS-CoV-2 pseudovirus with spike mutation E484D

doi: 10.1128/jvi.01230-24

Figure Lengend Snippet: EGTA blocks ACE2-independent entry into Huh-7 cells. ( A ) Expression and N-glycosylation of ASGR1 in Huh-7 cells. Untransfected Huh-7 cells and 293T and BHK21 cells transfected with EV or ASGR1 encoding vector were control treated or treated with PNGaseF and analyzed for ASGR1 expression by immunoblot. Expression of β-actin served as loading control. Similar results were obtained in two (all cell lines) to four (293T, BHK21 cells) independent experiments. ( B ) Huh-7 cells were preincubated with medium alone (control) or medium containing anti-ACE2 antibody, EGTA, or anti-ACE2 antibody combined with EGTA for 1 h followed by inoculation with pseudotypes bearing the indicated S proteins. Luciferase activity in cell lysates was quantified at 16–20 h postinoculation. The results ± SD of a single experiment performed with technical quadruplicates are shown. Similar results were obtained in a separate experiment.

Article Snippet: Particles bearing EBOV-GP, VSV-G, or no viral surface protein served as controls. (ii) In order to assess ACE2-independent entry into cell lines, Huh-7, Li-7, and NCI-H522 cells were preincubated (30 min, 37°C) with medium containing 7 µg/mL of anti-ACE2 neutralizing mouse monoclonal antibody (10108-MM36, Sino Biological), before addition of equal volumes of pseudotyped particles.

Techniques: Expressing, Transfection, Plasmid Preparation, Control, Western Blot, Luciferase, Activity Assay

Journal: Journal of Advanced Research

Article Title: Key β1-4 galactosylated glycan receptors of SARS-CoV-2 and its inhibitor from the galactosylated glycoproteins of bovine milk

doi: 10.1016/j.jare.2024.12.010

Figure Lengend Snippet: Glycan profiles of the SARS-CoV-2 S1 and the ACE2 receptor. (A) Schematic diagram illustrating the process of preparing antibody-overlay lectin microarrays. (B, C) Scanned images were obtained for the analysis of glycopatterns from the SARS-CoV-2-S1 (B) and ACE2 (C). HEK293-expressing recombinant proteins of S1 and ACE2 were incubated with lectin microarrays. Subsequently, the microarrays were incubated with biotin-labeled primary antibodies and Cy3-labeled streptavidin. The representative lectins that recognized β1-4 galactosylated glycans (ECA and MAL-I), agalactosylated glycans (GSL-II and STL), bisected and bi-antennary N-glycans (PHA-E), oligo-mannose type N-glycans (ConA and HHL), fucosylation (PSA and LCA) and α-2,3 linked sialic acid (MAL-II) were marked with white frames. (D, E) Analysis of glycopatterns on S1 (D) and ACE2 (E). The lectins were classified according to their glycan binding preferences. The NFIs of each lectin were obtained from three biological replicates. The proportion of main types of glycans was calculated by diverging the sum of the NFIs of the lectins that recognized this type of glycan by the total NFIs of all lectins. Blue square: GlcNAc; yellow circle: galactose; yellow square: GalNAc; green circle: mannose; red triangle: fucose; purple diamond: sialic acid. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The primary antibodies used were as follows: a mouse monoclonal antibody against ACE2 (Proteintech, China), a rabbit polyclonal antibody against the SARS-CoV-2 S protein (ABclonal, China), and a mouse monoclonal antibody against GAPDH (Abways, China).

Techniques: Glycoproteomics, Expressing, Recombinant, Incubation, Labeling, Binding Assay

Journal: Journal of Advanced Research

Article Title: Key β1-4 galactosylated glycan receptors of SARS-CoV-2 and its inhibitor from the galactosylated glycoproteins of bovine milk

doi: 10.1016/j.jare.2024.12.010

Figure Lengend Snippet: Role of N-glycans in the interaction between S1 and ACE2. (A) Schematic diagram illustrating the process of manufacturing the SRAS-CoV-2-related recombinant protein microarrays. (B, C) The N-glycans on S1 of SARS-CoV-2/1 and ACE2 were removed by PNGase F glycosidase. The roles of N-glycans in the interaction between the SARS-CoV-2-S1 /ACE2 (B) and the SARS-CoV-1-S1/ACE2 (C) were evaluated using protein microarrays. Statistical analysis of the relative fluorescence intensities was conducted by comparing the PNGase F-treated S1 and ACE2 to the intact glycosylated protein using one-way ANOVA with Dunnett multiple comparisons. The data were obtained from three biological replicates and presented as the mean ± SD (error bars), and the p values were indicated. (D) MD simulation of the interaction between the trimeric S protein and ACE2. The distances between the N-glycosites and the center of the binding interface (represented by the green globule) within 50 Å were marked with red spheres. Other N-glycosites were marked with yellow spheres. (E) The interactions of glycans at specific sites and GRDs (marked with a red frame) may be involved in the binding of the S protein to ACE2. (F) MD simulated the interactions of glycans at specific sites and GRDs. The distances between the terminal glycans on these sites and the three GRDs on the ACE and S1 subunit were monitored during a 100 ns MD simulation. The distances of N546-GRD1, N322-GRD2, and N53-GRD2 fluctuated between 1 and 15 Å, while the distances of N343-GRD3 and N165-GRD3 fluctuated between 20 and 35 Å. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The primary antibodies used were as follows: a mouse monoclonal antibody against ACE2 (Proteintech, China), a rabbit polyclonal antibody against the SARS-CoV-2 S protein (ABclonal, China), and a mouse monoclonal antibody against GAPDH (Abways, China).

Techniques: Recombinant, Fluorescence, Binding Assay

Journal: Journal of Advanced Research

Article Title: Key β1-4 galactosylated glycan receptors of SARS-CoV-2 and its inhibitor from the galactosylated glycoproteins of bovine milk

doi: 10.1016/j.jare.2024.12.010

Figure Lengend Snippet: β1-4 galactosylated N-glycans of ACE2 mediated the binding of S1 of SARS-CoV-2 and its variants. (A) Molecular docking analysis of S1 and ACE2 with various saccharides. The potential binding capacities of S1 of SARS-CoV-2 (Wuhan-Hu-1 strain, wild type) and its variants (Delta and Omicron), as well as ACE2, to various saccharides were predicted by molecular docking analysis. The saccharides were listed in columns, S1 and ACE2 were listed in rows. The different binding abilities were represented by the values of binding free energy, which were indicated by the color of each square: red: high affinity, blue: low affinity, Xyl: xylose, Glc: glucose; Man: mannose; GlcNAc: N-acetylglucosamine, GalNAc: N-acetylgalactosamine; SA: sialic acid. (B) Validation of β1-4 galactosylation level in intact and de-β1-4galactosylated ACE2. After β1-4 galactosidase treatment, the level of β1-4 galactosylation on ACE2 was detected by lectin blotting of MAL-I. The protein level of ACE2 served as the control. (C) Scanning images of protein microarrays incubated with 1 μg of intact or de-β1-4galactosylated ACE2. (D) Effect of β1-4 galactosylation of ACE2 on the binding of S1 to ACE2. The relative fluorescence intensities were statistically analyzed by comparing the de-β1-4galactosylated ACE2 to intact ACE2 using an unpaired t test with Welch's correction. The data were obtained from three biological replicates and presented as the mean ± SD (error bars), and the p values were indicated. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The primary antibodies used were as follows: a mouse monoclonal antibody against ACE2 (Proteintech, China), a rabbit polyclonal antibody against the SARS-CoV-2 S protein (ABclonal, China), and a mouse monoclonal antibody against GAPDH (Abways, China).

Techniques: Binding Assay, Biomarker Discovery, Control, Incubation, Fluorescence

Journal: Journal of Advanced Research

Article Title: Key β1-4 galactosylated glycan receptors of SARS-CoV-2 and its inhibitor from the galactosylated glycoproteins of bovine milk

doi: 10.1016/j.jare.2024.12.010

Figure Lengend Snippet: Evaluation of the ability of free saccharides to block S1 and ACE2 binding. (A, B) Scanning images of protein microarrays. ACE2 was mixed with GalNAc (A) or Galβ1-3GalNAc (B), and the inhibitory effect of saccharides was evaluated using protein microarrays. (C, D) Effect of GalNAc (C) and Galβ-1,3GalNAc (D) on the interaction between S1 of SARS-CoV-2/1 and ACE2. The binding signals were extracted, and the relative fluorescence intensities were compared with those of the controls using one-way ANOVA with Dunnett multiple comparisons. The data were obtained from three biological replicates and presented as the mean ± SD (error bars), and the p values were indicated.

Article Snippet: The primary antibodies used were as follows: a mouse monoclonal antibody against ACE2 (Proteintech, China), a rabbit polyclonal antibody against the SARS-CoV-2 S protein (ABclonal, China), and a mouse monoclonal antibody against GAPDH (Abways, China).

Techniques: Blocking Assay, Binding Assay, Fluorescence

Journal: Journal of Advanced Research

Article Title: Key β1-4 galactosylated glycan receptors of SARS-CoV-2 and its inhibitor from the galactosylated glycoproteins of bovine milk

doi: 10.1016/j.jare.2024.12.010

Figure Lengend Snippet: Evaluation of isolated glycoproteins for the inhibition of S1 and ACE2 binding. (A) The scanned image was obtained from the lectin microarray analysis of glycoproteins isolated from bovine milk. The representative lectins that recognized β1-4 galactosylated glycans (ECA and MAL-I), agalactosylated glycans (GSL-II), bisected N-glycans (PHA-E), high-mannose glycans (ConA), fucosylation (AAL, PSA, and LCA), α2-3 linked sialic acid (MAL-II), and α2-6 linked sialic acid (SNA) were marked with white frames. (B) Analysis of glycopatterns on isolated glycoproteins. The lectins were classified according to their glycan binding preferences. The NFIs of each lectin were obtained from three biological replicates. The proportion of galactosylated glycans was calculated by diverging the sum of the NFIs of the lectins that recognized Gal/GalNAc by the total NFIs. (C, D) Evaluation of the effect of intact and de-sialylated isolated glycoproteins on the interaction between S1 of SARS-CoV-2/1 and ACE2. The intact isolated glycoproteins (C) or de-sialylated isolated glycoproteins (D) were mixed with ACE2 and incubated with protein microarrays. The relative binding intensities of each group were compared with those of the control group, and any significant differences between groups were determined using one-way ANOVA with Dunnett multiple comparisons. The data were obtained from three biological replicates and presented as the mean ± SD (error bars), and the p values were indicated. (E) Inhibition curves for intact isolated glycoproteins (upper) and de-sialylated isolated glycoproteins (lower). Four-parameter inhibition curves were generated, and the particular IC50 values for intact isolated glycoproteins and de-sialylated isolated glycoproteins were indicated in this graph. The data were obtained from three biological replicates and presented as the mean ± SD (error bars).

Article Snippet: The primary antibodies used were as follows: a mouse monoclonal antibody against ACE2 (Proteintech, China), a rabbit polyclonal antibody against the SARS-CoV-2 S protein (ABclonal, China), and a mouse monoclonal antibody against GAPDH (Abways, China).

Techniques: Isolation, Inhibition, Binding Assay, Microarray, Glycoproteomics, Incubation, Control, Generated

Journal: Frontiers in cellular and infection microbiology

Article Title: The antipsychotic drug lurasidone inhibits coronaviruses by affecting multiple targets.

doi: 10.3389/fcimb.2024.1487604

Figure Lengend Snippet: FIGURE 1 Effect of lurasidone on SARS-CoV-2 replication and inactivation. ToA experiments: 5 µM lurasidone was added to Huh-7-ACE2 cells for 1 hour before infection (1 h PRE), for 1 hour during infection (CO), or after 1, 4, 18 hours after virus inoculum (POST). Cells were treated with an equivalent volume of DMSO as a control (DMSO). Subsequently, supernatants were collected at 24 hours post-infection, and virus samples were titrated. Viral titers are expressed as mean PFU/mL and shown as average values with standard deviation and p-values, measured with a paired two-tailed t-test. Significant p-values, calculated from at least 2 independent experiments, are indicated by asterisks (**p < 0.01; *p < 0.05).

Article Snippet: The membranes were incubated overnight at 4°C with an anti-ACE2 AC18Z mouse monoclonal antibody (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-b-actin mouse monoclonal antibody (1:5000, Sigma Aldrich).

Techniques: Infection, Virus, Control, Standard Deviation, Two Tailed Test

Journal: Frontiers in cellular and infection microbiology

Article Title: The antipsychotic drug lurasidone inhibits coronaviruses by affecting multiple targets.

doi: 10.3389/fcimb.2024.1487604

Figure Lengend Snippet: FIGURE 2 Effect of Lurasidone on pseudoviral transduction. (A) Percentage of GFP-positive HEK293-ACE2 cells pre-incubated for 4 h with 1 or 5 µM lurasidone and infected with pseudovirus particles exposing SARS-CoV-2 Wuhan, B.1.1.7 UK or B.1.351 South Africa (SA) Spike protein. Control cells were pre-incubated with an equivalent volume of DMSO (Vehicle). Data are the mean ± SD of GFP-positive cells compared to the control. *** p < 0.001 vs the corresponding vehicle according to one-way ANOVA and Bonferroni’s post hoc test. (B) Representative fluorescence microscopy images of cells treated or not with lurasidone and infected with pseudovirus displaying the different SARS-CoV-2 Spike isoforms on the envelope. Scale bar = 100 µm.

Article Snippet: The membranes were incubated overnight at 4°C with an anti-ACE2 AC18Z mouse monoclonal antibody (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-b-actin mouse monoclonal antibody (1:5000, Sigma Aldrich).

Techniques: Transduction, Incubation, Infection, Control, Microscopy

Journal: Frontiers in cellular and infection microbiology

Article Title: The antipsychotic drug lurasidone inhibits coronaviruses by affecting multiple targets.

doi: 10.3389/fcimb.2024.1487604

Figure Lengend Snippet: FIGURE 3 Effect of lurasidone on ACE2 expression and ACE2 binding. (A) Quantitative analysis of representative Western blot performed on HEK293-ACE2 cell lysates incubated for 3, 6 or 24 hours with 5 µM lurasidone. Control cells were treated with an equivalent volume of DMSO (Vehicle). ACE2 quantification is expressed as the percentage of the mean volume of the ACE2 band immunoreactivity/activity of the vehicle at the same time point. Data are the mean ± SD of three separate experiments. (B) Direct binding of lurasidone to rhACE2. SPR sensorgram revealed no binding signal after the injection of 10 µM lurasidone on immobilized rhACE2 (blue line) compared with the curve obtained after 10 nM SARS-CoV-2 Spike RBD Wuhan injection, used as a positive control (black line). The results shown are corrected with the data obtained on the reference channel.

Article Snippet: The membranes were incubated overnight at 4°C with an anti-ACE2 AC18Z mouse monoclonal antibody (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-b-actin mouse monoclonal antibody (1:5000, Sigma Aldrich).

Techniques: Expressing, Binding Assay, Western Blot, Incubation, Control, Activity Assay, Injection, Positive Control

Journal: Frontiers in cellular and infection microbiology

Article Title: The antipsychotic drug lurasidone inhibits coronaviruses by affecting multiple targets.

doi: 10.3389/fcimb.2024.1487604

Figure Lengend Snippet: FIGURE 4 Modeling of lurasidone in the interface between Spike-ACE2. SARS-CoV-2 RBD complex (PDB 6M0J) showing (A) lurasidone poses docked with DiffDock-L (rank 1 in black) highlighting the putative binding site at the interface between Spike (in green) and ACE2 (in white). A secondary low- ranked site is on the side of Spike protein (gray); (B) overview of the putative binding site as seen from the RBD: ACE2 interface. The 10 top ranked DiffDock poses are shown, with rank 1 in black. The residues characterizing the WT, B.1.1.7 and B.1.351 SARS-CoV-2 strains are shown in red (K417 and E484) or in bright green (N501).

Article Snippet: The membranes were incubated overnight at 4°C with an anti-ACE2 AC18Z mouse monoclonal antibody (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-b-actin mouse monoclonal antibody (1:5000, Sigma Aldrich).

Techniques: Binding Assay