Journal: iScience

Article Title: SLC38A9 regulates SARS-CoV-2 viral entry

doi: 10.1016/j.isci.2024.110387

Figure Lengend Snippet: The multibasic motif of SARS-CoV-2 S1, which is critical for its endolysosome de-acidifying effect, interacts with SLC39A9 (A) Recombinant SARS-CoV-2 S1 (S1 w/multibasic motif, 50 ng/mL) but not mutant S1 lacking the multibasic motif (S1 w/o multibasic motif, 50 ng/mL), induces endolysosome de-acidification in U87MG cells ( n = 3 repeats, ∗∗∗∗ p < 0.0001). (B) S1w/multibasic motif and mutant S1 w/o multibasic motif exhibit similar inhibition on the binding of spike protein receptor binding domain to ACE2 ( n = 3 repeats, ∗ p <0.05, ∗∗ p <0.01). (C) Fluorescent labeled SARS-CoV-2 S1 co-localizes with SLC38A9-RFP in endolysosomes (LysoTracker) in U87MG cells (scale bar, 5 μm). (D) Using biotin-labeled SARS-CoV-2 S1 proteins as bait proteins, SLC38A9, but not TLR3, was pulled down from U87MG cell lysates. (E) Using SLC38A9 antibodies as bait proteins that pull down SLC38A9 from U87MG cell lysates, S1 was detected. (F) Using SLC38A9 antibodies as bait proteins that pull down SLC38A9 from U87MG cell lysate, mutant S1 lacking the multibasic motif was not detected.

Article Snippet: ACE2: Spike S1 RBD inhibitor screening assay , BPS Bioscience , 79936.

Techniques: Recombinant, Mutagenesis, Inhibition, Binding Assay, Labeling

Journal: iScience

Article Title: SLC38A9 regulates SARS-CoV-2 viral entry

doi: 10.1016/j.isci.2024.110387

Figure Lengend Snippet:

Article Snippet: ACE2: Spike S1 RBD inhibitor screening assay , BPS Bioscience , 79936.

Techniques: Labeling, Recombinant, Lysis, Mutagenesis, Fluorescence, Protease Inhibitor, Screening Assay, Luciferase, shRNA, Control, Software

Journal: iScience

Article Title: SLC38A9 regulates SARS-CoV-2 viral entry

doi: 10.1016/j.isci.2024.110387

Figure Lengend Snippet: The multibasic motif of SARS-CoV-2 S1, which is critical for its endolysosome de-acidifying effect, interacts with SLC39A9 (A) Recombinant SARS-CoV-2 S1 (S1 w/multibasic motif, 50 ng/mL) but not mutant S1 lacking the multibasic motif (S1 w/o multibasic motif, 50 ng/mL), induces endolysosome de-acidification in U87MG cells ( n = 3 repeats, ∗∗∗∗ p < 0.0001). (B) S1w/multibasic motif and mutant S1 w/o multibasic motif exhibit similar inhibition on the binding of spike protein receptor binding domain to ACE2 ( n = 3 repeats, ∗ p <0.05, ∗∗ p <0.01). (C) Fluorescent labeled SARS-CoV-2 S1 co-localizes with SLC38A9-RFP in endolysosomes (LysoTracker) in U87MG cells (scale bar, 5 μm). (D) Using biotin-labeled SARS-CoV-2 S1 proteins as bait proteins, SLC38A9, but not TLR3, was pulled down from U87MG cell lysates. (E) Using SLC38A9 antibodies as bait proteins that pull down SLC38A9 from U87MG cell lysates, S1 was detected. (F) Using SLC38A9 antibodies as bait proteins that pull down SLC38A9 from U87MG cell lysate, mutant S1 lacking the multibasic motif was not detected.

Article Snippet: The binding of SARS-CoV-2 S1 or mutant S1 lacking multibasic motif with ACE2 was assessed using the ACE2: Spike S1 RBD (SARS-CoV-2) inhibitor screening assay kit (BPS Bioscience, #79936).

Techniques: Recombinant, Mutagenesis, Inhibition, Binding Assay, Labeling

Journal: iScience

Article Title: SLC38A9 regulates SARS-CoV-2 viral entry

doi: 10.1016/j.isci.2024.110387

Figure Lengend Snippet:

Article Snippet: The binding of SARS-CoV-2 S1 or mutant S1 lacking multibasic motif with ACE2 was assessed using the ACE2: Spike S1 RBD (SARS-CoV-2) inhibitor screening assay kit (BPS Bioscience, #79936).

Techniques: Labeling, Recombinant, Lysis, Mutagenesis, Fluorescence, Protease Inhibitor, Screening Assay, Luciferase, shRNA, Control, Software

Journal: iScience

Article Title: High-throughput screening identifies broad-spectrum Coronavirus entry inhibitors

doi: 10.1016/j.isci.2024.110019

Figure Lengend Snippet: Production of high titer VSVΔG pseudoviruses and quantification of single infection events (A) A schematic representation of the VSVΔG pseudoviruses production process. Viral glycoproteins (yellow) are expressed by plasmid transfection on cell surface. Transfected cells are subsequently infected with recombinant VSV in which the endogenous G glycoprotein (VSV-G) was replaced with a fluorescent reporter (VSVΔG) and complemented with VSV-G (VSVΔG-G). 1 h post-infection, the residual VSVΔG-G is thoroughly washed and the culture is replenished with medium. This results in the production of pseudovirus particles capable of a single round of infection, baring the desired glycoproteins on their surface. Nevertheless, all downstream experiments are performed in the presence of a neutralizing anti-VSV-G antibody (α-G). (B) A widefield image of cells infected with VSVΔG pseudoviruses expressing a fluorescent reporter (GFP; green). Infected cells become round after infection due to the virus-induced cytopathic effect (CPE). (C) (Left) A high magnification overlay image showing the infected GFP-positive cells (green) and nuclei (blue). (Right) The respective segmentation showing infected cells (yellow) and nuclei (cyan). (D–F) Pseudoviral titers in infectious units/mL. To remove any residual infections from VSVΔG-G, the neutralizing α-G antibody was added to all pseudoviruses, ensuring accurate titration. The activity of α-G was tested using VSVΔG-G in the presence and absence of α-G. Two-tailed unpaired t tests were used to evaluate the statistical significance of α-G activity ( p ∗∗≤ 0.01, ∗∗∗∗≤ 0.0001. N experiments = 3, n repeats = 9. (D) Titer of VSVΔG-S W in different cell types showing infection improves upon over-expression of the innate receptor ACE2 and host protease TMPRSS2. (E) Titer of VSVΔG-S W in HEK-293T-ACE2-TMPRSS2 cells showing the effect of modifications to the cytosolic tail of S W . (F) Titer of VSVΔG-G, Wuhan (S W ), Alpha (S α ), Delta (S δ ), Omicron (S ο ), and MERS-CoV S (S M ) showing similar infection levels, with and without DPP4. VSVΔG-G pseudoviruses infected all cell lines at similar levels (D and F). (G) Pseudoviral infectious units/mL of VSVΔG RFP -G and VSVΔG GFP -S W separately or simultaneously result in equivalent titer measurements. (Right) A high magnification overlay image of a well showing VSVΔG RFP -G and VSVΔG GFP -S W infected cells (white and green, respectively). Scale bar is 100 μM (B, C, and G). Error bars represent the SEM.

Article Snippet: SARS-CoV-2 Spike S1 RBD: ACE2 Inhibitor Screening Assay Kit , BPS Bioscience , Cat# 79936.

Techniques: Infection, Plasmid Preparation, Transfection, Recombinant, Expressing, Virus, Titration, Activity Assay, Two Tailed Test, Over Expression

Journal: iScience

Article Title: High-throughput screening identifies broad-spectrum Coronavirus entry inhibitors

doi: 10.1016/j.isci.2024.110019

Figure Lengend Snippet: The pseudovirus-based HTS platform demonstrates high reproducibility (A) Schematic of the high content screening pipeline: plating, imaging, and analysis. Compounds were pre-plated, and pseudoviruses, pre-incubated with α-G to neutralize any residual VSVΔG-G infection, were added in individual wells before introducing HEK-293T cells stably overexpressing SARS-CoV-2 receptor ACE2 and protease TMPRSS2. After 24 h, the nuclei were stained, and the plates were imaged. Images were subsequently segmented and quantified. (B) Overview image of a portion of a 384-well screening plate (orange box in A). The neutral control (yellow wells) indicates 100% infection, and the positive control (magenta wells) signifies 0% infection or 100% inhibition. The green well depicts an example of a compound with ∼90% inhibition. (C) Scatterplot of 2,489 compounds tested as single point, on two different days. Blue line is the fit for the compounds that inhibited >35%. The inhibitions were reproducible with a high correlation fit (R 2 = 0.85). Black line represents the fit for the rest of the compounds (R 2 = 0.60). (Right) Examples of two inhibitors showing similar inhibition values after normalization and despite having different total cell and infection counts.

Article Snippet: SARS-CoV-2 Spike S1 RBD: ACE2 Inhibitor Screening Assay Kit , BPS Bioscience , Cat# 79936.

Techniques: High Content Screening, Imaging, Incubation, Infection, Stable Transfection, Staining, Control, Positive Control, Inhibition

Journal: iScience

Article Title: High-throughput screening identifies broad-spectrum Coronavirus entry inhibitors

doi: 10.1016/j.isci.2024.110019

Figure Lengend Snippet:

Article Snippet: SARS-CoV-2 Spike S1 RBD: ACE2 Inhibitor Screening Assay Kit , BPS Bioscience , Cat# 79936.

Techniques: Virus, Recombinant, Screening Assay, Software, Modification, Saline, Transfection, Infection

Journal: Virus Research

Article Title: Peroxide derivatives as SARS-CoV-2 entry inhibitors

doi: 10.1016/j.virusres.2023.199295

Figure Lengend Snippet: Inhibition of RBD-ACE2 binding by compound 21 and 29 a , ELISA was used to determine inhibition of RBD-ACE2 binding. Concentrations of 0, 25, 50, 100 and 200 μM were used for both compounds. Technical triplicate was performed. b , Immunofluorescence staining was used to determine the ability of the compounds to inhibit RBD binding to cell surface ACE2. RBD was incubated with HEK293T transfected to overexpress ACE2-EGFP fusion protein in the presence of 0 (control), 25, 50 and 100 μM of compound. The RBD bound to cell surface was visualized with anti-His-tag antibody conjugated to Alexa Fluor 647 (AF647) fluorophore. One-way ANOVA followed by Tukey's test for comparison between groups was used to determine statistical significance between groups in a . P value for comparison with the 0 μM group was shown. *: p <0.0; **: p <0.01; ***: p <0.001; ****: p <0.0001; non-significant results were not labeled.

Article Snippet: The ACE2 SARS-CoV-2 Spike Inhibitor Screening Assay Kit (Cat: 79936) was purchased from BPS Biosciences (San Diego, US).

Techniques: Inhibition, Binding Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Incubation, Transfection, Control, Comparison, Labeling