Journal: Pathogens and Immunity

Article Title: Immune Dysregulation in Acute SARS-CoV-2 Infection

doi: 10.20411/pai.v7i2.537

Figure Lengend Snippet: Analysis of ACE2 inhibition by anti-S1(A) and anti-RBD (B) IgG antibodies by disease severity. Quantitative plasma anti-S had a correlation of r = 0.885 ( P < 0.001) and anti-RBD had a correlation of r = 0.75 ( P < 0.001) to ACE2 neutralization.

Article Snippet: The detection antibody used for this assay is R&D Systems Human ACE2 Biotinylated Antibody, (catalog BAF933).

Techniques: Inhibition, Clinical Proteomics, Neutralization

Journal: Pathogens and Immunity

Article Title: Immune Dysregulation in Acute SARS-CoV-2 Infection

doi: 10.20411/pai.v7i2.537

Figure Lengend Snippet: Proinflammatory dysregulation between cytokine microenvironment and antibody neutralization capacity. Correlation analysis of inflammatory marker plasma concentration and anti-spike antibody ACE2 neutralization disease severity. Elevations in ICAM ( P < 0.0001), IL-1β ( P = 0.0233), IL-4 ( P < 0.0001), IL-6 ( P = 0.0001), TNFα ( P = 0.001), and Syndecan ( P = 0.00) all showed statistically significant correlations to antibody neutralization capacity amongst PCR-positive COVID-19 disease cohorts.

Article Snippet: The detection antibody used for this assay is R&D Systems Human ACE2 Biotinylated Antibody, (catalog BAF933).

Techniques: Neutralization, Marker, Clinical Proteomics, Concentration Assay

Journal: Slas Discovery

Article Title: Identification of potent small molecule inhibitors of SARS-CoV-2 entry

doi: 10.1016/j.slasd.2021.10.012

Figure Lengend Snippet: High-throughput of ReFRAME, Pathogen Box, TargetMol and Cathepsin L drug libraries for SARS-CoV-2 antiviral compounds. A. Schematic of the spike protein of SARS-CoV-2. RBD: receptor binding domain. B. Schematic of the High-throughput assay. Compounds were pre-spotted in 1536-well plates. Next, 2000 HEK293T-ACE2 cells were added to each well and pre-incubated with each compound for 1 h, followed by infection with MLV reporter luciferase virus pseudotyped with the SARS-CoV-2 Spike protein (SARS2-S) or VSV-G protein (VSV-G). Luciferase was measured 48 h later. C. Summary of the ReFRAME library results. Conc.: concentration. D. Distribution of Z-Score for primary screens of each library. Scatter plot of Z_Score for all samples tested from the ReFrame library ( N = 1; circle) and other libraries ( N = 3; Cathepsin L: square; Pathogen Box: cross; TargetMol: filled circle). Total of 16,320 samples. Positive controls: orange; Negative control: cyan; Hit compounds: red; non-hit compounds: black. E. Summary of the 3 other libraries results. F. ReFrame library screening against different targets: SARS2-S, 3CLpro and PLpro. Venn diagram analysis of comparison between hits from SARS2-S entry, 3CLpro and PLpro assay against ReFRAME library results. There are 419 compounds that are SARS2-entry specific potential inhibitors. G. Robustness in terms of Z’ score of each screen for each library.

Article Snippet: Vero E6 cells were stained with Goat anti-Human Phycoerythrin-conjugated ACE2 Polyclonal Antibody (R&D Systems) for 30 min at 4 °C in dark.

Techniques: High Throughput Screening Assay, Binding Assay, Incubation, Infection, Luciferase, Virus, Concentration Assay, Negative Control, Library Screening, Comparison

Journal: Slas Discovery

Article Title: Identification of potent small molecule inhibitors of SARS-CoV-2 entry

doi: 10.1016/j.slasd.2021.10.012

Figure Lengend Snippet: Summary of the selected Cathepsin L, Pathogen box and TargetMol compounds in this study. Activity of the selected compounds against the different MLV pseudotyped viruses in HEK293-ACE2 cells and their respective cytotoxicity. Values for SARS2-S, VSV-G and toxicity are mean ± SEM of 2–4 independent experiments. TI: therapeutic index. * n = 1.

Article Snippet: Vero E6 cells were stained with Goat anti-Human Phycoerythrin-conjugated ACE2 Polyclonal Antibody (R&D Systems) for 30 min at 4 °C in dark.

Techniques: Activity Assay

Journal: Slas Discovery

Article Title: Identification of potent small molecule inhibitors of SARS-CoV-2 entry

doi: 10.1016/j.slasd.2021.10.012

Figure Lengend Snippet: Targets of the selected compounds and SARS-CoV-2 wild type infection. A. Description of the targets of the different hits from all the studied libraries. B. Antiviral activity of the 2 best hits in the SARS-CoV-2-induced CPE assay. Vero E6 cells treated with test compounds for two hours were infected with SARS-CoV-2 at an MOI of 0.05, then incubated for three days in the presence of compound. Cell viability (protection from virus-induced CPE) was measured with CellTiter-Glo. C and D. Antiviral effect was measured with a subset of Vero E6 cells expressing a low (C) and high (D) level of ACE2. E. Cytotoxicity of selected compounds in Vero E6 cells. Cytotoxicity was tested in the same conditions with cell culture media instead of the virus. F. Virus yield reduction activity of selected compounds. Vero E6 cells infected with SARS-CoV-2 at an MOI of 0.05 were cultured in the presence of test compound (5 µM) and the supernatant was harvested after 24 and 48 h of incubation. The Progeny virus was enumerated with a plaque assay using an Avicel overlay in fresh Vero E6 cells. N = 3 experiments were performed for infectivity assays and n = 2 for the cytotoxicity assays. **** P < 0.0001, Two-way ANOVA with Dunnett's multiple comparisons test against DMSO control.

Article Snippet: Vero E6 cells were stained with Goat anti-Human Phycoerythrin-conjugated ACE2 Polyclonal Antibody (R&D Systems) for 30 min at 4 °C in dark.

Techniques: Infection, Activity Assay, Incubation, Virus, Expressing, Cell Culture, Plaque Assay

Journal: Slas Discovery

Article Title: Identification of potent small molecule inhibitors of SARS-CoV-2 entry

doi: 10.1016/j.slasd.2021.10.012

Figure Lengend Snippet: SR-914 “calpeptin” specifically blocks SARS-CoV entry. A. Its activity against SARS2-S in HEK293T-ACE2-TMPRSS2 cells. Cells were incubated with different concentrations of drugs, then infected with SARS2-S or VSV-G. Luciferase was measured 48 h later, using Bright-Glo. Shown is the mean ± SEM of n = 2 to 4 independent experiments. B. Time of drug addition experiment schematic. Infection was performed for 1 h with or without drugs, Vero CCL81 cells were then washed, and fresh media was added with or without drugs. C. Time of drug addition experiment result. SR-914 was used at 10 µM. E64d at 20 µM. Calp.: calpeptin = SR-914. NI: not infected. Shown is the mean ± SEM of 4 to 6 independent experiments. D. Luciferase complementation assay schematic. The reporter consists of a split Firefly luciferase protein connected by a cleavable peptide for the tested protease. Upon cleavage of the peptide, the luciferase protein undergoes dimerization for an active state. DnaE intein helps in this dimerization. E. Its activity against SARS2-S Entry, 3CLpro and PLpro. C-: negative control. C+; positive control. Shown is the mean ± SD of 3 independent experiments. One-way ANOVA followed by Tukey's post-test were used for statistical comparisons. *, P < 0.01; **, P < 0.001; ***, P < 0.0001.

Article Snippet: Vero E6 cells were stained with Goat anti-Human Phycoerythrin-conjugated ACE2 Polyclonal Antibody (R&D Systems) for 30 min at 4 °C in dark.

Techniques: Activity Assay, Incubation, Infection, Luciferase, Negative Control, Positive Control

Journal: Slas Discovery

Article Title: Identification of potent small molecule inhibitors of SARS-CoV-2 entry

doi: 10.1016/j.slasd.2021.10.012

Figure Lengend Snippet: Breath of activity of calpeptin against various SARS-CoVs. A. Its activity against SARS1-S in HEK293T-ACE2 cells. HEK293T-ACE2 cells were incubated with different concentrations of calpeptin, then infected with SARS1-S. Luciferase was measured 48 h later, using Bright-Glo. Shown is the mean ± SEM of n = 2 independent experiments. B. Schematic of the substituted residues in the S protein of the highest threat of SARS-CoV-2 strains. C. Evolution of the S protein residues at the position 417, 484, 501 and 614 from 2019 to February 2021. Modified figure from https://nextstrain. org/ncov/global?branchLabel=none& c =gt-S_417,484,501,614& l =clock. D. Activity of the new emergent variants. HEK293T-ACE2 cells were infected with different mutants of SARS2-S. The day after, a medium change was performed. Luciferase was measured 48 h later, using Bright-Glo. Shown is the mean ± SEM of n = 3 independent experiments. WT: wild type, SA: South Africa, UK: United Kingdom. E. Activity of calpeptin activity against crucial mutations present in the S protein of the new emergent strains. Similar experiment than D but calpeptin was added during infection and after medium change. Shown is the mean ± SEM of n = 2–5 independent experiments. Two-way ANOVA followed by Dunnett's post-test were used for statistical comparisons. *, P < 0.01; **, P < 0.001; ***, P < 0.0001.

Article Snippet: Vero E6 cells were stained with Goat anti-Human Phycoerythrin-conjugated ACE2 Polyclonal Antibody (R&D Systems) for 30 min at 4 °C in dark.

Techniques: Activity Assay, Incubation, Infection, Luciferase, Modification

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Deficiency of the Angiotensinase Aminopeptidase A Increases Susceptibility to Glomerular Injury

doi: 10.1681/ASN.2016111166

Figure Lengend Snippet: RAS peptidase expression does not change in APA-KO mice after chronic AngII infusion. Representative images depicting the effect of chronic infusion of AngII on expression of NEP, ACE2, and APN as examined by immunofluorescence. Glomerular parietal epithelial and tubular epithelial cell distribution was noted for all three enzymes, with faint podocyte localization. No difference in degree of staining was observed across treatment groups. Ctrl, control; wt, wild type. Scale bars, 200 μm.

Article Snippet: Primary antibodies were goat anti-APA (anti–BP-1; Abcam Laboratories, Cambridge, MA), 1:167 (mouse specimens) and 1:150 (rat and human specimens); mouse anti-NEP (anti-CD10; Vector Laboratories), 1:50; goat anti-ACE2 (Santa Cruz Biotechnology, Santa Cruz, CA), 1:50; rabbit anti-APN (anti-CD13; Origene, Rockville, MD), 1:250; and mouse anti-GLEPP-1 (gift from Roger Wiggins, University of Michigan, Ann Arbor, MI), 1:50.

Techniques: Expressing, Immunofluorescence, Staining

Journal: Scientific Reports

Article Title: ACE2 protein expression within isogenic cell lines is heterogeneous and associated with distinct transcriptomes

doi: 10.1038/s41598-021-95308-9

Figure Lengend Snippet: Human ACE2 antibodies tested in this study.

Article Snippet: 7 , Cell Signaling , 4355S , "near C-terminus" , Rabbit , −−− , nd.

Techniques: