Journal: Cell Reports

Article Title: Antiviral drug screen identifies DNA-damage response inhibitor as potent blocker of SARS-CoV-2 replication

doi: 10.1016/j.celrep.2021.108940

Figure Lengend Snippet: Berzosertib inhibits SARS-CoV-2, SARS-CoV-1, and MERS-CoV replication in human cells and is synergistic with remdesivir (A and B) Graphs show an eight-point dose-response curve of berzosertib (A) or remdesivir (B) in SARS-CoV-2-infected Calu-3 cells. Contrasted with cell viability of mock-infected cells. (C and D) Antiviral effect of berzosertib on SARS-CoV-1 (C) and MERS-CoV (D). (E) Graphs show antiviral activity measured with a SARS-CoV-2 immunostaining signal used for identification of infected A549-ACE2 cells. IC 50 values were calculated by non-linear regression sigmoidal dose-response analysis using the GraphPad Prism 7 software package. (F) Graph shows synergistic effect of berzosertib and remdesivir in infected A549-ACE2 cells. Dose-response curves obtained with mixtures of remdesivir and berzosertib, remdesivir alone (thick black line), and berzosertib alone (thick pink line) are shown. (G) Isobologram of drug combinations is depicted. (H) Combinatorial data were analyzed for inhibitory, additive, or synergistic effects (upper triangle, dotted line, and lower triangle, respectively) by using the Compusyn software package.

Article Snippet: Synergistic effect of berzosertib and remdesivir in infected A549-ACE2 cells are investigated as follows: A549-ACE2 cells were treated with a mixture of berzosertib and remdesivir (concentrations specified in the top and bottom, respectively) prepared in a 7-by-7 concentration matrix, which generated 49 combinations ranging from 0.014 μM - 10 μM for berzosertib, and 6.8 nM - 5 μM for remdesivir.

Techniques: Infection, Activity Assay, Immunostaining, Software

Journal: Cell Reports

Article Title: Antiviral drug screen identifies DNA-damage response inhibitor as potent blocker of SARS-CoV-2 replication

doi: 10.1016/j.celrep.2021.108940

Figure Lengend Snippet:

Article Snippet: Synergistic effect of berzosertib and remdesivir in infected A549-ACE2 cells are investigated as follows: A549-ACE2 cells were treated with a mixture of berzosertib and remdesivir (concentrations specified in the top and bottom, respectively) prepared in a 7-by-7 concentration matrix, which generated 49 combinations ranging from 0.014 μM - 10 μM for berzosertib, and 6.8 nM - 5 μM for remdesivir.

Techniques: Produced, Recombinant, Electron Microscopy, Blocking Assay, MTT Cell Proliferation, Proliferation Assay, Software

Journal: Microbiology and immunology

Article Title: N-glycosylation of the SARS-CoV-2 spike protein at Asn331 and Asn343 is involved in spike-ACE2 binding, virus entry, and regulation of IL-6.

doi: 10.1111/1348-0421.13121

Figure Lengend Snippet: FIGURE 1 N‐linked glycosylation of RBD protein is required for binding to ACE2. (a) Binding activity of SARS‐CoV‐2 RBD and S2 proteins derived from HEK293 cells or Escherichia coli. Binding was measured using an ACE2 binding assay, and nucleocapsid protein (N) and HIV p24 (P24) were used as negative controls. (b) Binding activity of RBD expressed either in HEK293 cells or in E. coli to titrated concentrations of ACE2. (c) Detection of purified and deglycosylated recombinant RBD and ACE2 proteins by SDS‐PAGE. From left to right: untreated proteins, proteins deglycosylated (dg) under native conditions, proteins deglycosylated under reducing conditions. (d) Detection of RBD and ACE2 binding using the ACE2 binding assay under conditions where either or both RBD and ACE2 were deglycosylated (dg). (e) RBD was deglycosylated with individual enzymes: PNGase F, O‐glycosidase, α2‐3,6,8,9 neuraminidase A, β1‐4 galactosidase S, or β‐N‐acetylhexosaminidasef, prior to the ACE2 binding assay. Statistical significance was tested by T test for all experiments. ****p < 0.0001. n ≥3 for all experiments.

Article Snippet: Two cell lines with high ACE2 expression (Vero and A549) and were purchased from the ATCC.

Techniques: Glycoproteomics, Binding Assay, Activity Assay, Derivative Assay, Recombinant, SDS Page

Journal: Microbiology and immunology

Article Title: N-glycosylation of the SARS-CoV-2 spike protein at Asn331 and Asn343 is involved in spike-ACE2 binding, virus entry, and regulation of IL-6.

doi: 10.1111/1348-0421.13121

Figure Lengend Snippet: FIGURE 2 Glycosylation of RBD at Asn343 is essential for interaction with ACE2. (a) Detection of the binding of RBD wild type, or its N331Q, N343Q, and N331Q/N343Q mutated derivatives to ACE2 using the ACE2 binding assay. 40 ng/mL ACE2 was used in all assays. (b) Immunoblot analysis of recombinant SARS‐CoV‐2 RBD wild type or N331Q, N343Q, and N331Q/N343Q double mutated derivatives expressed within purified pseudovirus particles propagated in BHK21 cultured cells. (c) Detection of luciferase activity in A549 cultured cells infected with pseudovirus harboring S1 wild type, or its N331Q, N343Q, and N331Q/N343Q mutated derivatives. (d) Detection of luciferase activity in Vero cultured cells infected with pseudovirus harboring S1 wild type, or its N331Q, N343Q, and N331Q/N343Q mutated derivatives. (e) ACE2 expression levels (protein) in four cell lines, NIH3T3, A431, A549, and Vero, were detected by immunoblot. (f) The ACE2 binding was measured in the presence of blocking antibodies, including anti‐RBD (1H9; 4A9; 1F9), anti‐ S2, and anti‐N, to assess the specificity of ACE2 binding to RBD. Statistical significance was tested by T test for all experiments. ****p < 0.001. n = 4/5 for all experiments.

Article Snippet: Two cell lines with high ACE2 expression (Vero and A549) and were purchased from the ATCC.

Techniques: Glycoproteomics, Binding Assay, Western Blot, Recombinant, Cell Culture, Luciferase, Activity Assay, Infection, Expressing, Blocking Assay

Journal: Microbiology and immunology

Article Title: N-glycosylation of the SARS-CoV-2 spike protein at Asn331 and Asn343 is involved in spike-ACE2 binding, virus entry, and regulation of IL-6.

doi: 10.1111/1348-0421.13121

Figure Lengend Snippet: FIGURE 3 Identification of glycan binding epitope in the N‐Glycan mutant site of RBD protein. (a) Identification of glycan recognition domains in the N331Q and N343Q mutants of RBD protein using a glycan array. Data represents two independent experiments. Data were analyzed using unpaired Tukey's test *p < 0.02 for statistical significance. (b) Schematic model of interaction inhibition in between double mutant (N331Q and N343Q) RBD mutant and WT RBD protein. Adapted from BioRender.com (2021). (c) N‐Glycan distribution on sites N90 and N322 of human ACE2 receptor, which are proximal to the RBD binding region of S1 protein (red arrows show the glycans with terminal galactose). Modified from Shajahan and colleagues, with permission.23

Article Snippet: Two cell lines with high ACE2 expression (Vero and A549) and were purchased from the ATCC.

Techniques: Glycoproteomics, Binding Assay, Mutagenesis, Inhibition

Journal: Microbiology and immunology

Article Title: N-glycosylation of the SARS-CoV-2 spike protein at Asn331 and Asn343 is involved in spike-ACE2 binding, virus entry, and regulation of IL-6.

doi: 10.1111/1348-0421.13121

Figure Lengend Snippet: FIGURE 6 Proposed model: Mutations in N‐glycosylation sites at N331 and N343 residues of RBD protein inhibit S‐ACE2 binding, IL‐6 expression and cytotoxicity. I confirm and agree with the possibility that my figure images might be selected and used as a journal cover image at issue publication.

Article Snippet: Two cell lines with high ACE2 expression (Vero and A549) and were purchased from the ATCC.

Techniques: Glycoproteomics, Binding Assay, Expressing

Journal: ImmunoHorizons

Article Title: Targeting 2-Oxoglutarate–Dependent Dioxygenases Promotes Metabolic Reprogramming That Protects against Lethal SARS-CoV-2 Infection in the K18-hACE2 Transgenic Mouse Model

doi: 10.4049/immunohorizons.2300048

Figure Lengend Snippet: SARS-CoV-2 infection maintains mitochondrial function early following infection. ( A ) hACE2-A549 cells were infected with SARS-CoV-2 (MOI of 0.01) or mock (uninfected) and assessed for viral burden by TCID 50 . ( B and C ) Extracellular flux analysis of mitochondrial function was performed to determine the ATP-linked oxygen consumption rate (OCR) after injection of oligomycin (B) and maximal respiration was calculated after injection of FCCP (C). ( D ) Glycolysis was determined using the glycolysis stress test. ( E ) Cell death as determined by extracellular LDH activity over time following SARS-CoV-2 infection. Data are shown as mean ± SEM from four pooled experiments ( n = 4) with six technical replicates per group. * p < 0.05, *** p < 0.001 indicate significance between mock and SARS-CoV-2 infection by a Student t test or one-way ANOVA followed by a Dunnett multiple comparison test. ECAR, extracellular acidification rate.

Article Snippet: ACE2 was quantified in A549 cell lysates (using anti–ACE-2 Ab, catalog no. 4355S, Cell Signaling Technology) using a ProteinSimple Wes microcapillary Western blot.

Techniques: Infection, Injection, Activity Assay, Comparison

Journal: ImmunoHorizons

Article Title: Targeting 2-Oxoglutarate–Dependent Dioxygenases Promotes Metabolic Reprogramming That Protects against Lethal SARS-CoV-2 Infection in the K18-hACE2 Transgenic Mouse Model

doi: 10.4049/immunohorizons.2300048

Figure Lengend Snippet: DMOG treatment overcomes SARS-CoV-2 manipulation of cellular bioenergetics. hACE2-A549 cells were infected with SARS-CoV-2 (MOI of 0.01) or uninfected (mock) and treated with increasing doses of DMOG. ( A ) Intracellular HIF-1α levels were measured by ELISA. ( B – G ) Mitochondrial or glycolytic stress tests via extracellular flux analysis were also performed at 24 h postinfection to determine (B) glycolytic extracellular acidification rate (ECAR), (C) basal respiration, (D) ATP-dependent oxygen consumption rate (OCR), (E) proton leak, (F) maximal respiration, and (G) nonmitochondrial respiration rates. DMOG (0 mM) represents the baseline effects of infection or mock infection on cells after 24 h. ( H ) IC 50 of DMOG inhibition of OCR on mock- or SARS-CoV-2–infected cells. For calculation of the IC 50 , each individual data point represents an experimental repeat. Data are shown as mean ± SEM from four pooled experiments ( n = 4) with six technical replicates per group. # p < 0.05, ## p < 0.01, ### p < 0.001 indicate significance relative to the baseline (0 mM DMOG) using a one-way ANOVA followed by a Dunnett multiple comparison test. * p < 0.05, ** p < 0.01 indicates significance between mock (uninfected) or SARS-CoV-2–infected groups using a Student t test.

Article Snippet: ACE2 was quantified in A549 cell lysates (using anti–ACE-2 Ab, catalog no. 4355S, Cell Signaling Technology) using a ProteinSimple Wes microcapillary Western blot.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Inhibition, Comparison

Journal: ImmunoHorizons

Article Title: Targeting 2-Oxoglutarate–Dependent Dioxygenases Promotes Metabolic Reprogramming That Protects against Lethal SARS-CoV-2 Infection in the K18-hACE2 Transgenic Mouse Model

doi: 10.4049/immunohorizons.2300048

Figure Lengend Snippet: DMOG treatment limits SARS-CoV-2–induced cell death independent of viral burden. ( A – D ) hACE2-A549 cells were infected with SARS-CoV-2 (MOI of 0.01) and then treated with increasing doses of DMOG; cell death (A–C) or viral burden (D) was assessed after 72 h. The percent cell death by microscopic enumeration of NucGreen-positive cells was calculated. (A) Representative images of A549 cell cultures used to calculate (B). (C) Extracellular LDH activity as a surrogate marker of cell death. (D) Viral titer measure by TCID 50 . Data are shown as mean ± SEM from three pooled experiments ( n = 3) with three technical replicates per group. The dotted lane represents the background cell death levels in uninfected cultures without DMOG treatment. * p < 0.05, ** p < 0.01, *** p < 0.001 indicate significance relative to 0 (untreated) cells infected with SARS-CoV-2 using one-way ANOVA followed by a Dunnett multiple comparison test.

Article Snippet: ACE2 was quantified in A549 cell lysates (using anti–ACE-2 Ab, catalog no. 4355S, Cell Signaling Technology) using a ProteinSimple Wes microcapillary Western blot.

Techniques: Infection, Activity Assay, Marker, Comparison