Journal: Frontiers in Microbiology

Article Title: Identification of a Broad-Spectrum Viral Inhibitor Targeting a Novel Allosteric Site in the RNA-Dependent RNA Polymerases of Dengue Virus and Norovirus

doi: 10.3389/fmicb.2020.01440

Figure Lengend Snippet: Superimposition of complex structures of DENV-RdRp/JF-31-MG46 (PDB ID 5F3T) and hNV-RpRp/PPNDS (PDB ID 4LQ3). N-pocket inhibitor JF-31-MG46 and B-site ligand PPNDS are shown as cyan and magenta stick representation, respectively. The priming loop in DENV RdRp and C-terminal in hNV RdRp are highlighted in cyan and magenta loops, respectively. DENV and hNV RdRps share an overall structural architecture with fingers (blue), palm (yellow), and thumb domains (red).

Article Snippet: In order to identify DENV and hNV RdRp inhibitors through drug repurposing, we screened compounds from TargetMol Approved Drug Library.

Techniques:

Journal: Frontiers in Microbiology

Article Title: Identification of a Broad-Spectrum Viral Inhibitor Targeting a Novel Allosteric Site in the RNA-Dependent RNA Polymerases of Dengue Virus and Norovirus

doi: 10.3389/fmicb.2020.01440

Figure Lengend Snippet: Overall workflow of the structure based virtual screening for identification of inhibitors targeting DENV and hNV RdRps.

Article Snippet: In order to identify DENV and hNV RdRp inhibitors through drug repurposing, we screened compounds from TargetMol Approved Drug Library.

Techniques:

Journal: Frontiers in Microbiology

Article Title: Identification of a Broad-Spectrum Viral Inhibitor Targeting a Novel Allosteric Site in the RNA-Dependent RNA Polymerases of Dengue Virus and Norovirus

doi: 10.3389/fmicb.2020.01440

Figure Lengend Snippet: Results of the virtual screening of the TargetMol Database against DENV and hNV RdRps. Bars represent numbers of compounds with predicted free energies of binding in the indicated 1 kcal/mol bins.

Article Snippet: In order to identify DENV and hNV RdRp inhibitors through drug repurposing, we screened compounds from TargetMol Approved Drug Library.

Techniques: Binding Assay

Journal: Frontiers in Microbiology

Article Title: Identification of a Broad-Spectrum Viral Inhibitor Targeting a Novel Allosteric Site in the RNA-Dependent RNA Polymerases of Dengue Virus and Norovirus

doi: 10.3389/fmicb.2020.01440

Figure Lengend Snippet: Inhibitory Effects of RAI-13 against DENV RdRp activity and RNA replication. (A) The inhibitory effect of RAI-5, RAI-13, and RAI-14 on the activity of DENV RdRps were measured by DENV-RdRp inhibition assay in vitro at a concentration of 40 μM. DMSO and JF-31-MG46 were selected as negative and positive control, respectively. The experiments were performed in triplicate. (B) Anti-DENV activity in cell culture. Two-fold serially diluted RAI-13 (from 0.08 to 20 μM) were incubated with DENV2 in A549 cells for 48 h. The inhibition of RAI-13 against DENV2 replication was measured by detecting DENV2 RNA through qRT-PCR analysis. EC50 values were obtained by non-linear regression analysis using GraphPad 5.0. The Data are representative of three independent experiments and values are expressed in mean ± SD. (C,D) DENV2 infected cells were treated with 2 μM RAI-13. The levels of relative plus-strand and minus-strand DENV2 RNA were measured by qRT-PCR. Error bars indicate standard deviations ( n = 3). (E) The cytotoxicity of the RAI-13 was measured using a Cell Counting Kit-8 by following the manufacturer’s instructions, CC50 values were obtained by non-linear regression analysis using GraphPad 5.0. Data shown are means ± s.d. n = 3. P values were calculated using a two-sided paired t -test. The significance of differences is indicated in the figures (* P < 0.05; ** P < 0.01; *** P < 0.001; and n.s., not significant).

Article Snippet: In order to identify DENV and hNV RdRp inhibitors through drug repurposing, we screened compounds from TargetMol Approved Drug Library.

Techniques: Activity Assay, Inhibition, In Vitro, Concentration Assay, Positive Control, Cell Culture, Incubation, Quantitative RT-PCR, Infection, Cell Counting

Journal: Frontiers in Microbiology

Article Title: Identification of a Broad-Spectrum Viral Inhibitor Targeting a Novel Allosteric Site in the RNA-Dependent RNA Polymerases of Dengue Virus and Norovirus

doi: 10.3389/fmicb.2020.01440

Figure Lengend Snippet: BLI measurement of the interaction between RAI-13 and hNV RdRp (A) or DENV RdRp (B) . Biotinylated hNV or DENV RdRp was immobilized on SSA biosensors and incubated with 2-fold serially diluted RAI-13 (from 3.91 to 125 μM). Double reference subtraction method was processed to subtract the effect of baseline drift and non-specific binding. KD acquired from fitting into 1:1 binding model by global fitting of multiple kinetic traces and analyzed by Data Analysis 9.0 software. Data shown are representative of three independent experiments.

Article Snippet: In order to identify DENV and hNV RdRp inhibitors through drug repurposing, we screened compounds from TargetMol Approved Drug Library.

Techniques: Incubation, Binding Assay, Software

Journal: Frontiers in Microbiology

Article Title: Identification of a Broad-Spectrum Viral Inhibitor Targeting a Novel Allosteric Site in the RNA-Dependent RNA Polymerases of Dengue Virus and Norovirus

doi: 10.3389/fmicb.2020.01440

Figure Lengend Snippet: Structural properties of RAI-13 bound to DENV or hNV RdRps during MD simulation over 10 ns. (A) Root-mean square deviation (RMSD) values for the backbone atoms. Black and red lines represent complexes of DENV and hNV RdRps, respectively. (B) Superimposition of the most representative complex structures of DENV and hNV RdRp bound with RAI-13. The ligand is shown as magentas and orange sticks, respectively. The priming loop in DENV RdRp and C-terminal in hNV RdRp are highlighted in yellow and green loops, respectively.

Article Snippet: In order to identify DENV and hNV RdRp inhibitors through drug repurposing, we screened compounds from TargetMol Approved Drug Library.

Techniques:

Journal: Frontiers in Microbiology

Article Title: Identification of a Broad-Spectrum Viral Inhibitor Targeting a Novel Allosteric Site in the RNA-Dependent RNA Polymerases of Dengue Virus and Norovirus

doi: 10.3389/fmicb.2020.01440

Figure Lengend Snippet: Predicted binding sites for RAI-13 bound to DENV RdRp (A,B) and hNV RdRp (C,D) . Left panel: The polymerases are shown in cartoon representation with the backbone atoms depicted in yellow (DENV RdRp) and green (hNV RdRp). RAI-13 and the important residues for the polymerase-ligand interactions were shown as sticks. For clarity, only polar hydrogen atoms were depicted in the figures. Right panel: the 2D ligand-polymerase interaction diagrams. Residues within 4 Å of RAI-13 are displayed. Hydrogen bond is represented by the arrows with the distance between the donor and the acceptor. The ⊓⁣−⁣−⁣⊓ stacking and salt bridge are shown in green and purple lines, respectively. Here the color code is that dark blue is positive charged, red is negative charged, light blue is polar, green is hydrophobic, and white is glycine.

Article Snippet: In order to identify DENV and hNV RdRp inhibitors through drug repurposing, we screened compounds from TargetMol Approved Drug Library.

Techniques: Binding Assay

Journal: Frontiers in Microbiology

Article Title: Identification of a Broad-Spectrum Viral Inhibitor Targeting a Novel Allosteric Site in the RNA-Dependent RNA Polymerases of Dengue Virus and Norovirus

doi: 10.3389/fmicb.2020.01440

Figure Lengend Snippet: The inhibitory effects of RAI-13 on HCV and HIV-1. (A) Jc1-Gluc HCVcc was used to infect Huh7.5.1 cells, which were treated with 1 um RAI-13. Levels of HCV infection were measured by detecting the Gluc activity in cell culture supernatants at 48 hpi. (B) VSVG/HIV-Fluc was used to infect HEK293T cells in the presence of 2 μM RAI-13 for 48 h, and then cells were harvested and lysed. Infection of HIV-1 was measured by detecting Fluc activity in the cell lysates. (C) Superimposition of the RdRp structures of DENV (yellow) and HCV (blue). (D) The polymerase structure of HIV-1 RT-P66 (PDB ID 1HMV). The fingers, palm, and thumb domains were shown in blue, yellow, and red respectively. Data are representative of at least three independent experiments, and values are expressed as means ± SD. Data are normalized to the control group, with the control value arbitrarily set to 1. * P < 0.05; ** P < 0.01; *** P < 0.001; and n.s., not significant.

Article Snippet: In order to identify DENV and hNV RdRp inhibitors through drug repurposing, we screened compounds from TargetMol Approved Drug Library.

Techniques: Infection, Activity Assay, Cell Culture, Control

Journal: Applied Microbiology and Biotechnology

Article Title: Development and characterization of mouse monoclonal antibodies targeting to distinct epitopes of Zika virus envelope protein for specific detection of Zika virus

doi: 10.1007/s00253-021-11364-1

Figure Lengend Snippet: Alignment of the binding epitopes of mAbs A1 ( a ), B1 ( b ), C1 ( c ), and 9E-1 ( d ) with other flavivirus E proteins. DENV, dengue virus; WNV, West Nile virus; YFV, yellow fever virus; JEV, Japanese encephalitis virus. The NCBI accession numbers of the E proteins derived for the indicated virus strains are provided in the parentheses. e The recombinant E proteins of ZIKV (2 μg), DENV2 (1.75 μg), DENV3 (0.42 μg), and DENV4 (0.78 μg) were separated on SDS-PAGE and then transferred to a PVDF membrane for performing the WB analysis with mAbs Anti-His, A1, B1, C1, or 9E-1, respectively. The numbers labeled on the left of the designated ticks are the molecular weight standards in kDa unit

Article Snippet: The recombinant DENV serotype 2 (DENV2) E protein (GenBank: AAC59274.1), serotype 3 (DENV3) E protein (GenBank: AAA99437.1), and serotype 4 (DENV4) E protein (GenBank: AAX48017.1) were purchased from Sino Biological Inc. (Catalog Number: 40471-V08B, 40532-V08H1, and 40533-V08B2, respectively).

Techniques: Binding Assay, Virus, Derivative Assay, Recombinant, SDS Page, Membrane, Labeling, Molecular Weight

Journal: Emerging Microbes & Infections

Article Title: Convalescent patient-derived monoclonal antibodies targeting different epitopes of E protein confer protection against Zika virus in a neonatal mouse model

doi: 10.1080/22221751.2019.1614885

Figure Lengend Snippet: Binding and neutralizing activities of E-targeted mAbs.

Article Snippet: DENV1 EDIII protein (Cat. no. 40531-V08B), DENV2 EDIII protein (Cat. no. 40471-V08Y3), and DENV4 E protein (Cat. no. 40533-V08B2) were also purchased from Sino Biological Inc.

Techniques: Binding Assay, Neutralization

Journal: Emerging Microbes & Infections

Article Title: Convalescent patient-derived monoclonal antibodies targeting different epitopes of E protein confer protection against Zika virus in a neonatal mouse model

doi: 10.1080/22221751.2019.1614885

Figure Lengend Snippet: Binding and neutralizing activities of E-targeted mAbs.

Article Snippet: DENV1 EDIII protein (Cat. no. 40531-V08B), DENV2 EDIII protein (Cat. no. 40471-V08Y3), and DENV4 E protein (Cat. no. 40533-V08B2) were also purchased from Sino Biological Inc.

Techniques: Binding Assay, Neutralization

Journal: Science signaling

Article Title: Amino acid starvation enhances vaccine efficacy by augmenting neutralizing antibody production

doi: 10.1126/scisignal.aav4717

Figure Lengend Snippet: (A) Experimental outline of HF or vehicle control DMSO treatment and DENV-2 envelope domain III (DENVrEDIII) protein immunization. (B) Proliferation analysis by 3H thymidine incorporation in antigen specific T-cells from splenocytes of mice 28 days after immunization that were restimulated with DENVrEDIII protein. Data are means ± SEM of 12 mice per treatment group from 2 independent experiments.(C to J) Flow cytometry analysis of DENVrEDIII-specific CD8+(C to F) and CD4+(G to J) T-cell responses in blood (D and H), spleen (E and I) and lymph node (F and J) after treatment and immunization, as indicated. FACS plots (C and G) are representative of 2 independent experiments. Quantification of the percentage of IFN-γ producing T-cells (D to F, and H to J) are means ± SEM of 12 mice per treatment group from 2 experiments. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by two tailed unpaired student t test (B) and Mann-Whitney U test (D, E, F, H, I and J).

Article Snippet: Purification of recombinant DENV envelope domain III protein (DENVrEDIII) The optimized envelope domain III sequence for all the four DENV serotypes (DENV-1, 2, 3 and 4) were cloned into bacterial expression vector pET-28a by Genescript (USA).

Techniques: Control, Flow Cytometry, Two Tailed Test, MANN-WHITNEY

Journal: Science signaling

Article Title: Amino acid starvation enhances vaccine efficacy by augmenting neutralizing antibody production

doi: 10.1126/scisignal.aav4717

Figure Lengend Snippet: (A) Flow cytometry analysis of antigen-specific CD8+IFN-γ+ andCD4+IFN-γ+ T-cell kinetics in PBMCs of mice treated with DMSO or HF and immunized with DENV-2 envelope domain III after 14 and 28 days. Data are means ± SEM of 12 mice/group from 2 independent experiments. (B to D) Flow cytometry analysis of DENVrEDIII-specific polyfunctional CD8+ (B) and CD4+(C) T-cells in blood, spleen and lymph node of immunized mice. The frequency of double cytokine (IFN-γ and IL-2) producing cells with means (bar) ± SEM (B and C) and pie charts of the frequency of all cytokine producing T-cells (D) are from 12 mice/group from 2 independent experiments.*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001by 2-way ANOVA with Bonferroni post-hoc between DENVrEDIII+DMSO and DENVrEDIII+HF immunized groups(A), Mann-Whitney U test(B and C)

Article Snippet: Purification of recombinant DENV envelope domain III protein (DENVrEDIII) The optimized envelope domain III sequence for all the four DENV serotypes (DENV-1, 2, 3 and 4) were cloned into bacterial expression vector pET-28a by Genescript (USA).

Techniques: Flow Cytometry, MANN-WHITNEY

Journal: Science signaling

Article Title: Amino acid starvation enhances vaccine efficacy by augmenting neutralizing antibody production

doi: 10.1126/scisignal.aav4717

Figure Lengend Snippet: ELISA analysis of the amounts of IFN-γ, IL-12p40 and TNF-α produced bysplenocytes and lymph node cells from immunized mice after re-stimulation in vitro with DENVrEDIII for 72hr. Spleen and lymph nodes were collected from mice at 28th day post-secondary immunization. Data are means ± SEM from 12 mice per treatment group from 2 independent experiments. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by two tailed unpaired student t test.

Article Snippet: Purification of recombinant DENV envelope domain III protein (DENVrEDIII) The optimized envelope domain III sequence for all the four DENV serotypes (DENV-1, 2, 3 and 4) were cloned into bacterial expression vector pET-28a by Genescript (USA).

Techniques: Enzyme-linked Immunosorbent Assay, Produced, In Vitro, Two Tailed Test

Journal: Science signaling

Article Title: Amino acid starvation enhances vaccine efficacy by augmenting neutralizing antibody production

doi: 10.1126/scisignal.aav4717

Figure Lengend Snippet: (A) ELISA analysis of DENVrEDIII specific total IgG, IgG2a, IgG2b, and IgG1 in the serum of mice14 days after primary immunization and 28 days after secondary immunization. Data are means ± SEM of 12 mice/group from 2 independent experiments. (B and C) BIAcore SPR analysis of DENVrEDIII protein binding by pooled serum samples from mice preconditioned with HF or DMSO 28 days after immunization. Sensogram trace of the DENVrEDIII-specific antibody binding affinity (B) and correlation of the Maximal Response Unit (RUmax) with the dissociation constant (C, upper) are representative of 2 independent experiments. Quantified avidity scores(C, lower) are means ± SEM from 2 independent experiments on pooled serum samples from 10 mice/group assayed in triplicate.(D) DENV-2 virus neutralization assay on serum samples collected from mice 28 days after immunization. The 50% focus reduction neutralization titre (FRNT50) data are means ± SEM of 10 mice from 2 independent experiments assayed in triplicate.*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, by one-way ANOVA with Bonferroni post-hoc test (A), two tailed unpaired student t test(C) and Mann-Whitney U test (D).

Article Snippet: Purification of recombinant DENV envelope domain III protein (DENVrEDIII) The optimized envelope domain III sequence for all the four DENV serotypes (DENV-1, 2, 3 and 4) were cloned into bacterial expression vector pET-28a by Genescript (USA).

Techniques: Enzyme-linked Immunosorbent Assay, Protein Binding, Binding Assay, Virus, Neutralization, Two Tailed Test, MANN-WHITNEY

Journal: Science signaling

Article Title: Amino acid starvation enhances vaccine efficacy by augmenting neutralizing antibody production

doi: 10.1126/scisignal.aav4717

Figure Lengend Snippet: (A)ELISA of total IgG specific for all four DENV serotypes in the serum of mice immunized with a tetravalent combination of DENVrEDIII protein 14 days after primary immunization and 28 days after secondary immunization. Data are means ± SEMof10 mice/group from 2 independent experiments. (B)DENV serotype neutralization assay on serum from tDENVrEDIII immunized mice. The 50% focus reduction neutralization titre (FRNT50) data are means ± SEM of 10 mice from 2 independent experiments. *P<0.05, **P<0.01 by one-way ANOVA with Bonferroni post-hoc test (A) and Mann-Whitney U test (B).

Article Snippet: Purification of recombinant DENV envelope domain III protein (DENVrEDIII) The optimized envelope domain III sequence for all the four DENV serotypes (DENV-1, 2, 3 and 4) were cloned into bacterial expression vector pET-28a by Genescript (USA).

Techniques: Enzyme-linked Immunosorbent Assay, Neutralization, MANN-WHITNEY

Journal: Science signaling

Article Title: Amino acid starvation enhances vaccine efficacy by augmenting neutralizing antibody production

doi: 10.1126/scisignal.aav4717

Figure Lengend Snippet: (A and B) Confocal microscopy imaging of the GL-7+(red), B220+(green) IgG+(blue) GC B-cells in lymph node sections from mice treated with HF or DMSO and immunized with DENVrEDIII protein. Images (A) are representative of 2 independent experiments. Quantified data (B) are means ± SEM of 8 mice/condition from all experiments. (C) Flow cytometry analysis of lymph node GC-B cells frequency in DENVrEDIII immunized mice at the indicated time-points. Data are means ± SEM of 8 mice per group at each time-point from 2 independent experiments.(D) ELISA analysis of IL-10 production by splenocytes and lymph node cells from mice 28 days after secondary immunization that were restimulated with DENVrEDIII. Data are means ± SEM of 10 mice/group from 2 independent experiments. (E) qRT-PCR analysis of the indicated gene expression in lymph node cells restimulated with DENVrEDIII for 24hr. Heatmap of statistically significant changes are from the analysis of 10 biological replicates from 2 independent experiments. *P<0.05, **P< 0.01, ***P<0.001, ****P<0.0001by two tailed unpaired student t test (B and D) and 2-way ANOVA with Bonferroni Post-hoc test (C).

Article Snippet: Purification of recombinant DENV envelope domain III protein (DENVrEDIII) The optimized envelope domain III sequence for all the four DENV serotypes (DENV-1, 2, 3 and 4) were cloned into bacterial expression vector pET-28a by Genescript (USA).

Techniques: Confocal Microscopy, Imaging, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Gene Expression, Two Tailed Test