ns3 residues  (Addgene inc)

Journal: International Journal of Molecular Sciences

Article Title: Discovery of Novel Druggable Sites on Zika Virus NS3 Helicase Using X-ray Crystallography-Based Fragment Screening

doi: 10.3390/ijms19113664

Figure Lengend Snippet: Overall structure (two views related by 180° rotation, black arrow) of ZIKV NS3 protein, including the helicase domains I (magenta surface), II (cyan surface), and III (green surface), as well as the protease domain (grey ribbons). The latter is approximated from the homologous DENV NS3 full-length structure (PDB code 2VBC), which has been superposed on the ZIKV NS3-Hel structure. The binding positions of RNA and ATP are also shown. The fragment-binding sites, A and B, discovered here, are indicated using the ball representation for fragments 1 and 3 , respectively.

Article Snippet: ZIKV NS3-Hel (residues 171–617 of the NS3 protein) was overexpressed in a pET30+ plasmid with a cleavable, N-terminal 6× His-tagged SUMO tag using a Rosetta 2(DE3) pLysS E. coli strain (EMD Biosciences, San Diego, CA, USA) and Studier’s ZYP-5052 auto-induction medium [ ].

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Discovery of Novel Druggable Sites on Zika Virus NS3 Helicase Using X-ray Crystallography-Based Fragment Screening

doi: 10.3390/ijms19113664

Figure Lengend Snippet: Saturation transfer difference (STD) studies. ( A ) Spectra illustrating the binding of four fragments at 1 mM concentration to ZIKV NS3-Hel. For each fragment, reference 1 H NMR spectrum is shown on top, and the STD spectrum is shown at the bottom. ( B ) STD amplification factors A STD plotted vs the ligand concentration for fragments 1 and 4 . The plots are based on singlet peaks which arise from the protons, indicated with a star on the structural diagrams (top).

Article Snippet: ZIKV NS3-Hel (residues 171–617 of the NS3 protein) was overexpressed in a pET30+ plasmid with a cleavable, N-terminal 6× His-tagged SUMO tag using a Rosetta 2(DE3) pLysS E. coli strain (EMD Biosciences, San Diego, CA, USA) and Studier’s ZYP-5052 auto-induction medium [ ].

Techniques: Binding Assay, Concentration Assay, Amplification

Journal: International Journal of Molecular Sciences

Article Title: Discovery of Novel Druggable Sites on Zika Virus NS3 Helicase Using X-ray Crystallography-Based Fragment Screening

doi: 10.3390/ijms19113664

Figure Lengend Snippet: Residues forming the binding sites A and B in flaviviral NS3 helicases.

Article Snippet: ZIKV NS3-Hel (residues 171–617 of the NS3 protein) was overexpressed in a pET30+ plasmid with a cleavable, N-terminal 6× His-tagged SUMO tag using a Rosetta 2(DE3) pLysS E. coli strain (EMD Biosciences, San Diego, CA, USA) and Studier’s ZYP-5052 auto-induction medium [ ].

Techniques: Binding Assay

Journal: Antiviral research

Article Title: Identification of novel small molecule inhibitors against NS2B/NS3 serine protease from Zika virus

doi: 10.1016/j.antiviral.2016.12.016

Figure Lengend Snippet: (A) Schematics of the ZIKV and HCV polyproteins with cleavage sites. (B) Aligned X-ray structures of Zika NS2B-NS3pro (PDB:5LC0) and HCV NS4A-NS3pro (PDB:1CU1). The catalytic triad residues (S135, H51, and D75) of Zika NS2B-NS3pro are well aligned with those of HCV (S139, H57, and D81). (C) Sequence alignment of NS2B/NS3 proteases from ZIKV, WNV, DENV2, and HCV. Catalytic site residues are shown in green.

Article Snippet: Plasmid construction and purification of Zika NS2B-NS3 pro The gene of ZIKV NS3 protease (residues 1-187) with a His-tag at the N-terminus and NS2B (residues 46-99) were codon-optimized, synthesized (BioBasic Inc.), and cloned into a pET15b vector between NdeI and BamHI.

Techniques: Sequencing

Journal: Antiviral research

Article Title: Identification of novel small molecule inhibitors against NS2B/NS3 serine protease from Zika virus

doi: 10.1016/j.antiviral.2016.12.016

Figure Lengend Snippet: Comparison of kinetic parameters of His6-tagged and native NS2B-NS3 pro from ZIKV.

Article Snippet: Plasmid construction and purification of Zika NS2B-NS3 pro The gene of ZIKV NS3 protease (residues 1-187) with a His-tag at the N-terminus and NS2B (residues 46-99) were codon-optimized, synthesized (BioBasic Inc.), and cloned into a pET15b vector between NdeI and BamHI.

Techniques:

Journal: Antiviral research

Article Title: Identification of novel small molecule inhibitors against NS2B/NS3 serine protease from Zika virus

doi: 10.1016/j.antiviral.2016.12.016

Figure Lengend Snippet: (A) Schematic of compound test and hit validation process. (B) Bar graphs of 10 selected compound inhibitory activity (IC50) and binding affinity (KD) to ZIKV NS2B-NS3 protease. (C) Sensorgrams of compound 3 at a series of increasing concentrations. (D) Steady-state fitting curve of compound 3 using steady-state affinity equation embedded in Biaevaluation v3.0.

Article Snippet: Plasmid construction and purification of Zika NS2B-NS3 pro The gene of ZIKV NS3 protease (residues 1-187) with a His-tag at the N-terminus and NS2B (residues 46-99) were codon-optimized, synthesized (BioBasic Inc.), and cloned into a pET15b vector between NdeI and BamHI.

Techniques: Activity Assay, Binding Assay

Journal: Antiviral research

Article Title: Identification of novel small molecule inhibitors against NS2B/NS3 serine protease from Zika virus

doi: 10.1016/j.antiviral.2016.12.016

Figure Lengend Snippet: (A) Surface of the Zika NS2B-NS3pro in pre-open conformation (5T1V) with the surface of NS2B in black and the surface of the C terminus of NS3 in red. (B) Surface of the Dengue Virus NS2B-NS3pro in open conformation (2FOM) with the surface of the NS2B in black and the surface of the C terminus of NS3 in red. (C) Surface of the Zika virus NS2B-NS3pro in closed conformation in complex with a boronate inhibitor (5LC0) with the surface of the NS2B in black and the surface of the C terminus of NS3 in red. (D) Electrostatic surface of the Zika NS2B-NS3pro in pre-open conformation (5T1V). (E) Electrostatic surface of the Dengue Virus NS2B-NS3pro in open conformation (2FOM). (F) Electrostatic surface of the Zika virus NS2B-NS3pro in closed conformation in complex with a boronate inhibitor (5LC0).

Article Snippet: Plasmid construction and purification of Zika NS2B-NS3 pro The gene of ZIKV NS3 protease (residues 1-187) with a His-tag at the N-terminus and NS2B (residues 46-99) were codon-optimized, synthesized (BioBasic Inc.), and cloned into a pET15b vector between NdeI and BamHI.

Techniques:

Journal: Cell Research

Article Title: Existing drugs as broad-spectrum and potent inhibitors for Zika virus by targeting NS2B-NS3 interaction

doi: 10.1038/cr.2017.88

Figure Lengend Snippet: The DENV2 NS2B/NS3 SLC assays. (A) The DENV2 NS2B/NS3 SLC constructs. NLuc aa 1-416-NS2B (named as NLuc-NS2B), NS2B-NLuc aa 1-416 (NS2B-NLuc), NLuc416-NS2B aa 49-66 (NLuc-E66stop), NS2B (aa 49-66)-NLuc416 (E66stop-NLuc), GST-NS3-CLuc aa 398-550 (GNC), and GST-CLuc (aa 398-550)-NS3 (GCN). (B) The positions of NLuc and CLuc are important. Equal concentrations (100 nM) of each pair of NS2B/NS3 constructs were mixed (or alone) and incubated with luciferin substrate. ***P < 0.001. In all bar graphs, means and SD from triplicate experimental data were shown, unless otherwise specified. (C) Effects of detergent at 0.05% concentration. **P < 0.01; ***P < 0.001. (D) Dose-response of NLuc-E66stop/GCN pair. Nluc-E66stop at 20 nM was included in each experiment. Concentration of GCN was varied as indicated. ***P < 0.001. (E) The DENV2 MBP-NS3 fusion protein specifically inhibited the SLC by NLuc-E66stop and GCN (80 nM each). MBP-NS3 or MBP were at 3.25 μM each. ***P < 0.001. (F) Dose-response inhibition of the SLC signals from NLuc-E66stop and GCN by “cold” MBP-NS3. Experimental data were fitted using the sigmoidal function with the Origin6.0 software. (G) NS2B mutations greatly reduced SLC. GCN was paired with equal molar of NLuc-E66stop or NLuc-E66stop mutants (L51A, L53A, and V59A). ***P < 0.001.

Article Snippet: The PCR product representing the NS3 residues 1-185 was used as a megaprimer for PCR with an in-house His-MBP-Bcl10 constructed in a pDEST-His-MBP vector (Addgene).

Techniques: Construct, Incubation, Concentration Assay, Inhibition, Software

Journal: Cell Research

Article Title: Existing drugs as broad-spectrum and potent inhibitors for Zika virus by targeting NS2B-NS3 interaction

doi: 10.1038/cr.2017.88

Figure Lengend Snippet: HTS assay identified potent orthosteric protease inhibitors. (A) Dose-response inhibition of the SLC signals from NLuc-E66stop and GCN by SK-12. Experimental data were fitted using the sigmoidal function with the Origin6.0 software. (B) HTS parameters using purified NLuc-E66stop and GCN. NLuc-E66stop and GCN at 100 nM were used with DMSO or SK-12 (40 μM). n = 8. ***P < 0.001. (C) NS3 pockets were accessible to small molecule inhibitor when NS2B and NS3 were co-expressed. Plasmids of NLuc-E66stop and GCN were co-transformed into Escherichia coli BL21 (DE3). Cells were grown to OD600 of 0.6 and were induced by IPTG. Cells were continuously grown for 2 h, collected and resuspended in luciferase assay buffer. About 100 μl of cells was dispensed into a 96-well plate, incubated with 1% DMSO or SK-12 (40 μM) for 2 h, then mixed with substrate luciferin n = 8. ***P < 0.001. (D) Summary of HT screening of the NCGC Pharmaceutical Collection in all plates. Statistics were generated by averaging those of 20 plates that were calculated from 32 wells in each plate. (E) SDS-PAGE analysis of purified His-MBP-NS3 (lane 2) and His-NS2B (Lane 3). Lane 1, Bio-Rad broad range molecular weight (MW) standard. (F) CD spectrum of purified His-MBP-NS3. (G) Sigmoidal curve fittings of dose-response inhibitions of the His-NS2B/His-MBP-NS3 protease activities by drugs. Inset: schematic representations of identified drugs.

Article Snippet: The PCR product representing the NS3 residues 1-185 was used as a megaprimer for PCR with an in-house His-MBP-Bcl10 constructed in a pDEST-His-MBP vector (Addgene).

Techniques: HTS Assay, Inhibition, Software, Purification, Transformation Assay, Luciferase, Incubation, Generated, SDS Page, Molecular Weight

Journal: Cell Research

Article Title: Existing drugs as broad-spectrum and potent inhibitors for Zika virus by targeting NS2B-NS3 interaction

doi: 10.1038/cr.2017.88

Figure Lengend Snippet: Drugs directly bind to the NS3 protease domain and disrupt interactions between NS2B and NS3. (A) GST pull-down assay. GST-NS3 or the GST-tag (10 μg) was immobilized on the Glutathione sepharose-4B affinity beads (GE HealthCare). The FLAG-tagged NS2B (10 μg) was incubated with the beads for 2 h, and subjected to western blots (WB), using anti-FLAG (Genscript) and anti-GST antibodies (GE HealthCare). (B) Dose-dependent inhibition of NS2B-NS3 interactions by drugs, using the GST pull-down assay. The assay was performed the same as in A, except that two-fold dilution series of drugs were incubated with the GST-NS3 beads overnight prior to incubation with the FLAG-NS2B. Bottom panels showed normalized binding of FLAG-tag NS2B to GST-NS3. The binding of NS2B to NS3 in the absence of each drug (DMSO control) was set as 100%. The relative binding of NS2B to NS3 in the presence of each drug was normalized to the DMSO control. n = 3. (C) PTSA for binding of drugs to the MBP-NS3 protein. ΔTm was defined as Tm−drug−TmDMSO. (D) SPR sensorgrams of kinetic data for the binding of drugs to refolded NS3. The refolded His-NS3 was coupled to a ProteOn GLH sensor chip (∼15 000 RU). Each drug with three-fold dilutions was injected. Global fitting of data to a 1:1 binding model is shown in dark black.

Article Snippet: The PCR product representing the NS3 residues 1-185 was used as a megaprimer for PCR with an in-house His-MBP-Bcl10 constructed in a pDEST-His-MBP vector (Addgene).

Techniques: Pull Down Assay, Incubation, Western Blot, Inhibition, Binding Assay, FLAG-tag, Injection

Journal: Cell Research

Article Title: Existing drugs as broad-spectrum and potent inhibitors for Zika virus by targeting NS2B-NS3 interaction

doi: 10.1038/cr.2017.88

Figure Lengend Snippet: Drugs inhibit viral polyprotein precursor (PP) processing. (A) Lineweaver-Burk plot of kinetics experimental data for inhibition of the His-NS2B/His-MBP-NS3 protease complex by drugs. The DENV2 MBP-NS3 (100 nM) was mixed with temoporfin (3, 1.5, and 0.75 μM), niclosamide (30, 15, and 7.5 μM), or nitazoxanide (30, 15, and 7.5 μM) for 30 min. The DENV2 His-NS2B (1 μM) was added together with the Abz substrate at various concentrations (800-25 μM in two-fold dilutions). (B-D) Western blots (WB) analysis of dose-dependent inhibition of ZIKV NS3 expression by temoporfin (B), niclosamide (C), and nitazoxanide (D) using the GTX133309 ZIKV α-NS3 antibody (GeneTex) (left panel), respectively. The experiment was performed at the 48 h time point. Middle panel, NS3 expression (lower bands) normalized to the GAPDH loading control. Right panel, accumulated PP normalized to the DMSO control. **P < 0.01; ***P < 0.001. (E) MS/MS spectra obtained from the fragmentation of the precursor ion at m/z corresponding to representative ZIKV peptides. Fragment ions corresponding to y- and b-ions were observed (red lines).

Article Snippet: The PCR product representing the NS3 residues 1-185 was used as a megaprimer for PCR with an in-house His-MBP-Bcl10 constructed in a pDEST-His-MBP vector (Addgene).

Techniques: Inhibition, Western Blot, Expressing, Tandem Mass Spectroscopy