Journal: Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology

Article Title: Cross-talk Between Glia, Neurons and Mast Cells in Neuroinflammation Associated with Parkinson’s Disease

doi: 10.1007/s11481-017-9766-1

Figure Lengend Snippet: MPP+ and mast cell proteases-induce neuronal degeneration. Primary mouse neurons from Wt mice and GMF-KO mice fetal brains were cultured for 2 weeks. These cells were incubated with MPP+ (10 μM) or mast cell proteases (MMCP-6 or MMCP-7 at 100 ng/ml) for 24 h. MAP-2 ICC was performed to analyze the neuronal morphology. (A) MPP+, MMCP-6 and MMCP-7-induced significant neuronal degeneration (arrows) in the neurons obtained from Wt mice fetal brains as compared to non-treated control neuronal cells. (B) GMF-KO neurons showed less neuronal degeneration when compared to the extent of neurodegeneration observed in the neurons grown from Wt mice fetal brains. Control neuronal cells did not show any degeneration. Scale bar = 100 μm. (C, D) The total neuronal outgrowth length was measured in the whole photomicrographs using MetaMorph software to determine the neuronal degeneration. MPP+, MMCP-6 and MMCP-7 significantly reduced the total neurite length as compared with control cells as shown in the bar graphs (One-way ANOVA and Tukey-Kramer post hoc, n=3, *p<0.05). Neurodegeneration is relatively higher in Wt neurons than in GMF-KO neurons.

Article Snippet: Recombinant mouse mast cell protease-6/Mcpt6, Recombinant mouse tryptase beta-1/Mcpt7, tryptase/BSSP-4, Enzyme-linked immunosorbent assay (ELISA) kits for mouse CCL2, MMP-3, tryptase/BSSP-4, monoclonal anti-mouse CD40L/TNFSF5 Phycoerythrin conjugated Rat IgG2A antibodies and flow cytometry reagents were purchased from R&D Systems (Minneapolis, MN).

Techniques: Cell Culture, Incubation, Software

Journal: Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology

Article Title: Cross-talk Between Glia, Neurons and Mast Cells in Neuroinflammation Associated with Parkinson’s Disease

doi: 10.1007/s11481-017-9766-1

Figure Lengend Snippet: MMCP-6 -induces CCL2 release from Wt mouse glia/neurons. (A) Wt mouse glia/neurons were incubated with MMCP-6 for dose-response (5 to 200 ng/ml) and time-course studies (6 to 48 h), and CCL2 levels in the culture media were determined by ELISA (n=3). MMCP-6 induced significant CCL2 release from 6 h (at 25 ng/ml concentration) onwards from glia/neurons. Results were presented as mean ± SEM (*p<0.05 control vs MMCP-6 treated cells, One-way ANOVA and Tukey-Kramer post hoc). (B) Wt glia/neurons were incubated with MMCP-7 (100 ng/ml) for 24 h and CCL2 release was measured by ELISA (n=5). MMCP-7 induced significant CCL2 release from glia/neurons as compared to untreated control cells (*p<0.05 control vs cells incubated with MMCP-7, t-test).

Article Snippet: Recombinant mouse mast cell protease-6/Mcpt6, Recombinant mouse tryptase beta-1/Mcpt7, tryptase/BSSP-4, Enzyme-linked immunosorbent assay (ELISA) kits for mouse CCL2, MMP-3, tryptase/BSSP-4, monoclonal anti-mouse CD40L/TNFSF5 Phycoerythrin conjugated Rat IgG2A antibodies and flow cytometry reagents were purchased from R&D Systems (Minneapolis, MN).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology

Article Title: Cross-talk Between Glia, Neurons and Mast Cells in Neuroinflammation Associated with Parkinson’s Disease

doi: 10.1007/s11481-017-9766-1

Figure Lengend Snippet: MMCP-6 on CCL2 release from GMF-KO mouse glia/neurons. Glia/neurons obtained from GMF-KO mice were incubated with MMCP-6 (100 ng/ml) for 6, 24 and 48 h. CCL2 release was measured in the culture media by ELISA (n=4). MMCP-6 significantly released CCL2 only at 48 h when compared to untreated control cells. Results were presented as mean ± SEM (*p<0.05 control vs MMCP-6 treated cells, t-test).

Article Snippet: Recombinant mouse mast cell protease-6/Mcpt6, Recombinant mouse tryptase beta-1/Mcpt7, tryptase/BSSP-4, Enzyme-linked immunosorbent assay (ELISA) kits for mouse CCL2, MMP-3, tryptase/BSSP-4, monoclonal anti-mouse CD40L/TNFSF5 Phycoerythrin conjugated Rat IgG2A antibodies and flow cytometry reagents were purchased from R&D Systems (Minneapolis, MN).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay

Journal: Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology

Article Title: Cross-talk Between Glia, Neurons and Mast Cells in Neuroinflammation Associated with Parkinson’s Disease

doi: 10.1007/s11481-017-9766-1

Figure Lengend Snippet: MMCP-6 and MMCP-7-induce CCL2 release from Wt mouse astrocytes. Wt mouse astrocytes were grown and stimulated with MMCP-6 and MMCP-7 for 6 h and 24 h, and CCL2 release was measured in the culture media by ELISA (n=3–8). Both (A) MMCP-6 and (B) MMCP-7 induced CCL2 release in a dose-dependent manner from mouse astrocytes when compared with non-treated control cells. Results were presented as mean ± SEM (*p<0.05 control vs MMCP-6 or MMCP-7 treated cells, One-way ANOVA and Tukey-Kramer post hoc).

Article Snippet: Recombinant mouse mast cell protease-6/Mcpt6, Recombinant mouse tryptase beta-1/Mcpt7, tryptase/BSSP-4, Enzyme-linked immunosorbent assay (ELISA) kits for mouse CCL2, MMP-3, tryptase/BSSP-4, monoclonal anti-mouse CD40L/TNFSF5 Phycoerythrin conjugated Rat IgG2A antibodies and flow cytometry reagents were purchased from R&D Systems (Minneapolis, MN).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology

Article Title: Cross-talk Between Glia, Neurons and Mast Cells in Neuroinflammation Associated with Parkinson’s Disease

doi: 10.1007/s11481-017-9766-1

Figure Lengend Snippet: MMCP-6, MMCP-7, and MPP+ -induce CCL2 release from mouse neurons. We examined whether MMCP-6, MMCP-7 and MPP+ activate mouse neurons to release CCL2. Mouse neuronal cells grown from Wt mice as well as from GMF-KO mice were incubated with MMCP-6 (200 ng/ml), MMCP-7 (200 ng/ml) or MPP+ (20 μM) for 24 h and the CCL2 release was assayed in the culture media by ELISA (n=3). Neurons obtained from Wt mice significantly released CCL2 after incubation with MMCP-6, MMCP-7, and MPP+ when compared to control non-treated cells. However, only MPP+ significantly released CCL2 from neuronal cultures obtained from GMF-KO mice. Results were presented as mean ± SEM (*p<0.05 control vs treated cells, One-way ANOVA and Tukey-Kramer post hoc).

Article Snippet: Recombinant mouse mast cell protease-6/Mcpt6, Recombinant mouse tryptase beta-1/Mcpt7, tryptase/BSSP-4, Enzyme-linked immunosorbent assay (ELISA) kits for mouse CCL2, MMP-3, tryptase/BSSP-4, monoclonal anti-mouse CD40L/TNFSF5 Phycoerythrin conjugated Rat IgG2A antibodies and flow cytometry reagents were purchased from R&D Systems (Minneapolis, MN).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay

Journal: Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology

Article Title: Cross-talk Between Glia, Neurons and Mast Cells in Neuroinflammation Associated with Parkinson’s Disease

doi: 10.1007/s11481-017-9766-1

Figure Lengend Snippet: GMF, MPP+, MMCP-6, MMCP-7 and tryptase release MMP-3 from Wt brain cells or mast cells. (A, B) BMMCs grown from Wt mice were incubated with GMF (100 ng/ml) or MPP+ (20 μM) for 24 h and MMP-3 release in the cell culture media was measured by ELISA. (A) GMF (n=3) and (B) MPP+ (n=4) significantly increased the release of MMP-3 when compared with non-treated control BMMCs (*p<0.05; t test, control vs treated). (C) Wt mouse BMMCs plus glia/neurons incubated with MPP+ significantly induced MMP-3 release as compared to non-treated control cells (n=3, *p<0.05; t test, control vs treated). (D) Wt mouse glia/neurons were then incubated with MPP+ (15 μM) or GMF (100 ng/ml) for 24 h and the release of MMP-3 was measured in the culture media (n=3). MPP+ and GMF significantly induced the release of MMP-3 from glia/neurons. Wt glia/neurons and BMMCs were co-cultured and then stimulated with GMF. GMF augments the release of MMP-3 in this co-culture system as compared to single glia/neurons culture condition (*p<0.05; t test, control vs treated). (E, F, G) Wt mouse astrocytes or glia/neurons were incubated with MMCP-6, MMCP-7, MPP+ or tryptase/BSSP-4 and the release of MMCP-3 in the culture media was measured by ELISA. MMCP-7 (n=6), MMP-6 (n=6), MPP+ (n=6) and tryptase/BSSP-4 (n=3)-induced significantly increased MMP-3 release as compared to control cells. Results were presented as mean ± SEM (*p<0.05 control vs treated cells, One-way ANOVA and Tukey-Kramer post hoc).

Article Snippet: Recombinant mouse mast cell protease-6/Mcpt6, Recombinant mouse tryptase beta-1/Mcpt7, tryptase/BSSP-4, Enzyme-linked immunosorbent assay (ELISA) kits for mouse CCL2, MMP-3, tryptase/BSSP-4, monoclonal anti-mouse CD40L/TNFSF5 Phycoerythrin conjugated Rat IgG2A antibodies and flow cytometry reagents were purchased from R&D Systems (Minneapolis, MN).

Techniques: Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay, Co-Culture Assay

Journal: Journal of Medicinal Chemistry

Article Title: Discovery of S-217622, a Noncovalent Oral SARS-CoV-2 3CL Protease Inhibitor Clinical Candidate for Treating COVID-19

doi: 10.1021/acs.jmedchem.2c00117

Figure Lengend Snippet: Binding modes of 3CL pro inhibitors, their interactions, and defined pharmacophore filters for virtual screening. (a) Crystal structures of GC376 (PDB code: 6WTT), (b) 3-aminopyridine-like compound of the Postera COVID moonshot project (PDB code: 5RH2), and (c) ML188 (PDB code: 7L0D). The common H-bond acceptors are circled in red; the common hydrophobic features are circled in blue. (d) Common pharmacophore shared with inhibitors A–C. Red and green spheres represent H-bond acceptors and lipophilic features, respectively.

Article Snippet: The reaction was initiated by adding 5 μL of 12 nM 3CL protease (R&D Systems, Inc.) in an inhibition buffer and incubated at room temperature for 3 h. The following operations were the same as those described in the Biological Screening .

Techniques: Binding Assay

Journal: Journal of Medicinal Chemistry

Article Title: Discovery of S-217622, a Noncovalent Oral SARS-CoV-2 3CL Protease Inhibitor Clinical Candidate for Treating COVID-19

doi: 10.1021/acs.jmedchem.2c00117

Figure Lengend Snippet: X-ray costructure of hit compound 1 and 3CL pro (PDB code: 7VTH). (a) Close-up view of 1 (cyan) in the binding pocket. Water molecules are shown as red spheres. Hydrogen bonds are indicated as yellow dashed lines; π–π stacking is indicated as a cyan dashed line. (b–d) Comparison of a docking pose and X-ray crystal structure of 1 . Near the S2 pocket, the side chain of His41 was rotated to form a face-to-face π interaction with the 3,4,5-trifluorobenzene moiety of 1 . Docked structure is in lime green, and the X-ray structure is in cyan ( 1 ) and orange (protein residues).

Article Snippet: The reaction was initiated by adding 5 μL of 12 nM 3CL protease (R&D Systems, Inc.) in an inhibition buffer and incubated at room temperature for 3 h. The following operations were the same as those described in the Biological Screening .

Techniques: Binding Assay, Comparison

Journal: Journal of Medicinal Chemistry

Article Title: Discovery of S-217622, a Noncovalent Oral SARS-CoV-2 3CL Protease Inhibitor Clinical Candidate for Treating COVID-19

doi: 10.1021/acs.jmedchem.2c00117

Figure Lengend Snippet: X-ray costructure of S-217622 ( 3 ) and 3CL pro (PDB code: 7VU6). 3 is colored in orange and the protein is colored in gray. Water molecules are shown as red spheres. Hydrogen bonds are indicated as yellow dashed lines; π–π stacking is indicated as a cyan dashed line.

Article Snippet: The reaction was initiated by adding 5 μL of 12 nM 3CL protease (R&D Systems, Inc.) in an inhibition buffer and incubated at room temperature for 3 h. The following operations were the same as those described in the Biological Screening .

Techniques:

Journal: European Journal of Medicinal Chemistry

Article Title: Novel dithiocarbamates selectively inhibit 3CL protease of SARS-CoV-2 and other coronaviruses

doi: 10.1016/j.ejmech.2023.115186

Figure Lengend Snippet: SARS-CoV-2 3CL pro expression and characterization. (a) SDS-PAGE of 3CL pro ; the calculated molecular weight of the 3CL pro is 33,796 Da. (b) Michaelis-Menten plot of 15 nM 3CL pro with various concentrations of FRET substrate (c) Initial velocity plot of enzyme reaction with various concentrations of 3CL pro . (d) Principle of the enzymatic FRET-based assay used to monitor 3CL pro activity. Once the fluorogenic substrate is cleaved by the enzyme, the fluorophore (Edans) and the fluorescence quencher (Dabcyl) are spatially separated, resulting in an increase in fluorescence which is proportional to the enzyme activity. (e) SARS-CoV-2 3CL pro inhibition by reference compounds. 3CL pro was pre-incubated 30 min with various concentration of GC-376 or boceprevir before FRET substrate addition and initial velocity measurement. Data are shown as mean ± SD of duplicates from representative experiments.

Article Snippet: SARS-CoV 3CL protease was purchased from Boston Biochem.

Techniques: Expressing, SDS Page, Molecular Weight, Activity Assay, Fluorescence, Inhibition, Incubation, Concentration Assay

Journal: European Journal of Medicinal Chemistry

Article Title: Novel dithiocarbamates selectively inhibit 3CL protease of SARS-CoV-2 and other coronaviruses

doi: 10.1016/j.ejmech.2023.115186

Figure Lengend Snippet: Left. Overview of the screening. SARS-CoV-2 3CL pro inhibition was reported (%) for each incubate. Green dots: tested compounds at 30 μM, Red dots: positive controls (incubations with vehicle), Blue dots: negative controls (incubations w/o enzyme), Orange dots: reference compound Boceprevir at 40 μM, Light orange dots: Boceprevir at 4 μM. Right. Workflow of the screening campaign.

Article Snippet: SARS-CoV 3CL protease was purchased from Boston Biochem.

Techniques: Inhibition

Journal: European Journal of Medicinal Chemistry

Article Title: Novel dithiocarbamates selectively inhibit 3CL protease of SARS-CoV-2 and other coronaviruses

doi: 10.1016/j.ejmech.2023.115186

Figure Lengend Snippet: (a) SARS-CoV-2 3CL pro inhibition by hit compounds 1 . 3CL pro was pre-incubated 60 min or not with various concentration of compound before FRET substrate addition and initial velocity measurement. Inhibitions (%) are shown as duplicate from a representative experiment. (b) SARS-CoV-2 3CL pro inhibition by hit compounds in buffer containing (2 mM) or not GSH and THP. 3CL pro was pre-incubated 60 min with various concentration of compound before FRET substrate addition and initial velocity measurement. Inhibitions (%) are shown as duplicate from a representative experiment.

Article Snippet: SARS-CoV 3CL protease was purchased from Boston Biochem.

Techniques: Inhibition, Incubation, Concentration Assay

Journal: European Journal of Medicinal Chemistry

Article Title: Novel dithiocarbamates selectively inhibit 3CL protease of SARS-CoV-2 and other coronaviruses

doi: 10.1016/j.ejmech.2023.115186

Figure Lengend Snippet: Assessment of the irreversible/reversible mechanism of inhibition with compound 1 . SARS-CoV-2 3CL pro was incubated with compound 1 in a jump dilution assay. Initial rates are measured just after the compound dilution with substrate and enzymatic inhibitions (%) are calculated as mean ± SD of triplicate. Data are representative of three other experiments.

Article Snippet: SARS-CoV 3CL protease was purchased from Boston Biochem.

Techniques: Inhibition, Incubation, Dilution Assay

Journal: European Journal of Medicinal Chemistry

Article Title: Novel dithiocarbamates selectively inhibit 3CL protease of SARS-CoV-2 and other coronaviruses

doi: 10.1016/j.ejmech.2023.115186

Figure Lengend Snippet: Evaluation of thermal stabilization of SARS-CoV-2 3CL pro by Thermal Shift Assay (TSA) in presence of Boceprevir, GC-376 or Cpd 1 (40 μM). Thermal shift (ΔTm) was calculated by subtracting reference melting temperature of the protease from the Tm in presence of the compound. Values presented are the means of ΔTm ± SD of the eight independent TSA experiments.

Article Snippet: SARS-CoV 3CL protease was purchased from Boston Biochem.

Techniques: Thermal Shift Assay

Journal: European Journal of Medicinal Chemistry

Article Title: Novel dithiocarbamates selectively inhibit 3CL protease of SARS-CoV-2 and other coronaviruses

doi: 10.1016/j.ejmech.2023.115186

Figure Lengend Snippet: Interaction of SARS-CoV-2 3CL pro with compound 1 assessed by NMR spectroscopy. a) The variations in NMR resonance intensities (top) and chemical shift perturbations (CSP) (bottom) induced upon cpd 1 addition are displayed along the main protease sequence. b) The CSPs, shown in (a, bottom), have been color coded (from light yellow to red) and are displayed on the structure of the dimeric 3CL pro , with the two protomers shown in dark grey and white, respectively. The side chains of both catalytic His41 and Cys145 are shown in green. c) Overlaid 2D 1 H, 15 N-TROSY-HSQC spectra acquired on 2 H, 15 N-labelled 3CL pro (100 μM) in the absence (in blue) or in the presence (in red) of cpd 1 (target concentration of 2 mM). The final DMSO‑ d 6 concentration was 3%. The spectra were acquired at 305 K on a 900 MHz NMR spectrometer.

Article Snippet: SARS-CoV 3CL protease was purchased from Boston Biochem.

Techniques: Spectroscopy, Sequencing, Concentration Assay

Journal: European Journal of Medicinal Chemistry

Article Title: Novel dithiocarbamates selectively inhibit 3CL protease of SARS-CoV-2 and other coronaviruses

doi: 10.1016/j.ejmech.2023.115186

Figure Lengend Snippet: Crystallographic structure of the SARS-CoV-2 3CL pro bound to the N-(pyridin-3-ylmethyl)thioformamide moiety (in pink) from the compound 1 (PDB ID: 7NTQ ) (a). The 2 Fo-Fc electron-density map, contoured at 1.5 σ, is shown as light grey mesh. (b) Interaction of the ligand (cpd 1 after Cys145 binding) with the 3CL pro S1 pocket. The analysis of the interaction was made using LigPlot+ (v2.2.4). 3CL pro and ligand bonds are shown in brown and purple, respectively. Water molecules are displayed as cyan spheres. Dashed green lines and dashed red lines represent hydrogen bonds and hydrophobic interactions, respectively. The 3CL pro residues that are involved in hydrogen bonds have their name displayed in green whereas the residues making hydrophobic contact(s) are indicated with their name surrounded by red spikes. Atoms involved in hydrophobic contact(s) are surrounded by red spikes.

Article Snippet: SARS-CoV 3CL protease was purchased from Boston Biochem.

Techniques: Binding Assay

Journal: European Journal of Medicinal Chemistry

Article Title: Novel dithiocarbamates selectively inhibit 3CL protease of SARS-CoV-2 and other coronaviruses

doi: 10.1016/j.ejmech.2023.115186

Figure Lengend Snippet: SARS-CoV-2 3CL pro inhibition of hit compound 1 and analogs 1A-D, 1x.

Article Snippet: SARS-CoV 3CL protease was purchased from Boston Biochem.

Techniques: Inhibition

Journal: European Journal of Medicinal Chemistry

Article Title: Novel dithiocarbamates selectively inhibit 3CL protease of SARS-CoV-2 and other coronaviruses

doi: 10.1016/j.ejmech.2023.115186

Figure Lengend Snippet: Activity of compounds against 3CL pro of different human coronaviruses.

Article Snippet: SARS-CoV 3CL protease was purchased from Boston Biochem.

Techniques: Activity Assay

Journal: European Journal of Medicinal Chemistry

Article Title: Novel dithiocarbamates selectively inhibit 3CL protease of SARS-CoV-2 and other coronaviruses

doi: 10.1016/j.ejmech.2023.115186

Figure Lengend Snippet: Selectivity profile of compounds against human cysteine proteases.

Article Snippet: SARS-CoV 3CL protease was purchased from Boston Biochem.

Techniques:

Journal: European Journal of Medicinal Chemistry

Article Title: Novel dithiocarbamates selectively inhibit 3CL protease of SARS-CoV-2 and other coronaviruses

doi: 10.1016/j.ejmech.2023.115186

Figure Lengend Snippet: F1G cells were infected with SARS-CoV-2 in presence of P-gp inhibitor (CP-100356) and increasing concentrations of compound 1 (top) or GC-376 (bottom). 16 h later, cells were fixed in presence of Hoechst 33342. Infected cells (GFP positive nuclei) and the total number of cells (number of nuclei, Hoechst staining) were determined. Results are presented as the percentage of the control and are the average of four independent experiments. Error bars represent the SEM.

Article Snippet: SARS-CoV 3CL protease was purchased from Boston Biochem.

Techniques: Infection, Staining

Journal: ACS Medicinal Chemistry Letters

Article Title: One Step Beyond: Design of Substrates Spanning Primed Positions of Zika Virus NS2B-NS3 Protease

doi: 10.1021/acsmedchemlett.8b00316

Figure Lengend Snippet: Cleavage rates of peptide ABZ-Val-Lys-Lys-Arg-Ala-Ala-Trp-Tyr(3-NO2)-NH2 incubated with ZIKV protease (1) and West Nile Virus protease (2). Enzymatic hydrolysis was performed in PBS (pH 7.4) at 37 °C for 30 min.

Article Snippet: Each reaction was monitored via the measurement of the fluorescence increase at 37 °C over 30 min. Substrate Hydrolysis by West Nile Virus Protease The WNS NS3 protease was purchased from R&D Systems, USA.

Techniques: Incubation, Virus