Journal: International Journal of Biological Sciences

Article Title: Gut Microbiota-Derived Tyrosol Alleviates Radiation-Induced Intestinal Injury via Targeting SCD1-MUFA Axis to Suppress ER Stress

doi: 10.7150/ijbs.126269

Figure Lengend Snippet: Tyrosol inhibits VCP-mediated degradation of SCD1 in HIECs. (A) Western blot analysis of SCD1 levels in the irradiated HIECs treated with tyrosol at indicating time points after IR; cells pretreated with CHX for 2 h prior to IR; right: quantification of SCD1 relative to β-tubulin, normalized to 0 h. (B) Western blot analysis of SCD1 levels at 12 h post-IR following treatment with CHX plus MG132 or Leupeptin in HIECs; bottom, quantification normalized to β-tubulin (relative to NR). (C) Western blot analysis of SCD1 at 12 h post-IR after CHX with MG132 or TAK-243; bottom, quantification normalized to β-tubulin (relative to NR). (D) Co-IP showing ubiquitination of SCD1 at 12 h post-IR, with MG132 and tyrosol pretreatment. (E) Western blot analysis of VCP and SCD1 levels at 12 h post-IR in HIECs transfected with VCP siRNA or control siRNA; bottom, quantification normalized to β-tubulin (relative to NR). (F) Co-IP demonstrating interaction between SCD1 and VCP with or without 24 h tyrosol treatment prior to IR; bottom, quantification normalized to vinculin (relative to NR). (G) Immunofluorescence staining of SCD1 and PDI, following 24 h tyrosol or 2 h VCP inhibitor (NMS-873) pretreatment before IR (8 Gy). Scale bar = 20 μm. Right: Manders' coefficient-based quantification of SCD1-ER colocalization in HIECs, 50 cells per group. (H) Violin plots presenting SCD1 metabolic products (OA, POA) and substrates (SA, PA) measured by LC-MS in HIECs pretreated with tyrosol prior to IR (n=3 per group). Bars represent mean ± SD. IR, irradiation; Ty, tyrosol; CHX, cycloheximide; SA, stearic acid; OA, oleic acid; PA, palmitic acid; POA, palmitoleic acid; VCPinh, VCP inhibitor. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, no significant difference.

Article Snippet: To assess protein stability, cells were treated with Cycloheximide (CHX; T1225, TargetMol, protein synthesis inhibitor, 10 μM), MG132 (HY-13259, MCE, proteasome inhibitor, 10 μM), Leupeptin (HY-18234, MCE, lysosomal protease inhibitor, 10 μM), or TAK-243 (T16974, TargetMol, ubiquitin-activating enzyme inhibitor, 10 μM) 2 h prior to irradiation or cell collection.

Techniques: Western Blot, Irradiation, Co-Immunoprecipitation Assay, Ubiquitin Proteomics, Transfection, Control, Immunofluorescence, Staining, Liquid Chromatography with Mass Spectroscopy

Journal: Computational and Structural Biotechnology Journal

Article Title: Potential Drug Discovery for COVID-19 Treatment Targeting Cathepsin L Using a Deep Learning-Based Strategy

doi: 10.1016/j.csbj.2022.05.023

Figure Lengend Snippet: Prediction dataset validation with a cell-free CTSL activity detection system. A, Among the available molecules, the top 50 molecules from the prediction dataset were chosen for verifying the inhibition effect against CTSL in a cell-free system at a single dose of 100 μM. Twelve of the 50 predicted molecules displayed over 50% inhibition against CTSL, and the top 5 were Mg-132, Z-FA-FMK, leupeptin hemisulfate, Mg-101 and calpeptin, with inhibition efficiencies greater than 90%. The data are expressed as the mean of three individual trials. B-F, Five predicted CTSL inhibitors, Mg-132(B), Z-FA-FMK(C), leupeptin hemisulfate(D), Mg-101(E) and calpeptin(F), with inhibition efficiencies greater than 90% at 100 μM were further tested for determination of the half maximal inhibitory concentration (IC 50 ) in the cell-free system. Corresponding molecular structure was drawn by Chemdraw. Non-linear fit to a variable response curve from one representative experiment with three replicates is shown (black lines). The data are expressed as the mean ± s.e.m.

Article Snippet: Mg-132 (Cat. No. S2619), Z-FA-FMK (Cat. No. S7391), leupeptin hemisulfate (Cat. No. S7380), Mg-101 (Cat. No. S7386), calpeptin (Cat. No. S7396), daptomycin (Cat. No. S1373), beta-lapachone (Cat. No. S7261) and other molecules and drugs were purchased from Selleck (Selleckchem, Houston, TX, USA).

Techniques: Activity Assay, Inhibition, Concentration Assay

Journal: Computational and Structural Biotechnology Journal

Article Title: Potential Drug Discovery for COVID-19 Treatment Targeting Cathepsin L Using a Deep Learning-Based Strategy

doi: 10.1016/j.csbj.2022.05.023

Figure Lengend Snippet: The predicted CTSL inhibitors from bioactive compounds prevent SARS-CoV-2 pseudovirus infection in Huh7 cells in vitro. A, Schematic of the predicted CTSL inhibitor assay setup. Huh7 cells were pretreated with different drugs 1 hour(h) before infection with SARS-CoV-2 pseudovirus or SARS-CoV-2 B.1.351 (Beta) variant pseudovirus at the same dose (1.3×10 4 TCID 50 /ml). Pseudovirus infection and cell viability were evaluated 24 h later by a luciferase activity and MTT assay, respectively. B-F, Inhibition of pseudovirus infection by different doses of Mg-132 (B), Z-FA-FMK (C), Leupeptin Hemisulfate (D), Mg-101 (E), and Calpeptin (F) and viability of Huh7 cells treated with different doses of the drugs as indicated. Non-linear fit to a variable response curve from one representative experiment with four replicates is shown (red lines). Cytotoxic effect on Huh7 cells exposed to increasing concentrations of drugs in the absence of virus is also shown (blue lines). The CC 50 , EC 50 , and SI values of this graph are indicated. n=4. The data are expressed as the mean ± s.e.m. G-K, Inhibition of SARS-CoV-2 B.1.351(Bata) variant pseudovirus infection by different doses of Mg-132 (G), Z-FA-FMK (H), Leupeptin Hemisulfate (I), Mg-101 (J), and Calpeptin (K) and viability of Huh7 cells treated with different doses of the drugs as indicated. Non-linear fit to a variable response curve from one representative experiment with four replicates is shown (purple lines). Cytotoxic effect on Huh7 cells exposed to increasing concentrations of drugs in the absence of virus is also shown (blue lines). The CC 50 , EC 50 , and SI values of this graph are indicated. CC 50 : 50% cytotoxic concentration. EC 50 : half maximal effective concentration. SI: the selectivity index, which is calculated as the ratio of CC 50 and EC 50 . n=4. The data are expressed as the mean ± s.e.m.

Article Snippet: Mg-132 (Cat. No. S2619), Z-FA-FMK (Cat. No. S7391), leupeptin hemisulfate (Cat. No. S7380), Mg-101 (Cat. No. S7386), calpeptin (Cat. No. S7396), daptomycin (Cat. No. S1373), beta-lapachone (Cat. No. S7261) and other molecules and drugs were purchased from Selleck (Selleckchem, Houston, TX, USA).

Techniques: Infection, In Vitro, Variant Assay, Luciferase, Activity Assay, MTT Assay, Inhibition, Concentration Assay

Journal: Computational and Structural Biotechnology Journal

Article Title: Potential Drug Discovery for COVID-19 Treatment Targeting Cathepsin L Using a Deep Learning-Based Strategy

doi: 10.1016/j.csbj.2022.05.023

Figure Lengend Snippet: The predicted CTSL inhibitors from bioactive compounds prevent live SARS-CoV-2 infection in Huh7 cells in vitro. A, Schematic of the predicted CTSL inhibitor assay setup. Huh7 cells were pretreated with different drugs 1 h before infection with live SARS-CoV-2 at the same dose (0.5 moi), followed by changing to fresh medium with the indicated concentrations of drugs 1 h later. The detection of infected cells was performed 48 h later by using an immunofluorescence assay. B-D, Inhibition of live SARS-CoV-2 infection by different doses of Mg-132 (B), Z-FA-FMK (C), and Leupeptin Hemisulfate (D). Non-linear fit to a variable response curve from one representative experiment with three replicates is shown (blue lines). The EC 50 value of this graph is indicated. n = 3. The data are expressed as the mean ± s.e.m.

Article Snippet: Mg-132 (Cat. No. S2619), Z-FA-FMK (Cat. No. S7391), leupeptin hemisulfate (Cat. No. S7380), Mg-101 (Cat. No. S7386), calpeptin (Cat. No. S7396), daptomycin (Cat. No. S1373), beta-lapachone (Cat. No. S7261) and other molecules and drugs were purchased from Selleck (Selleckchem, Houston, TX, USA).

Techniques: Infection, In Vitro, Immunofluorescence, Inhibition

Journal: Computational and Structural Biotechnology Journal

Article Title: Potential Drug Discovery for COVID-19 Treatment Targeting Cathepsin L Using a Deep Learning-Based Strategy

doi: 10.1016/j.csbj.2022.05.023

Figure Lengend Snippet: Molecular docking results of CTSL inhibitors in the crystal structure of human CTSL (5MQY). A-G, 3D structure of the human CTSL (5MQY) showing the main residues involved in the protein-ligand interaction of Compound 35 (A), Mg-132 (B), Z-FA-FMK (C), Leupeptin Hemisulfate (D), Calpeptin (E), Mg-101 (F) and Daptomycin (G). Compound 35 is a covalent inhibitor cocrystallized with human CTSL protein in 5MQY, used here as a positive control. Short intermolecular contacts with distances of < 4.0 Å between the ligand fragment (gray) and protein residues (dark green) are shown as dashed yellow lines. Structure visualization was by PyMol.

Article Snippet: Mg-132 (Cat. No. S2619), Z-FA-FMK (Cat. No. S7391), leupeptin hemisulfate (Cat. No. S7380), Mg-101 (Cat. No. S7386), calpeptin (Cat. No. S7396), daptomycin (Cat. No. S1373), beta-lapachone (Cat. No. S7261) and other molecules and drugs were purchased from Selleck (Selleckchem, Houston, TX, USA).

Techniques: Positive Control