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

Journal: Frontiers in Physiology

Article Title: Estrogen receptor α regulates phenotypic switching and proliferation of vascular smooth muscle cells through the NRF1-OMI-mitophagy signaling pathway under simulated microgravity

doi: 10.3389/fphys.2022.1039913

Figure Lengend Snippet: ERα-NRF1-OMI involved in regulating parkin expression. (A,B) Western blot analysis of OMI in A7r5 cells transfected with different OMI of adenoviruses (A) or siRNA (B) expressing OMI. (C,D) Western blot analysis of Parkin and OMI protein levels in total cell lysis (left), cytoplasm (middle) and mitochondria (right) of A7r5 cells treated with FBS, transfected with adenoviruses expressing OMI or noncoding control (C) and with adenoviruses expressing OMI or noncoding control (D) . (E) Western blot analysis of parkin and OMI protein levels in HEK293 cells. The cells were transfected with Parkin and OMI vector, and treated with MG132 and/or leupeptin. (F,G) Expression of OMI promoted Parkin stability via the proteasomal pathway. HEK293 Cells were transfected with Parkin alone [ (F) ; top panel], Parkin and OMI [ (F) ; middle panel], or Parkin and OMI with MG132 treatment [ (F) ; bottom panel], and levels of Parkin were detected by western blot analysis. The representative bolt (F) and quantitation of 3 independent experiments are shown (G) . * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. control.

Article Snippet: For inhibitor treatment, 3-MA (10 mM), FCCP (5 μM), MG132 (5 μM) and leupeptin (50 μM) were purchased from MedChemExpress Co., Ltd. (New Jersey, United States).

Techniques: Expressing, Western Blot, Transfection, Lysis, Control, Plasmid Preparation, Quantitation Assay