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 second model training and the identification of daptomycin. For drug repurposing screening for COVID-19 from the FDA-approved drug library, we trained the second model by adding the experimentally validated molecules from bioactive compounds aforementioned to the initial training dataset. A, The ROC-AUC plot evaluating the second model performance after training. Blue is the mean of twenty folds (grey). B, Rank-ordered prediction scores of the FDA-approved drug library that were not present in the training dataset. C, Visualization of all molecules from the second training dataset (green) and the second prediction dataset(orange) using t-SNE, revealing chemical relationships between these libraries. D, Among the available drugs, the top 50 drugs from the FDA-approved drug library were chosen for verifying the inhibition effect against CTSL in the cell-free system at a single dose of 100 μM. Four of the 50 predicted drugs displayed over 50% inhibition against CTSL, and the top 2 were daptomycin and beta-lapachone, with inhibition efficiencies greater than 90%. The data are expressed as the mean of three individual trials. E-F, Daptomycin(E) and beta-lapachone(F) were further tested for determination of IC 50 in the cell-free system. These 2 drugs were used at a concentration ranging from 8 nM and 80nM to 100 μM, respectively. The IC 50 value of this graph is indicated. n=3. The data are expressed as the mean ± s.e.m. G-H, Inhibition of pseudovirus infection by different doses of Daptomycin (G), and beta-lapachone (H) 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. I, Inhibition of pseudovirus infection by different doses of Daptomycin, and viability of A549 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 A549 cells exposed to increasing concentrations of drugs in the absence of virus is also shown (green lines). The CC 50 , EC 50 , and SI values of this graph are indicated. n = 5. The data are expressed as the mean ± s.e.m. J, Inhibition of SARS-CoV-2 B.1.351(Bata) variant pseudovirus infection by different doses of Daptomycin, 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. n = 5. The data are expressed as the mean ± s.e.m. K, Inhibition of SARS-CoV-2 B.1.351(Bata) variant pseudovirus infection by different doses of Daptomycin, and viability of Huh7 cells with TMPRSS2 overexpression. 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 as indicated is also shown (blue lines). The EC 50 values of this graph are indicated. n = 5. The data are expressed as the mean ± s.e.m. L, Inhibition of SARS-CoV-2 B.1.351(Bata) variant pseudovirus infection by different doses of Daptomycin, and viability of A549 cells with TMPRSS2 overexpression. Non-linear fit to a variable response curve from one representative experiment with four replicates is shown (purple lines). Cytotoxic effect on A549 cells exposed to increasing concentrations of drugs as indicated is also shown (green lines). The EC 50 values of this graph are indicated. n = 5. 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: Inhibition, Concentration Assay, Infection, Variant Assay, Over Expression

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:

Article Title: Selective killing of cancer cells by ?-lapachone: Direct checkpoint activation as a strategy against cancer

doi: 10.1073/pnas.0538044100

Figure Lengend Snippet: β-Lapachone selectively induced apoptosis of human cancer cells and not normal cells. (A) Multiple myeloma (MM) cells and proliferating peripheral blood mononuclear cells (PBMC) were treated with β-lapachone (0, 0.5, 2, 4, or 8 μM) for 24 h. Cell death was measured by the MTT assay. Results are the average of triplicates from one of the three independent experiments. MM.As (▴) is a cell line from a myeloma patient; MM.1R (○) is a myeloma cell line that is resistant to dexamethasone; PBMCs (●) were stimulated with PHA at 2 μg/ml for 72-h incubation before β-lapachone treatment. Control cells were treated with an equal volume of DMSO. (B) Human breast cancer cells (MCF-7) and nontransformed breast epithelial cells (MCF-10A) were treated with β-lapachone at the concentrations indicated for 4 h and incubated in drug-free media for 10–14 days. ▪, MCF-7; □, MCF-10A. Cell survival was determined by colony formation assay (8). Results are from one of two independent experiments. (C) Human colonic cancer cells (DLD1, SW480) and normal human colonic epithelial cells (NCM460) were treated with β-lapachone for 4 h. Cells were incubated in drug-free media for an additional 20 h and were harvested for flow cytometry analysis. Apoptosis was determined by the sub-G1 fraction (8). (D) Human colonic cancer cells (DLD1) were treated with β-lapachone at 4 μM for different times as indicated. Cytoplasmic cytochrome c was determined by immunoblotting with anti-cytochrome c. β-Actin was used as a loading control. (E) Human colon cancer (SW 480, lanes 1–4) and normal colonic epithelial cells (NCM 460, lanes 5–8) were treated with β-lapachone at 0 μM (lanes 1 and 5), 0.5 μM (lanes 2 and 6), 2 μM (lanes 3 and 7), or 4 μM (lanes 4 and 8) for 4 h, followed by incubation in drug-free media for an additional 4 h before cells were harvested for analysis of poly(ADP-ribose) polymerase by Western blot.

Article Snippet: Whole-cell extracts were prepared, and the E2F1 level was determined by Western blotting with a monoclonal antibody against E2F1. ( C ) Effects of β-lapachone on E2F2, -3, -4, and -5 were similarly determined from the same set of cell lysates of B with monoclonal antibodies from Santa Cruz Biotechnology. β-Actin was used as a loading control. ( D ) SW 480 cells (lanes 1–5) and NCM 460 cells (lanes 6–8) were treated with β-lapachone at 4 μM for 0.5 h (lanes 2 and 7), 1 h (lanes 3 and 8), 2 h (lane 4), or 4 h (lane 5).

Techniques: MTT Assay, Incubation, Colony Assay, Flow Cytometry, Western Blot

Journal:

Article Title: Selective killing of cancer cells by ?-lapachone: Direct checkpoint activation as a strategy against cancer

doi: 10.1073/pnas.0538044100

Figure Lengend Snippet: Activation of S-phase checkpoint by β-lapachone (A and B) is accompanied by indirect inhibition of cyclin A/CDK2 (C and D). DU145 prostate cancer cells were treated with vehicle control (A) or β-lapachone at 4 μM (B) for 4 h and incubated in drug-free medium for an additional 4 h. Cells were harvested for flow cytometry analysis (8). (C) Cyclin A/CDK2 was immunoprecipated from untreated cells, and the direct effect of β-lapachone on the kinase activity was determined with histone H1 as the substrate (8, 24). Lane 1, control; lane 2, 2 μM; lane 3, 4 μM. (D) DU145 prostate cancer cells were treated with or without β-lapachone for 4 h and incubated in drug-free medium for an additional 4 h. Nuclear extracts were prepared. Cylin A CDK2 were immunoprecipated, and the kinase activity was determined as described. Lane 1, control; lane 2, 0.5 μM; lane 3, 2 μM; lane 4, 4 μM.

Article Snippet: Whole-cell extracts were prepared, and the E2F1 level was determined by Western blotting with a monoclonal antibody against E2F1. ( C ) Effects of β-lapachone on E2F2, -3, -4, and -5 were similarly determined from the same set of cell lysates of B with monoclonal antibodies from Santa Cruz Biotechnology. β-Actin was used as a loading control. ( D ) SW 480 cells (lanes 1–5) and NCM 460 cells (lanes 6–8) were treated with β-lapachone at 4 μM for 0.5 h (lanes 2 and 7), 1 h (lanes 3 and 8), 2 h (lane 4), or 4 h (lane 5).

Techniques: Activation Assay, Inhibition, Incubation, Flow Cytometry, Activity Assay

Journal:

Article Title: Selective killing of cancer cells by ?-lapachone: Direct checkpoint activation as a strategy against cancer

doi: 10.1073/pnas.0538044100

Figure Lengend Snippet: β-Lapachone selectively induced elevation of E2F1 protein in human pancreatic cancer cells (A) and colon cancer cells (B), but not in normal colonic epithelial cells (C). (A) Human pancreatic cancer cells (Paca-2) were exposed to β-lapachone at 0 μM (lane 1), 0.5 μM (lane 2), 2 μM (lane 3), and 4 μM (lane 4) for 0.5 h and were harvested for determination of E2F1 level by Western blot. Monoclonal antibody against E2F1 was obtained from Santa Cruz Biotechnology. (B) SW480 and NCM460 were treated with β-lapachone at 2 μM and were harvested after 20 min (lane 2), 1 h (lane 3), 2 h (lane 4), or 4 h (lane 5). Control cells were treated with an equal volume of DMSO (lane 1). Whole-cell extracts were prepared, and the E2F1 level was determined by Western blotting with a monoclonal antibody against E2F1. (C) Effects of β-lapachone on E2F2, -3, -4, and -5 were similarly determined from the same set of cell lysates of B with monoclonal antibodies from Santa Cruz Biotechnology. β-Actin was used as a loading control. (D) SW 480 cells (lanes 1–5) and NCM 460 cells (lanes 6–8) were treated with β-lapachone at 4 μM for 0.5 h (lanes 2 and 7), 1 h (lanes 3 and 8), 2 h (lane 4), or 4 h (lane 5). Control cells were treated with an equal volume of DMSO (lanes 1 and 6). Nuclear extracts were prepared, and E2F1 activity was determined by the electromobility shift assay by using a 32P-labeled, 100-bp, double-stranded DNA fragment containing three E2F consensus sequences (18). Arrow denotes the location of different forms of E2F protein–DNA complexes. Results represent one of three independent experiments. (E) Expression of pRB and phosphorylation status in cell lines used. Lane 1, DLD1; lane 2, SW480; lane 3, NCM 460; lane 4, MCF7; lane 5, MCF10A. Whole-cell lysates were prepared, and Western blotting was used to analyze pRB by using a monoclonal antibody from Santa Cruz Biotechnology.

Article Snippet: Whole-cell extracts were prepared, and the E2F1 level was determined by Western blotting with a monoclonal antibody against E2F1. ( C ) Effects of β-lapachone on E2F2, -3, -4, and -5 were similarly determined from the same set of cell lysates of B with monoclonal antibodies from Santa Cruz Biotechnology. β-Actin was used as a loading control. ( D ) SW 480 cells (lanes 1–5) and NCM 460 cells (lanes 6–8) were treated with β-lapachone at 4 μM for 0.5 h (lanes 2 and 7), 1 h (lanes 3 and 8), 2 h (lane 4), or 4 h (lane 5).

Techniques: Western Blot, Activity Assay, Electro Mobility Shift Assay, Labeling, Expressing

Journal:

Article Title: Selective killing of cancer cells by ?-lapachone: Direct checkpoint activation as a strategy against cancer

doi: 10.1073/pnas.0538044100

Figure Lengend Snippet: Proposed apoptotic mechanism of β-lapachone.

Article Snippet: Whole-cell extracts were prepared, and the E2F1 level was determined by Western blotting with a monoclonal antibody against E2F1. ( C ) Effects of β-lapachone on E2F2, -3, -4, and -5 were similarly determined from the same set of cell lysates of B with monoclonal antibodies from Santa Cruz Biotechnology. β-Actin was used as a loading control. ( D ) SW 480 cells (lanes 1–5) and NCM 460 cells (lanes 6–8) were treated with β-lapachone at 4 μM for 0.5 h (lanes 2 and 7), 1 h (lanes 3 and 8), 2 h (lane 4), or 4 h (lane 5).

Techniques:

Journal: Assay and Drug Development Technologies

Article Title: Profiling the NIH Small Molecule Repository for Compounds That Generate H 2 O 2 by Redox Cycling in Reducing Environments

doi: 10.1089/adt.2009.0247

Figure Lengend Snippet: Screening the Library of Pharmacologically Active Compounds (LOPAC) set to identify redox cycling compounds (RCCs). (A) The 4 × 384-well plate overlay scatter plot of the % activation of H2O2 production data from the first iteration of the LOPAC screen. An ActivityBase™ HTS template was written to calculate the % activation based on the maximum (n = 32) and minimum (n = 24) plate controls for the HRP-PR H2O2 detection assay. A Spotfire® visualization of the % activation data from 4 × 384-well plates of the HRP-PR H2O2 production data for the first iteration of the LOPAC screen overlaid in a single scatter plot is presented; () maximum (100 μM H2O2 in 1% Dimethyl Sulfoxide (DMSO), n = 32) plate controls, () minimum (1% DMSO, n = 24) plate controls, () 50% (50 μM H2O2 in 1% DMSO, n = 8) plate controls, and () 10 μM compounds in 0.8 mM DTT (1% DMSO, n = 320). (B) Chemical structures, names, and PubChem substance identity numbers (SIDs) of the RCCs identified in the LOPAC set. The chemical structures of 2,3-bis(2-hydroxyethylthio)naphthalene-1,4-dione (NSC 95397, PubChem-SID 400206), and 2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione (β-lapachone, PubChem-SID 207115) are presented.

Article Snippet: β-Lapachone SID 207115 , UPDDI , 14 , 7 , MKP-1, MKP-3, YopH, SptP, L929-zVAD and Jurkat-FADD −/− TNFα necroptosis, and MEF cell death.

Techniques: Activation Assay, Detection Assay