Journal: Molecular Pharmaceutics

Article Title: NQO2 Is a Reactive Oxygen Species Generating Off-Target for Acetaminophen

doi: 10.1021/mp5004866

Figure Lengend Snippet: Acetaminophen binds to NQO2. (a) Structures of acetaminophen and thioacetaminophen coupled to affinity matrix. (b) SDS–PAGE separation of protein eluates from thioacetaminophen (+) and control (−) columns. NQO2 detection from Western blot of the eluates is shown at bottom. (c) Thermal shift assays of APAP and AMAP with NQO2 and APAP with NQO1. See panel d for controls. (d) NQO2 thermal shift assay of acetaminophen regioisomers (2 mM), nitroacetanilide (2 mM), and NQO2 inhibitors quercetin (1 μM) and resveratrol (10 μM). AOAP is acetyl- o -aminophenol. Dicoumarol (10 μM) control for NQO1 is shown ( N = 3–4 in panels c and d).

Article Snippet: Antibodies used were GAPDH (#5174) from Cell Signaling Technology, SOD1 (HPA001401) from Sigma-Aldrich, and NQO2 (NBP1-31563) from Novus Biologicals.

Techniques: SDS Page, Control, Western Blot, Thermal Shift Assay

Journal: Molecular Pharmaceutics

Article Title: NQO2 Is a Reactive Oxygen Species Generating Off-Target for Acetaminophen

doi: 10.1021/mp5004866

Figure Lengend Snippet: Cellular thermal shift assays of acetaminophen binding to NQO2. (a) Schematic presentation of CETSA. The interaction between drug and target protein increases the thermal stability of the protein, which can be measured by separating and quantifying nonaggregated levels of the target protein at different temperatures. (b) SOD1 levels in HeLa cells treated as for CETSA experiments. No significant changes in protein levels were seen in the used temperatures. (c) SOD1 protein levels in cellular thermal shift samples. SOD1 levels did not change significantly when HeLa cells were treated as in CETSA (left panel) or ITDRF CETSA (right panel) experiments. For the ITDRF CETSA experiment the second highest concentration of each chemical was used, as samples reached maximal NQO2 intensity at this concentration. GAPDH was used as a loading control. (d) HeLa cell count and membrane integrity after CETSA treatment. HeLa cells were treated as for CETSA, after which cells were treated with propidium iodide for 15 min before analyzing membrane integrity and cell count using flow cytometer. Values are normalized to samples treated at 37 °C. (e) Cellular thermal shift assay of NQO2. APAP (10 mM), AMAP (10 mM), and quercetin (100 μM) were incubated with HeLa cells for 1 h. Representative NQO2 Western blots of each sample are shown. NQO2 levels were normalized to SOD1 levels used as loading control (not shown). Statistical significances between DMSO and APAP are indicated. Note that the membrane integrity is not maintained at the highest temperatures (panel d), but NQO2 binding is observed already before this, indicating binding in intact cells. APAP and AMAP did not differ significantly. (f) Isothermal (at 72 °C) dose response fingerprints in HeLa cells after 1 h exposure to APAP, nitroacetanilide, resveratrol, and menadione. Representative NQO2 Western blots of each sample are shown. Statistical significance between APAP and nitroacetanilide with 0.5 mM concentration is indicated ( N = 3–4 in all panels).

Article Snippet: Antibodies used were GAPDH (#5174) from Cell Signaling Technology, SOD1 (HPA001401) from Sigma-Aldrich, and NQO2 (NBP1-31563) from Novus Biologicals.

Techniques: Binding Assay, Concentration Assay, Control, Cell Counting, Membrane, Flow Cytometry, Thermal Shift Assay, Incubation, Western Blot

Journal: Molecular Pharmaceutics

Article Title: NQO2 Is a Reactive Oxygen Species Generating Off-Target for Acetaminophen

doi: 10.1021/mp5004866

Figure Lengend Snippet: Acetaminophen binds to NQO2 active site. (a) In silico docking analysis of APAP to NQO2 (PDB: 1ZX1) indicates binding at the active site adjacent to the isoalloxazine ring of flavin adenine mononucleotide (FAD). (b) Suggested interacting amino acids in NQO2 based on in silico docking assays. (c) Electronic absorption spectroscopy of NQO2 with 0.5 mM APAP (red) and DMSO (blue). Inset on right top corner shows difference spectrum, and inset on right bottom shows spectra with free FAD. (d) NQO2 thermal shifts of APAP (10 mM) with known NQO2 active site binding molecules menadione (50 μM) and quercetin (1 μM) ( N = 4).

Article Snippet: Antibodies used were GAPDH (#5174) from Cell Signaling Technology, SOD1 (HPA001401) from Sigma-Aldrich, and NQO2 (NBP1-31563) from Novus Biologicals.

Techniques: In Silico, Binding Assay, Spectroscopy

Journal: Molecular Pharmaceutics

Article Title: NQO2 Is a Reactive Oxygen Species Generating Off-Target for Acetaminophen

doi: 10.1021/mp5004866

Figure Lengend Snippet: Acetaminophen functions as a weak substrate for NQO2. (a) NQO2 in vitro inhibition assay. NQO2 was incubated in the presence of compounds at the concentrations indicated and NQO2 activity measured with a colorimetric assay. (b) NQO2 substrate assay. Substrate activities of menadione (25 μM), APAP (2.5 mM), AMAP (2.5 mM), and control (DMSO) are shown. All substrate activities were abolished by addition of quercetin (10 μM). (c) Kinetic analysis of NQO2 substrate activity with menadione (left) and acetaminophen (right). Red lines represent 95% confidence intervals of the fitted ligand binding curves (black). NQO2 activity was measured with increasing substrate concentrations at fixed enzyme and cosubstrate (500 μM NMEH) concentrations. Michaelis constants and maximum reaction rates are displayed with standard errors. Note that acetaminophen and menadione were dissolved in the assay buffer in order to omit DMSO ( N = 3 in all panels).

Article Snippet: Antibodies used were GAPDH (#5174) from Cell Signaling Technology, SOD1 (HPA001401) from Sigma-Aldrich, and NQO2 (NBP1-31563) from Novus Biologicals.

Techniques: In Vitro, Inhibition, Incubation, Activity Assay, Colorimetric Assay, Control, Ligand Binding Assay

Journal: Molecular Pharmaceutics

Article Title: NQO2 Is a Reactive Oxygen Species Generating Off-Target for Acetaminophen

doi: 10.1021/mp5004866

Figure Lengend Snippet: Acetaminophen-like compounds display wide range of NQO2 substrate activity in vitro and correlating superoxide production in cultured HeLa cells. (a) Comparison of NQO2 thermal shifts and NQO2 substrate activities for acetaminophen analogues (2 mM). Structures of some of the analogues are shown for comparison. (b) Specificity of MitoSOX measurement. HeLa cells were treated with APAP (5 mM) for 2.5 h followed by 0.5 h treatment with superoxide scavenger, MnTBAP (200 μM). (c) Comparison of APAP and NAPQI mediated superoxide production. HeLa cells were treated with APAP (2 mM) or NAPQI (20 μM) for 8 h. Note that only a small fraction of APAP is metabolized to NAPQI, and higher NAPQI concentrations are lethal to cells. (d) Effect of P450 inhibition on APAP mediated superoxide production. HeLa cells were treated with P450 inhibitor disulfiram (2 μM) for 1 h followed by APAP treatment (5 mM) for 2 h. Data normalized to controls without APAP treatment for clarity. (e) Comparison of NQO2 substrate activity and cellular superoxide production by acetaminophen and its analogues. For MitoSOX measurements HeLa cells were treated with 2 mM chemicals for 1 h. See Figure S1 and Table S1 for structures and data, respectively ( N = 3 in all panels).

Article Snippet: Antibodies used were GAPDH (#5174) from Cell Signaling Technology, SOD1 (HPA001401) from Sigma-Aldrich, and NQO2 (NBP1-31563) from Novus Biologicals.

Techniques: Activity Assay, In Vitro, Cell Culture, Comparison, Analogues, Inhibition

Journal: Molecular Pharmaceutics

Article Title: NQO2 Is a Reactive Oxygen Species Generating Off-Target for Acetaminophen

doi: 10.1021/mp5004866

Figure Lengend Snippet: NQO2 protein and mRNA levels in normal human tissues. Tissue Western blot displays unknown NQO2 variants in skeletal muscle. For quantifications, mRNA and protein levels were normalized to NQO2 in liver. See also Figure S3.

Article Snippet: Antibodies used were GAPDH (#5174) from Cell Signaling Technology, SOD1 (HPA001401) from Sigma-Aldrich, and NQO2 (NBP1-31563) from Novus Biologicals.

Techniques: Western Blot

Journal: Molecular Pharmaceutics

Article Title: NQO2 Is a Reactive Oxygen Species Generating Off-Target for Acetaminophen

doi: 10.1021/mp5004866

Figure Lengend Snippet: NQO2 is involved in acetaminophen induced superoxide production. (a) APAP induced superoxide production (left) and intracellular Ca 2+ levels (right) after chemical inhibition of NQO2. HeLa cells were treated with control (DMSO), quercetin (10 μM), resveratrol (10 μM), or imatinib (20 μM) for 1 h followed by 4 h treatment with vehicle (DMSO), APAP (5 mM), or AMAP (5 mM). (b) APAP induced superoxide production after NQO2 RNAi. HeLa cells were treated with control or NQO2 siRNA for 48 h followed by 4 h treatment with vehicle (DMSO), APAP (5 mM), or AMAP (5 mM). (c) APAP induced superoxide production after NQO2 overexpression. HeLa cells were transfected with empty vector or NQO2, incubated for 36 h followed by 5 h treatment with vehicle (DMSO) or APAP (5 mM). In panels b and c representative Western blots of NQO2 levels are on the left, superoxide levels in the middle, and intracellular Ca 2+ levels on the right. Data is normalized to each control for clarity ( N = 3–4 in all panels).

Article Snippet: Antibodies used were GAPDH (#5174) from Cell Signaling Technology, SOD1 (HPA001401) from Sigma-Aldrich, and NQO2 (NBP1-31563) from Novus Biologicals.

Techniques: Inhibition, Control, Over Expression, Transfection, Plasmid Preparation, Incubation, Western Blot

Journal: Cancers

Article Title: Comprehensive Constitutional Genetic and Epigenetic Characterization of Lynch-Like Individuals

doi: 10.3390/cancers12071799

Figure Lengend Snippet: Results obtained from the characterization of LLS patients, including cases analyzed by NGS, MMR VUS carriers and epimutants. (*) Not previously reported MMR variant classified according to InSiGHT variant classification rules. (#) See details in Table S1 . (^) Up to date only EPCAM 3′UTR deletions has been associated with CRC predisposition, whereas point mutations cause an autosomal recessive congenital diarrhoea (OMIM:613217) unrelated to CRC.

Article Snippet: Immunohistochemistry of MSH3 protein was performed using anti-MSH3 antibody at dilution 1:150 (Novus Biologicals, Centennial, CO, USA).

Techniques: Variant Assay, Sequencing, Expressing

Journal: Cancers

Article Title: Comprehensive Constitutional Genetic and Epigenetic Characterization of Lynch-Like Individuals

doi: 10.3390/cancers12071799

Figure Lengend Snippet: Variants identified in non LS-associated genes and predicted pathogenic by in silico predictors . See Table S5 and S6 for futher details. Abbreviations: NP: not performed; NA: not available; DSS=Consensus Donor Splice Site; ASS=Consensus Aceptor Splice Site; D = Damaging; PrD = Probably Damaging; PsD = Possibly Damaging; T = Tolerated. Gain or Loss of Splice sites are considered when 4 of the 5 predictors are in agreement of their calculation. Inconclusive interpretation is given when 3 of the 5 predictors predicted changes. Less than 3 similar predictions are considered as no changes. (^) Up to date only EPCAM 3′UTR deletions has been associated with CRC predisposition, whereas point mutations cause an autosomal recessive congenital diarrhoea (OMIM:613217) unrelated to CRC.

Article Snippet: Immunohistochemistry of MSH3 protein was performed using anti-MSH3 antibody at dilution 1:150 (Novus Biologicals, Centennial, CO, USA).

Techniques: In Silico, Variant Assay, Mutagenesis