Journal: Drug metabolism and disposition: the biological fate of chemicals

Article Title: Discovery and characterization of novel, potent, and selective cytochrome P450 2J2 inhibitors.

doi: 10.1124/dmd.112.048264

Figure Lengend Snippet: Fig. 1. Representative IC50 plots for telmisartan (A) and flunarizine (B) inhibition of astemizole O-demethylation using recombinant CYP2J2 with astemizole concen- trations of 0.1–20 mM.

Article Snippet: CYP substrates, inhibitors, metabolite standards, and all other materials were obtained from the following sources: all compounds from Table 1, except olmesartan, that were used as inhibitors for the CYP2J2 and human liver microsome (HLM) inhibition studies, astemizole (AST), phenacetin, tolbutamide, bufuralol, omeprazole, 4’-hydroxytolbutamide, 1’- hydroxybufuralol, 6b-hydroxytestosterone, acetaminophen, dextrorphan, and nicotinamide adenine dinucleotide phosphate (NADPH) were purchased from Sigma-Aldrich (St. Louis, MO); testosterone was purchased from Acros Organics (Morris Plains, NJ); 5’-hydroxyomeprazole was purchased from Toronto Research Chemicals Inc. (North York, ON, Canada); olmesartan and O-desmethyl astemizole (DES-AST) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); potassium phosphate (monobasic and dibasic) and magnesium chloride hexahydrate (MgCl2) were purchased from Merck (Darmstadt, Germany); pooled HLMs and recombinant CYP enzyme were purchased from BD Gentest (Woburn, MA); and high-performance liquid chromatography (HPLC) grade dimethyl sulfoxide (DMSO), methanol, and formic acid used for liquid chromatography-tandem mass spectrometry (LC-MS/ MS) analysis were purchased from Fisher Scientific Co. (Pittsburgh, PA).

Techniques: Inhibition, Recombinant

Journal: Drug metabolism and disposition: the biological fate of chemicals

Article Title: Discovery and characterization of novel, potent, and selective cytochrome P450 2J2 inhibitors.

doi: 10.1124/dmd.112.048264

Figure Lengend Snippet: Fig. 2. Disappearance of astemizole (A), telmisartan (B), and flunarizine (C), measured from incubation with recombinant CYP2J2 in the presence of NADPH at different time points (n = 2).

Article Snippet: CYP substrates, inhibitors, metabolite standards, and all other materials were obtained from the following sources: all compounds from Table 1, except olmesartan, that were used as inhibitors for the CYP2J2 and human liver microsome (HLM) inhibition studies, astemizole (AST), phenacetin, tolbutamide, bufuralol, omeprazole, 4’-hydroxytolbutamide, 1’- hydroxybufuralol, 6b-hydroxytestosterone, acetaminophen, dextrorphan, and nicotinamide adenine dinucleotide phosphate (NADPH) were purchased from Sigma-Aldrich (St. Louis, MO); testosterone was purchased from Acros Organics (Morris Plains, NJ); 5’-hydroxyomeprazole was purchased from Toronto Research Chemicals Inc. (North York, ON, Canada); olmesartan and O-desmethyl astemizole (DES-AST) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); potassium phosphate (monobasic and dibasic) and magnesium chloride hexahydrate (MgCl2) were purchased from Merck (Darmstadt, Germany); pooled HLMs and recombinant CYP enzyme were purchased from BD Gentest (Woburn, MA); and high-performance liquid chromatography (HPLC) grade dimethyl sulfoxide (DMSO), methanol, and formic acid used for liquid chromatography-tandem mass spectrometry (LC-MS/ MS) analysis were purchased from Fisher Scientific Co. (Pittsburgh, PA).

Techniques: Incubation, Recombinant

Journal: Drug metabolism and disposition: the biological fate of chemicals

Article Title: Discovery and characterization of novel, potent, and selective cytochrome P450 2J2 inhibitors.

doi: 10.1124/dmd.112.048264

Figure Lengend Snippet: Fig. 3. IC50 determination of inhibition of CYP2J2-mediated astemizole O- demethylation by telmisartan (A) and flunarizine (B) in the presence and absence of NADPH. The inhibitors were preincubated with CYP2J2 for 30 minutes. The IC50 shift was calculated as IC50 in the absence of NADPH over IC50 in the presence of NADPH, to evaluate time-dependent inhibition.

Article Snippet: CYP substrates, inhibitors, metabolite standards, and all other materials were obtained from the following sources: all compounds from Table 1, except olmesartan, that were used as inhibitors for the CYP2J2 and human liver microsome (HLM) inhibition studies, astemizole (AST), phenacetin, tolbutamide, bufuralol, omeprazole, 4’-hydroxytolbutamide, 1’- hydroxybufuralol, 6b-hydroxytestosterone, acetaminophen, dextrorphan, and nicotinamide adenine dinucleotide phosphate (NADPH) were purchased from Sigma-Aldrich (St. Louis, MO); testosterone was purchased from Acros Organics (Morris Plains, NJ); 5’-hydroxyomeprazole was purchased from Toronto Research Chemicals Inc. (North York, ON, Canada); olmesartan and O-desmethyl astemizole (DES-AST) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); potassium phosphate (monobasic and dibasic) and magnesium chloride hexahydrate (MgCl2) were purchased from Merck (Darmstadt, Germany); pooled HLMs and recombinant CYP enzyme were purchased from BD Gentest (Woburn, MA); and high-performance liquid chromatography (HPLC) grade dimethyl sulfoxide (DMSO), methanol, and formic acid used for liquid chromatography-tandem mass spectrometry (LC-MS/ MS) analysis were purchased from Fisher Scientific Co. (Pittsburgh, PA).

Techniques: Inhibition

Journal: Drug metabolism and disposition: the biological fate of chemicals

Article Title: Discovery and characterization of novel, potent, and selective cytochrome P450 2J2 inhibitors.

doi: 10.1124/dmd.112.048264

Figure Lengend Snippet: Fig. 4. Inhibition assay against the enzymatic activity of recombinant CYP2J2. Nonlinear regression of the initial velocity at various substrate concentrations in the presence of telmisartan (A) and flunarizine (B) as the inhibitor with concentrations of 0.1–2 mM and 0.2–5 mM, respectively. Dixon plots with amplified insets for the enzyme kinetic study of CYP2J2-mediated astemizole O-demethylation in the presence of different concentrations of telmisartan (C) and flunarizine (D) as the inhibitor. Astemizole concentrations used were 0.05 (d), 0.1 (u), 0.15 (m), 0.3 ()), and 0.45 mM (,) (n = 4). The replots of the slope of Dixon plot versus reciprocal of substrate concentration for telmisartan (E) and flunarizine (F).

Article Snippet: CYP substrates, inhibitors, metabolite standards, and all other materials were obtained from the following sources: all compounds from Table 1, except olmesartan, that were used as inhibitors for the CYP2J2 and human liver microsome (HLM) inhibition studies, astemizole (AST), phenacetin, tolbutamide, bufuralol, omeprazole, 4’-hydroxytolbutamide, 1’- hydroxybufuralol, 6b-hydroxytestosterone, acetaminophen, dextrorphan, and nicotinamide adenine dinucleotide phosphate (NADPH) were purchased from Sigma-Aldrich (St. Louis, MO); testosterone was purchased from Acros Organics (Morris Plains, NJ); 5’-hydroxyomeprazole was purchased from Toronto Research Chemicals Inc. (North York, ON, Canada); olmesartan and O-desmethyl astemizole (DES-AST) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); potassium phosphate (monobasic and dibasic) and magnesium chloride hexahydrate (MgCl2) were purchased from Merck (Darmstadt, Germany); pooled HLMs and recombinant CYP enzyme were purchased from BD Gentest (Woburn, MA); and high-performance liquid chromatography (HPLC) grade dimethyl sulfoxide (DMSO), methanol, and formic acid used for liquid chromatography-tandem mass spectrometry (LC-MS/ MS) analysis were purchased from Fisher Scientific Co. (Pittsburgh, PA).

Techniques: Inhibition, Activity Assay, Recombinant, Amplification, Concentration Assay

Journal: Drug metabolism and disposition: the biological fate of chemicals

Article Title: Discovery and characterization of novel, potent, and selective cytochrome P450 2J2 inhibitors.

doi: 10.1124/dmd.112.048264

Figure Lengend Snippet: Fig. 7. Comparison of measured compound CYP2J2 inhibitory activities (IC50) with those reported in the literature (percentage activity remaining). Astemizole O- demethylation was used for evaluating the metabolic activity of CYP2J2; compounds with a measured IC50 value higher than 50 mM in our laboratory were treated as IC50 of 50 mM in the comparison; percentage activity remaining was obtained at single inhibitor concentration of 30 mM as reported in the literature, and only compounds with activity remaining less than 100% were included. Compounds included were amodiaquine, nicardipine, haloperidol, clozapine, lansoprazole, verapamil, fluoxetine, and omeprazole.

Article Snippet: CYP substrates, inhibitors, metabolite standards, and all other materials were obtained from the following sources: all compounds from Table 1, except olmesartan, that were used as inhibitors for the CYP2J2 and human liver microsome (HLM) inhibition studies, astemizole (AST), phenacetin, tolbutamide, bufuralol, omeprazole, 4’-hydroxytolbutamide, 1’- hydroxybufuralol, 6b-hydroxytestosterone, acetaminophen, dextrorphan, and nicotinamide adenine dinucleotide phosphate (NADPH) were purchased from Sigma-Aldrich (St. Louis, MO); testosterone was purchased from Acros Organics (Morris Plains, NJ); 5’-hydroxyomeprazole was purchased from Toronto Research Chemicals Inc. (North York, ON, Canada); olmesartan and O-desmethyl astemizole (DES-AST) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); potassium phosphate (monobasic and dibasic) and magnesium chloride hexahydrate (MgCl2) were purchased from Merck (Darmstadt, Germany); pooled HLMs and recombinant CYP enzyme were purchased from BD Gentest (Woburn, MA); and high-performance liquid chromatography (HPLC) grade dimethyl sulfoxide (DMSO), methanol, and formic acid used for liquid chromatography-tandem mass spectrometry (LC-MS/ MS) analysis were purchased from Fisher Scientific Co. (Pittsburgh, PA).

Techniques: Comparison, Activity Assay, Concentration Assay

Journal: Drug metabolism and disposition: the biological fate of chemicals

Article Title: Discovery and characterization of novel, potent, and selective cytochrome P450 2J2 inhibitors.

doi: 10.1124/dmd.112.048264

Figure Lengend Snippet: Fig. 8. Overlay of substrate (astemizole) and the inhibitor within the binding pocket of CYP2J2 for telmisartan (A) and flunarizine (B), respectively. The CYP2J2 protein is in cartoon representation and colored in rainbow spectrum; the heme is in stick and colored in orange; telmisartan, flunarizine, and astemizole are in stick and colored in yellow, magenta, and green, respectively.

Article Snippet: CYP substrates, inhibitors, metabolite standards, and all other materials were obtained from the following sources: all compounds from Table 1, except olmesartan, that were used as inhibitors for the CYP2J2 and human liver microsome (HLM) inhibition studies, astemizole (AST), phenacetin, tolbutamide, bufuralol, omeprazole, 4’-hydroxytolbutamide, 1’- hydroxybufuralol, 6b-hydroxytestosterone, acetaminophen, dextrorphan, and nicotinamide adenine dinucleotide phosphate (NADPH) were purchased from Sigma-Aldrich (St. Louis, MO); testosterone was purchased from Acros Organics (Morris Plains, NJ); 5’-hydroxyomeprazole was purchased from Toronto Research Chemicals Inc. (North York, ON, Canada); olmesartan and O-desmethyl astemizole (DES-AST) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); potassium phosphate (monobasic and dibasic) and magnesium chloride hexahydrate (MgCl2) were purchased from Merck (Darmstadt, Germany); pooled HLMs and recombinant CYP enzyme were purchased from BD Gentest (Woburn, MA); and high-performance liquid chromatography (HPLC) grade dimethyl sulfoxide (DMSO), methanol, and formic acid used for liquid chromatography-tandem mass spectrometry (LC-MS/ MS) analysis were purchased from Fisher Scientific Co. (Pittsburgh, PA).

Techniques: Binding Assay

Journal: Cell reports

Article Title: Pyruvate metabolism controls chromatin remodeling during CD4 + T cell activation.

doi: 10.1016/j.celrep.2023.112583

Figure Lengend Snippet: Figure 2. ACSS2 inhibition or ACLY deficiency does not affect epigenome remodeling during T cell activation (A) Immunoblots of ACLY, ACSS2, PDCE1, and actin from human T cells activated for 8, 16, 24, or 48 h with anti-CD3/CD28 stimulation. (B and C) CD25 protein expression was measured by flow cytometry (FACS) and IL2ra gene expression was determined by quantitative reverse transcription- polymerase chain reaction (qRT-PCR) in human CD4+ T cells activated with anti-CD3/CD28 in the presence and absence of ACSS2 inhibitor (15.6 mM).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER FCCP, mitochondrial oxidative phosphorylation uncoupler Abcam # ab120081 Rotenone Merck Millipore # R8875 UK-5099 (Synonyms: PF-1005023) Med Chem Express # HY-15475 3PO R98% (HPLC) Sigma-Aldrich # SML1343 Sodium acetate Sigma-Aldrich # S1429 DCA Tocris Bioscience # 2755 Etomoxir sodium salt hydrate Sigma-Aldrich # E1905 ACSS2 inhibitor Selleck # S8588 6,8-Bis(benzylthio)-octanoic acid Sigma-Aldrich # SML0404 Sodium Pyruvate Thermo Fisher # 11360070 SB204990 Med Chem Express # HY-16450 BMS303141 Sigma-Aldrich # SML0784 HEPES Sigma-Aldrich #H4034-100G EDTA Sigma-Aldrich #EDS-100G DL-Dithiothreitol solution (DTT) Sigma-Aldrich #43816-10ML Bovine serum albumin (BSA) Sigma-Aldrich #A9647-500G D-(+)-Glucose solution Sigma-Aldrich #G8769-100ML 13C6-glucose Cambridge Isotope Lab #CLM-1396 DMSO Sigma-Aldrich # D2650 Ficoll Paque Plus Sigma-Aldrich # GE17-1440-02 13C6-glucose Cambridge Isotope Lab #CLM-1396 Cell Trace Violet Invitrogen #C34557 Molecular ProbesTM 2-NBDG Invitrogen #N13195 SYBR Thermo fisher #S33102 iScript cDNA kit biorad #170-8891 Formaldehyde Sigma-Aldrich #252549-1L DAPI (1:2000) Invitrogen #D1306 Zombie NIRTM Fixable Viability Kit Biolegend #423106 Zombie GreenTM Fixable Viability Kit Biolegend #423112 7-AAD Invitrogen #A1310 D-(+)-Glucose solution Sigma-Aldrich #G8769-100ML GolgiPlugTM (Protein Transport Inhibitor) BD Biosciences #555029 Tween 20 Sigma-Aldrich #P7949-500ML HALT protease inhibitor Thermo scientific #78439 Powdered milk Carl Roth #T145.2 PierceTM Protein A/G Magnetic Beads thermo fisher #88802 Proteinase K, recombinant, PCR Grade Sigma #3115828001 RNeasy Mini Kit Qiagen #74106 Seahorse XF DMEM medium Agilent Technologies #103575-100 Critical commercial assays MagniSortTM Human CD4+ T cell Enrichment Kit eBioscience #8804-6811-74 CD4 (L3T4) MicroBeads, mouse Miltenyi # 130-117-043 Duolink In Situ Detection Reagents Red Sigma DUO92008 Duolink Green PLA Sigma # DUO92014 Foxp3/Transcription Factor Staining Buffer Set eBioscience #00-5523-00 BD Cytofix/Cytoperm Fixation/Permeabilization kit BD Biosciences #554714 seahorse XFe24 Flux Packs Agilent #1023-40-100 Acetyl-CoA Assay Kit Biovision, Milpitas CA #K317-100 (Continued on next page) Cell Reports 42, 112583, June 27, 2023 19

Techniques: Inhibition, Activation Assay, Western Blot, Expressing, Cytometry, Gene Expression, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR