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Nadph

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NADPH is a coenzyme that plays a crucial role in various cellular processes. It serves as an electron donor in numerous enzymatic reactions, particularly in the reduction of oxidized molecules. NADPH is an essential component in maintaining the redox balance within cells and supporting antioxidant defenses.

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Lab products found in correlation

Hydrochloric acid (hcl), by Thermo Fisher Scientific (1 mentions) Saccharolactone, by Merck Group (1 mentions) Reduced glutathione, by Thermo Fisher Scientific (1 mentions) Udpga, by Merck Group (1 mentions) Acs grade methanol, by Thermo Fisher Scientific (1 mentions) Alamethicin, by Thermo Fisher Scientific (1 mentions) Ticlopidine, by Tokyo Chemical Industry (1 mentions) Hydroxybupropion, by Cerilliant (1 mentions) Dipotassium phosphate, by Thermo Fisher Scientific (1 mentions) Monopotassium phosphate, by Thermo Fisher Scientific (1 mentions) Ammonium acetate, by Thermo Fisher Scientific (1 mentions) Optima hplc grade acetonitrile, by Thermo Fisher Scientific (1 mentions) 2 4 dichlorophenoxyacetic acid, by Merck Group (1 mentions) Acs grade acetone, by Thermo Fisher Scientific (1 mentions) 1 naphthol, by Thermo Fisher Scientific (1 mentions) Hydrogen peroxide h2o2, by Thermo Fisher Scientific (1 mentions) Mgcl2, by Merck Group (1 mentions) Potassium chloride (kcl), by Merck Group (1 mentions) Nadph, by Roche (1 mentions) Protease inhibitor cocktail mix, by Merck Group (1 mentions)

11 protocols using nadph

1

Integrated ADME Characterization Protocol

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Optima HPLC grade methanol, Optima HPLC grade water, Optima HPLC grade acetonitrile, American Chemical Society (ACS) grade acetone, ACS grade methanol, ACS grade pentane, hydrochloric acid, ammonium acetate, dipotassium phosphate, monopotassium phosphate, magnesium chloride (MgCl2) and reduced glutathione (GSH) were purchased from Fisher Chemical (Fair Lawn, NJ, USA); NADPH, 1-aminobenzotriazole, methimazole, 1-naphthol and hydroxyacetone from Acros Organics (Morris Plain, NJ, USA); UDPGA, saccharolactone and 2,4-dichlorophenoxyacetic acid from Sigma-Aldrich (St. Louis, MO, USA); bupropion, benzydamine and alamethicin from Alfa Aesar (Ward Hill, MA, USA); oxcarbazepine from European Pharmacopoeia Reference Standard (Strasbourg, France); ticlopidine from Tokyo Chemical Industry (Tokyo, Japan); hydroxybupropion from Cerilliant Corporation (Round Rock, Texas, USA); deuterated acetone (acetone-d6) from Cambridge Isotope Labs (Cambridge, MA, USA); hydrogen peroxide (50%) from Univar (Redmond, WA, USA); HLM, rat liver microsomes (RLM), DLM and human lung microsomes (HLungM) from Sekisui XenoTech (Kansas City, KS, USA); human recombinant CYP (rCYP) bactosomes expressed in Escherichia coli (E. coli) from Cypex (Dundee, Scotland); human recombinant flavin monooxygenase (rFMO) supersomes and human recombinant UGT (rUGT) supersomes expressed in insect cells from Corning (Woburn, MA, USA).
Gonsalves M.D., Colizza K., Smith J.L, & Oxley J.C. (2020). In vitro and in vivo studies of triacetone triperoxide (TATP) metabolism in humans. Forensic Toxicology, 39(1), 59-72.
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2

Microsomal Incubation of Compounds

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Liver microsomal incubations (1 mg microsomal protein/mL) with human (Corning Life Sciences, Oneonta, NY), rat, and mouse microsomes (Sekisui XenoTech, Kansas City, KS) were assessed for compounds 5m, 5t, 2a, azixa/verubulin, and verapamil (1 μg/mL) in the presence of NADPH (Acros Organics, Fair Lawn, NJ) (1 mM). At predefined times (0, 5, 10, 15, 30, 45, and 60 min), aliquots (50 μL) were removed and the reaction mixture was quenched by addition of 200 μL of ice-cold methanol containing an internal standard. Samples were briefly vortexed and centrifuged at 3200g for 5 min at 4 °C. Supernatants were collected and analyzed by LC–MS/MS. In vitro half-life and intrinsic clearance were assessed per standard procedures.84 (link)
Banerjee S., Mahmud F., Deng S., Ma L., Yun M.K., Fakayode S.O., Arnst K.E., Yang L., Chen H., Wu Z., Lukka P.B., Parmar K., Meibohm B., White S.W., Wang Y., Li W, & Miller D.D. (2021). X-ray Crystallography-Guided Design, Antitumor Efficacy, and QSAR Analysis of Metabolically Stable Cyclopenta-Pyrimidinyl Dihydroquinoxalinone as a Potent Tubulin Polymerization Inhibitor. Journal of medicinal chemistry, 64(17), 13072-13095.
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3

Peroxide Quantification and Handling

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All chemicals were of
reagent grade and were
used without additional purification. Tris was from VWR (West Chester,
PA). Tris, (2-carboxyethyl)phosphine hydrochloride (TCEP), tert-butyl hydroperoxide (tBOOH), cumene
hydroperoxide (CuOOH), saccharose, Trp, NaF, KCl, and MgCl2 were from Merck (Darmstadt, Germany). NADPH was obtained from Roche
(Basel, Switzerland), and dithiothreitol (DTT) and ammonium sulfate
were from Euromedex (Souffelweyersheim, France). Hydrogen peroxide
(H2O2) was from Acros Organics (Geel, Belgium).
Peroxide stock concentrations were measured accurately by the peroxidase
enzymatic coupled assay using Tsa1/Trx/Trx reductase/NADPH, following
the total NADPH consumption at 340 nm (ε340 = 6200
M–1 cm–1).
Kriznik A., Libiad M., Le Cordier H., Boukhenouna S., Toledano M.B, & Rahuel-Clermont S. (2020). Dynamics of a Key Conformational Transition in the Mechanism of Peroxiredoxin Sulfinylation. ACS Catalysis, 10(5), 3326-3339.
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4

Potassium Phosphate Metabolic Assay

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Potassium dihydrogen phosphate, di-potassium hydrogen phosphate, EDTA, sodium hydroxide, aminooxyacetic acid (AOAA), l-tryptophan (l-Try), indole-3-pyruvic acid (IPA), phenylpyruvic acid (PPA), α-Keto-γ-methylthiobutyric acid sodium salt (KMB), se-methylselenocysteine hydrochloride (MSC), dimethyl-2-oxoglutarate (α-KG), l-phenyl alanine (l-Phe), 2-amino-2-methyl-1,3-propanediol, pyridoxal 5′-phosphate hydrate (PLP), 2,4-dinitrophenylhydrazine (DNPH), Phenylmethanesulfonyl fluoride (PMSF), RIPA buffer, protease inhibitor cocktail mix, and N-N-dimethyl formamide, pLKO.1 vector were purchased from Sigma-Aldrich (Darmstadt, Germany). BFF-122 was purchased from Axon Medchem (Groningen, Netherlands), NADPH was purchased from Acros Organics (New Jersey, NJ, USA). Plasmid pEGFP-N1 (Clontech, Takara Bio USA, Inc, Mountain View, CA, USA) was a generous gift from Dr. Gildert Lauter from the Department for Biosciences and Nutrition, Karolinska Institutet, Stockholm, Sweden. HEPG2 cells and EMEM media were purchased from ATCC (Wesel, Germany).
Selvam A.K, & Björnstedt M. (2020). A Novel Assay Method to Determine the β-Elimination of Se-Methylselenocysteine to Monomethylselenol by Kynurenine Aminotransferase 1. Antioxidants, 9(2), 139.
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5

Purification of Recombinant Proteins

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E. coli Turbo Bl21 (DE3) chemically competent cells were purchased from Invitrogen. Purification was performed on an AKTA Start FPLC (GE Healthcare, Chicago, IL, USA). l-Orn, l-Lys, buffers, salts, kanamycin, NADPH, NADH, 96 well-plates, and Pierce hydrogen peroxide detection kits were purchased from Thermo-Fisher Scientific. Oxygen consumption assays were done on a Hansatech Oxygraph (King’ Lynn, Norfolk, UK).
Bufkin K, & Sobrado P. (2017). Characterization of the Ornithine Hydroxylation Step in Albachelin Biosynthesis. Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry, 22(10), 1652.
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6

Bacterial Cell Transformation and Cloning

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Escherichia coli NEB10β cells (Cat. # C3019I), DpnI (Cat. # R0176L), and BsaI-HF V2 (Cat. # R3733S) were from New England Biolabs. T4 DNA ligase (Cat. # EL0013) was ordered from Thermo Fisher Scientific. NADPH (Cat. # 44335000) was ordered from Oriental Yeast Co. Tamoxifen N-oxide (Cat. # FT27997) and benzydamine N-oxide (Cat. # FB18263) were purchased from Biosynth and all other chemicals were from Sigma-Aldrich.
Bailleul G., Yang G., Nicoll C.R., Mattevi A., Fraaije M.W, & Mascotti M.L. (2023). Evolution of enzyme functionality in the flavin-containing monooxygenases. Nature Communications, 14, 1042.
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7

Metabolite Quantification Protocol

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Acetonitrile (HPLC grade), formic acid (LC/MS grade), and water
(LC/MS grade) were purchased from Fisher Scientific (Fisher Scientific,
Pittsburgh, PA, USA); the standard compounds of NAD+, NADH, NADP,
NADPH, AMP, ADP, ATP, ribulose 5-phosphate (R 5-P), xylulose 5-phosphate
(X 5-P), fructose 6-phosphate (F 6-P), glyceraldehyde 3-phosphate
(G 3-P), phosphoenolpyruvate (PEP), lactate, pyruvate, citrate, (iso)citrate,
alpha-ketoglutarate (alpha-KG), succinate, malate, oxaloacetate (OAA),
aspartate, glutaminate, glutamine, reduced glutathione (GSH), oxidized
glutathione (GSSG), (iso)leucine, leucine, alanine, arginine, cysteine,
methionine, proline, serine, threonine, tyrosine, phenylalanine, valine,
histidine, N-acetylcysteine, and hydroxybutyric acid
were purchased from Sigma (Sigma-Aldrich Corp., Saint Louis, MO, USA).
All other reagents were ACS grade.
Lv H., Hung C.S, & Henderson J.P. (2014). Metabolomic Analysis of Siderophore Cheater Mutants Reveals Metabolic Costs of Expression in Uropathogenic Escherichia coli. Journal of Proteome Research, 13(3), 1397-1404.
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8

Screening Novel Inhibitors of AbFabI

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All compounds, including the hit compound 1, were custom synthesized by Life Chemicals through their CRO services with their identity and purity confirmed through mass spectrometry and NMR methods. All compounds tested herein have purity >95%. Compounds 1-29 are racemic mixtures. Crotonyl-CoA was purchased from Sigma Aldrich and Life Sciences Inc., while NADH and NADPH were from Fisher Scientific. Polymixin B nonapeptide was purchased from Sigma Aldrich. AbFabI was a generous gift from the Seattle Structural Genomics Center for Infectious Disease (SSGCID).
Kwon J., Mistry T., Ren J., Johnson M.E, & Mehboob S. (2017). A Novel Series of Enoyl Reductase Inhibitors Targeting the ESKAPE Pathogens, Staphylococcus aureus and Acinetobacter baumannii. Bioorganic & medicinal chemistry, 26(1), 65-76.
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9

Oxidative Stress Pathway Activation Assay

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AngII was purchased from Sigma-Aldrich (Amersham, UK); NADPH was purchased from Fisher Scientific (Loughborough, UK); dihydroethidium (DHE) was purchased from Invitrogen (Loughborough, UK); FITC-labelled wheat germ agglutinin (WGA, Catalogue No. L-4895) was from Sigma-Aldrich. Primary antibodies to p47phox, p22phox, Nox1, Nox2, Nox4, p38-MAPK, ERK1/2, phos-JNK (Thr183/Tyr185) and total JNK, phos-Akt (Ser473) and total Akt were purchased from Santa Cruz Biotechnology (Dallas, TX, USA); antibodies to β-actin, phos-MKK3(Ser189)/6(Ser207) and phos-ASK1 (Thr845), total MKK3/6, γH2AX (Ser139/Tyr142) were purchased from Cell Signalling Technology (London, UK); Antibodies to phos-p47phox (Ser359), phos-p38-MAPK (Thr180/Tyr182) and phos-ERK1/2 (Thr202/Tyr204) were purchased from Sigma-Aldrich. Nox2-ds-tat (Nox2tat, [H]-RKKRRQRRRCSTRVRRQL-[NH2]) were provided by PeptideSynthetics (PPR Ltd., Fareham, UK). Other reagents, chemicals and antibodies, unless specified, were purchased from Sigma-Aldrich.
Liu F., Fan L.M., Geng L, & Li J.M. (2021). p47phox-Dependent Oxidant Signalling through ASK1, MKK3/6 and MAPKs in Angiotensin II-Induced Cardiac Hypertrophy and Apoptosis. Antioxidants, 10(9), 1363.
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10

Enzyme Kinetics and Inhibition Assay

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Tris-hydrochloric acid, Tris base, bis–Tris, NaOH, NaCl, MgCl2 hexahydrate, dithiothreitol, NADP+ disodium salt, NADPH, KPhos dibasic, KCl, BL-21 Gold (DE3) competent cells, Luria-broth (LB)-Agar, kanamycin sulfate, Terrific broth, IPTG, EDTA-free protease inhibitor tablets, Ni-NTA resin, Dulbecco’s modified Eagle medium (DMEM), 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester, Dulbecco’s phosphate buffered saline (DPBS) and Triton X-100 were all obtained from Fisher Scientific (Hampton, NH). Isocitrate, αKG, and imidazole were obtained from Acros Organics (Fisher Scientific, Hampton, NH). Fetal bovine serum (FBS) was obtained from VWR (Radnor, PA). 5-(N-ethyl-N-isopropyl)amiloride was obtained from Sigma-Aldrich (St. Louis, MO). ESOM sodium salt was obtained from Apexbio (Houston, TX). Nigericin sodium was obtained from Tocris (Bristol, U.K.).
Luna L.A., Lesecq Z., White K.A., Hoang A., Scott D.A., Zagnitko O., Bobkov A.A., Barber D.L., Schiffer J.M., Isom D.G, & Sohl C.D. (2020). An acidic residue buried in the dimer interface of isocitrate dehydrogenase 1 (IDH1) helps regulate catalysis and pH sensitivity. The Biochemical journal, 477(16), 2999-3018.
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