2-methyl-1,4-naphthoquinone, 5 Menadione (Vitamin K3) 2-methyl-1,4-dihydronaphthalene-1,4-dione CHEMBL590 Menadione BDBM24778 cid_4055 Menadione (5d) Vitamin K3 Menadione, 9
Menadione, 11 4-cyclohexyl-2-phenylquinazoline MLS001049987 cid_23640938 4-Cyclohexyl-2-phenyl-quinazoline SMR000469604 BDBM53421
cid_23640941 Menadione, 10 2,4-Dicyclohexyl-6-methoxy-quinazoline SMR000469610 2,4-dicyclohexyl-6-methoxyquinazoline MLS001050047 BDBM53422
- ChEMBL_607766 (CHEMBL1068737) Inhibition of CBR-mediated NADPH-dependent reduction of menadione to menadiol
- ChEMBL_1634419 (CHEMBL3877211) Inhibition of recombinant human NQO2 using menadione/CMCDP as substrate/cofactor
- ChEMBL_607770 (CHEMBL1068741) Displacement of menadione from human recombinant CBR expressed in Escherichia coli BL21 (DE3)
- ChEMBL_2240213 (CHEMBL5154109) Inhibition of Mycobacterium smegmatis MC2 155 recombinant ndhA assessed as rate of NADH oxidation in the presence of an electron acceptor menadione
- ChEMBL_886534 (CHEMBL2215243) Binding affinity to human recombinant quinine reductase 2 expressed in Escherichia coli BL21 (DE3) using menadione and NMeH as substrates
- ChEMBL_982299 (CHEMBL2429517) Competitive inhibition of human quinone reductase 2 using menadione/N-methyldihydronicotinamide as substrate after 10 mins by double-reciprocal plot analysis
- ChEMBL_2240211 (CHEMBL5154107) Inhibition of Mycobacterium tuberculosis recombinant type II NADH dehydrogenase ndh expressed in Escherichia coli assessed as rate of NADH oxidation in the presence of an electron acceptor menadione
- ChEMBL_2240212 (CHEMBL5154108) Inhibition of Mycobacterium smegmatis MC2 155 recombinant type II NADH dehydrogenase ndh expressed in Escherichia coli assessed as rate of NADH oxidation in the presence of an electron acceptor menadione
- ChEMBL_1636786 (CHEMBL3879684) Inhibition of full length human His6/GST-tagged NQO2 expressed in Escherichia coli Tuner(DE3)pLysS using menadione as substrate and CCHP as co-substrate preincubated with enzyme followed by substrate addition by MTT dye based continuous spectrophotometric assay
- Inhibition of Steady-State Catalysis Assay To determine the IC50 value of each inhibitor, reactions were initiated by addition of 154 pM NQO2 to a reaction buffer containing 150 μM SCDP and 5 μM menadione with TBB (0.625−640 μM), TBBz (2.5−640 nM), or DMAT (2.5−640 nM).
- QR2 Inhibition Assay The activity of recombinant human QR2 under steady-state conditions was evaluated on a MolecularDevices SpectraMax Plus 384 UV-visible spectrophotometer by monitoring the decrease in absorbance of the enzyme co-substrate NMeH at 360 nm. The inhibition assays were initiated by adding the enzyme to a reaction mixture containing NMeH, menadione and different concentrations of inhibitor. The inhibitory concentration that produces 50% inhibition (IC50 value) for each inhibitor was calculated by curve-fitting. All initial velocity data were measured in triplicate, and the mean +/- SD.
- Inhibition of NQO1 (With 2 uM BSA) Recombinant human NQO1 was obtained from Sigma and diluted in 50 mM phosphate buffer to give an absorbance of 0.1 at 550 nm; 5 uL of this solution was then mixed with 495 uL of 50 mM phosphate buffer at pH 7.5 containing 200 uM NADH, 70 uM cytochrome c, 20 uM menadione, with 2 uM BSA, and various concentrations of the potential inhibitor dissolved in DMSO (final concentration 1.0% v/v). On some occasions, potential inhibitors were dissolved in 0.13 M NaOH. Reactions were carried out at 25 deg C and cytochrome c reduction was monitored at 550nm in a BeckmanDU650 spectrophotometer. IC50 values were determined using nonlinear curve fitting as implemented in the program Excel for which a 50% reduction of the initial rate was attained.
- AO Enzyme Assay Guinea pig AO activity was assayed spectrophotometrically using phenanthridine as a substrate at 322 nm. All spectrophotometric determinations were carried out at 25 °C using a PClinked perkin Elemer Lambda 25 UV/Vis spectrophotometer. The varying substrate concentrations (4-60 µM) were separately added into Sorenson's phosphate buffer (final concentration of 50 mM, pH 7.0) containing 0.1 mM of EDTA. Then, the reaction was started by addition of the enzyme and monitored for up to 2 min. The enzyme was also assayed in the presence of different concentrations of the compounds as potential inhibitors (1-200 µM) and constant concentration of substrate (40 µM) to calculation of IC50 values. The results were compared with the inhibitory effects of 1-100 µM menadione, a specific AO inhibitor. Before enzymatic reaction, all of the solutions were equilibrated at 25 °C, except the enzyme fraction which was kept on ice bath prior to addition to the incubation solution. All compounds were dis
- Enzymatic IDO Assay The IC50 values for each compound were determined by testing the activity of IDO in a mixture containing 50 mM potassium phosphate buffer at pH 6.5; 70 nM purified human IDO protein, 200 μM L-tryptophan, 20 mM ascorbate, 20 μM methylene blue, 0.1% DMSO. The inhibitors were initially diluted in DMSO at 100 mM and were diluted in potassium phosphate 50 mM, added to the reaction mixture at final concentrations raging from 1 mM to 5 nM and preincubated with the enzyme for 5 min at 25° C. The reaction was started by addition of L-tryptophan to 200 μM and incubated 15 min at 37° C. The reaction was stopped by addition of 0.5 vol of 30% trichloroacetic acid and incubated 30 min at 60° C. to hydrolyze N-formylkynurenine to kynurenine. The reaction was centrifuged at 3400 g for 5 min to remove precipitated protein and the supernatant was reacted with 2% (w/v) of p-dimethylaminobenzaldehyde in acetic acid. The reaction was incubated 10 min at 25° C. and read at 480 nm in a spectrophotometer. Control samples with no IDO inhibitor, or with no IDO enzyme or with the reference inhibitors 1-methyl-tryptophan (200 μM) and menadione (1.2 μM) were used as controls to set the parameters for the non-linear regressions necessary for determination of the IC50 for each compound.
- Enzymatic IDO Assay The IC50 values for each compound were determined by testing the activity of IDO in a mixture containing 50 mM potassium phosphate buffer at pH 6.5; 70 nM purified human IDO protein, 20 μM L-tryptophan, 20 mM ascorbate, 2 μM methylene blue, 0.1% DMSO. The inhibitors were initially diluted in DMSO at 100 mM and were diluted in potassium phosphate 50 mM, added to the reaction mixture at final concentrations raging from 1 mM to 5 nM and preincubated with the enzyme for 5 min at 25° C. The reaction was started by addition of L-tryptophan to 20 μM and incubated 15 min at 37° C. The reaction was stopped by addition of 0.5 vol of 30% trichloroacetic acid and incubated 30 min at 60° C. to hydrolyze N-formylkynurenine to kynurenine. The reaction was centrifuged at 3400 g for 5 min to remove precipitated protein and the supernatant was reacted with 2% (w/v) of p-dimethylaminobenzaldehyde in acetic acid. The reaction was incubated 10 min at 25° C. and read at 480 nm in a spectrophotometer. Control samples with no IDO inhibitor, or with no IDO enzyme or with the reference inhibitors 1-methyl-tryptophan (200 μM) and menadione (1.2 μM) were used as controls to set the parameters for the non-linear regressions necessary for determination of the IC50 for each compound. Nonlinear regressions and determination of the IC50 values were performed using the GraphPad Prism 4 software. Compounds with an IC50 of less than 50 μM were considered as active inhibitors in this assay.
- Enzymatic IDO Assay The IC50 values for each compound were determined by testing the activity of IDO in a mixture containing 50 mM potassium phosphate buffer at pH 6.5; 70 nM purified human IDO protein, 200 μM L-tryptophan, 20 mM ascorbate, 20 μM methylene blue, 0.1% DMSO. The inhibitors were initially diluted in DMSO at 100 mM and were diluted in potassium phosphate 50 mM, added to the reaction mixture at final concentrations raging from 1 mM to 5 nM and preincubated with the enzyme for 5 min at 25° C. The reaction was started by addition of L-tryptophan to 200 μM and incubated 15 min at 37° C. The reaction was stopped by addition of 0.5 vol of 30% trichloroacetic acid and incubated 30 min at 60° C. to hydrolyze N-formylkynurenine to kynurenine. The reaction was centrifuged at 3400 g for 5 min to remove precipitated protein and the supernatant was reacted with 2% (w/v) of p-dimethylaminobenzaldehyde in acetic acid. The reaction was incubated 10 min at 25° C. and read at 480 nm in a spectrophotometer. Control samples with no IDO inhibitor, or with no IDO enzyme or with the reference inhibitors 1-methyl-tryptophan (200 μM) and menadione (1.2 μM) were used as controls to set the parameters for the non-linear regressions necessary for determination of the IC50 for each compound. Nonlinear regressions and determination of the IC50 values were performed using the GraphPad Prism 4 software. Compounds with an IC50 of less than 500 μM were considered as active inhibitors in this assay.