| Assay Method Information | |
| | Evaluation of Compound Inhibition of Cytochrome P450 |
| Description: | Enzymatic experiments were performed to quantify the inhibition of CYP450 enzyme activity of each isoform of CYP450 by small molecule inhibitors through fluorescence generated by the oxidation of the substrate by cytochrome P450. The experiments were performed in 384-well plates (Corning, Cat #3575) using a reaction buffer of 142.86 mM Potassium Phosphate, pH 7.4. The Solution A components used in the experiments were: 26.13 mM NADP+(Sigma-aldrich, Cat #N0505) 65.77 mM G6P (J&K, Cat #968161) and 65.42 mM MgCl2 (Sigma-aldrich, Cat #M2670). The Solution B composition used for the experiment was: 40 U/mL G6PDH (Sigma-aldrich, Cat #G6378). The substrate mix was 0.05× Solution A, 0.01× Solution B, 50 mM Potassium Phosphate, 0.01 mM BOMCC/0.01 mM EOMCC/0.001 mM DBOMF. For CYP3A4 and CYP2C9, the reaction system was 50 μL or 20 μL, respectively, including 3 nM CYP3A4 or 120 nM CYP2C9, BOMCC substrate mixed solution and different concentrations of compounds to be tested. For CYP2C19, CYP2D6 and CYP1A2, the reaction system was 20 μL and included 12.5 nM CYP2C19, 80 nM CYP2D6 or 1 nM CYP1A2, EOMCC substrate mix and various concentrations of the compounds to be tested. For CYP2C8, the reaction system is 50 μL and includes 1.5 nM CYP2C8, DBOMF substrate mix and various concentrations of compound to be tested. After preincubation with the enzyme for 10 minutes, the substrate was added and the fluorescence signal was read at different wavelengths (BOMCC/EOMCC Ex430 nm/Em480 nm, DBOMF Ex490 nm/Em520 nm) using BMG PHERAStar depending on the substrate, with reaction intervals of 30 seconds or more (depending on the actual number of wells) and reaction times of 30 minutes. |
| Affinity data for this assay | |
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