| Assay Method Information | |
| | Recombinant CYP Inhibition Assay |
| Description: | Recombinant CYP Inhibition Assay: In a drug discovery program, a rapid screening for cyctochrome P450 (CYP450) inhibitors is a part of the existing standard for avoiding the development of drugs likely to give clinical pharmacokinetic drug-drug interactions and associated toxicities. A microtiter plate-based, direct fluorometric assay for the activities of the principal human drug-metabolizing enzymes, CYP2D6 and CYP3A4 can be used and these assays are rapid and compatible with existing high-throughput assay instrumentation. Fluorometric Enzyme Inhibition Assays: Test compounds were dissolved in 100% organic solvent (CH3CN or DMSO) to make 30 mM stock solutions. Quinidine (CYP2D6 assay, Sigma Aldrich) and ketoconazole (CYP3A4 assay, Sigma Aldrich) ran as positive controls and were dissolved in 100% acetonitrile to make 1 mM stock solutions. A 100 mM potassium phosphate buffer was prepared and adjusted to pH 7.4. The 30 mM stock solution of test and control compounds (1 mM) were further diluted in phosphate buffer (100 mM, pH 7.4) to ensure the final organic solvent content was <0.2% in the reaction. In a separate falcon tube, a 2× enzyme/substrate (E/S) solution was prepared in phosphate buffer. The final concentration of CYP2D6 (Corning) and AMMC was 10 nM and 4 μM, and CYP3A4 (Corning) and BFC was 20 nM and 40 μM, respectively. In a separate falcon tube, a 2× NADPH regenerating system (NRS) was prepared in phosphate buffer. The final concentration for each component in the assay was as follows:CYP2D6 assay=0.008 mM NADPH, 3.3 mM glucose 6-phosphate, 0.4 U of glucose-6-phosphate dehydrogenase/mL CYP3A4 assay=2.45 mM NADPH, 24.7 mM glucose 6-phosphate, 1.25 U of glucose-6-phosphate dehydrogenase/mL. |
| Affinity data for this assay | |
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