Cytochrome P450 (CYP) enzymes represent a heme containing protein superfamily metabolizing a broad variety of xenobiotics, including drugs and toxic chemicals. 11 CYP families are expressed in a human liver (CYP1A2, CYP2A6, CYP2B6, CYP2C8/9/18/19, CYP2D6, CYP2E1, and CYP3A4/5), and 5 of them (CYPs 1A2, 2C9, 2C19, 2D6 and 3A4) demonstratе broad substrate selectivity which accounts for about 95% of the known drug metabolism. Cytochrome P450s are of critical importance to the two of the most significant problems in clinical pharmacology: drug interactions and individual variability in drug metabolism. Most drugs undergo deactivation by CYPs, either directly or by facilitated excretion from the body. Some substances are bioactivated by CYPs to form pharmacologically active compounds. Also many drugs may increase or decrease the activity of various CYPs due to the ability of binding to them. Hence it is necessary to check possible drug candidates’ ability to interact with CYPs. During the drug discovery process, routine assessment to identify the following major CYP enzymes for potential metabolism-mediated interactions is recommended: CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A.
The potential for CYP inhibition is assessed by performing in vitro inhibition studies using luminogenic CYP substrates with a CYP enzyme and NADPH regeneration system (P450-Glo™ Screening Systems, Promega). The amount of light produced from a glow-type luciferase reaction is directly proportional to the cytochrome P450 activity. In the cases when the tested compounds interfere with the CYP enzyme-substrate reaction, the luminescent signal decreases, which is detected by Omega POLARStar microplate reader (BMG Labtech). For the rough estimate, point assays are typically performed for each compound at 20 µM concentration or another concentration stipulated by the customer. If a noticeable inhibition is detected, the IC50 values for the tested compounds can be determined upon request. For this purpose, 7-point, 2-fold serial dilution dose-response inhibition curves of the compounds are built, starting at 100µM compound concentrations. All test points are performed in quadruplicates. Based on this data, IC50 values for each compound for each of the CYP450 enzymes are calculated using Prism software (GraphPad). Reference inhibitors specific for each CYP enzyme are used to assess CYP inhibition in the control experiments for every batch of tested compounds. Test concentrations of the reference compounds correspond to approximately 4 x fold reported IC50 values for the corresponding cytochromes P450, which is expected to produce 80-100% inhibition in the properly performing assay. Control membranes (without CYPs) of the same total protein concentration represent the negative control (baseline).
Either point assay data for each compound at 20 µM concentration or IC50 values for the tested compounds based on 7-point, 2-fold serial dilution dose-response inhibition curves (upon request). Full study report is provided.
A minimal weighted amount of dry compound, 1 mg, or 200 µL of 10 mM stock DMSO solution, is required for this assay. For multiple assays, lesser amount of compound per assay may be sufficient, which should be discussed for each particular project.
1. Promega Technical Bulletin P450-Glo™ Screening Systems Instructions for Use of Products V9770, V9790, V9800, V9880, V9890, V9910 AND V9920.
2. P450-Glo™ Screening Systems
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