By Douglas S. Auld, Henrike Veith, James J. Cali (auth.), Ian R. Phillips, Elizabeth A. Shephard, Paul R. Ortiz de Montellano (eds.)
Cytochromes P450 (CYPs) contain a wide superfamily of proteins which are of critical significance within the detoxing or activation of a big variety of typical and artificial hydrophobic xenobiotics, together with many healing medicinal drugs, chemical cancer agents and environmental toxins. CYPs are very important in mediating interactions among an organism and its chemical surroundings and within the law of physiological techniques. Cytochrome P450 Protocols, 3rd variation focuses on high-throughput equipment for the simultaneous research of a number of CYPs, substrates or ligands. even if the emphasis is on CYPs of mammalian foundation, it displays an expanding curiosity in CYPs of bacterial species. additionally integrated are chapters on cytochrome P450 reductase (the redox associate of CYPs) and the flavin-containing monooxygenases (FMOs), and metabolomic and lipidomic methods for id of endogenous substrates of CYPs (‘de-orphanizing’ CYP substrates). Written within the profitable Methods in Molecular Biology™ sequence structure, chapters comprise introductions to their respective themes, lists of the mandatory fabrics and reagents, step by step, conveniently reproducible protocols, and notes on troubleshooting and heading off identified pitfalls.
Authoritative and simply obtainable, Cytochrome P450 Protocols, 3rd version provides a variety of options obtainable to researchers in fields as varied as biochemistry, molecular biology, pharmacology, toxicology, environmental biology and genetics.
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Extra info for Cytochrome P450 Protocols
J Chromatogr B Analyt Technol Biomed Life Sci 850:455–63 Walsky RL, Obach RS (2004) Validated assays for human cytochrome P450 activities. Drug Metab Dispos 32:647–60 Youdim KA, Lyons R, Payne L, Jones BC, Saunders K (2008) An automated, highthroughput, 384 well Cytochrome P450 cocktail IC50 assay using a rapid resolution LC-MS/ MS end-point. J Pharm Biomed Anal 48:92–9 Zientek M, Miller H, Smith D, Dunklee M, Heinle L, Thurston A, Lee C, Hyland R, Fahmi O, Burdette D (2008) Development of an in vitro drug-drug interaction assay to simultaneously monitor five cytochrome P450 isoforms and performance assessment using drug library compounds.
6. Repeat the serial dilution process (taking 20 mL from row B and mix into C, then C into D and so on) until row H. 7. On a separate 96-well dilution plate (“DP-2”) fill in all wells with 180 mL of water. 8. Perform a plate-stamping operation, transferring 20 mL from each well in DP-1 into DP-2, and mix thoroughly (see Note 5). 9. Save the DP-2 for use in the CYP inhibition assay described below. 5). 10. Repeat the dilution procedure for the next set of 11 test compounds and one reference inhibitor mixture so that two plates (24 compounds × 8 concentrations) can be assayed together.
Concurrent experiments utilizing reference inhibitors are critical to serve as references for the entire workflow. These reference inhibitors can be used for assay troubleshooting and ensuring consistency in the assay quality. High-Throughput Mass Spectrometric Cytochrome P450 Inhibition Screening 27 Fig. 1 An overview of the CYP inhibition assay. (a) Experimental design. (b) Implementation of the automated assay using a programmed liquid handler. Each test compound was evaluated using a panel of six recombinantly expressed CYP enzymes (1A2, 2C8, 2C9, 2C19, 2D6, and 3A4) and over a range of eight test concentrations separately.
Cytochrome P450 Protocols by Douglas S. Auld, Henrike Veith, James J. Cali (auth.), Ian R. Phillips, Elizabeth A. Shephard, Paul R. Ortiz de Montellano (eds.)