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Title: A direct, real time, fluorimetric assay for cytochrome P450 N-dealkylases

item Mayer, Richard

Submitted to: Drug Metabolism and Disposition
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/4/2006
Publication Date: 1/1/2007
Citation: Mayer, Richard T., Kurt E. Dolence, and Babriele E. Mayer. 2007. A direct, real time, fluorimetric assay for cytochrome P450 N-dealkylases. Drug Metabolism and Disposition. 35(1):103-109.

Interpretive Summary: The breakdown (metabolism) of drugs and other chemicals in the body are handled by different enzymes. One group of enzymes are called cytochromes P450 (CYP) which makeup a superfamily of enzymes. N-Dealkylation (removal of alkyl groups, i.e., methyl, ethyl, etc., from nitrogen atoms in compounds) constitutes a major metabolic pathway in the body. Because interference in the pathway (inhibition) can influence how long certain drugs remain active in the blood it is possible for individuals to suffer ill effects from combined consumption of drugs and foods. It is necessary then to know whether or not drugs and other chemicals are either metabolized by or inhibit the cytochromes P450 to prevent overdosing and other serious consequences. Measurement of N-dealkylases usually complicated because several processing and analytical steps are required after the enzyme reactions (extraction of products and analysis) and rarely can assay be done in real-time. A new assay procedure was developed that utilizes substrates that when N-dealkylated produce a highly fluorescent product (9-aminoacridine). This allows the reaction to be observed as it occurs (real-time). The new method is very sensitive, easy to use, and very selective for two specific forms of cytochromes P450 (i.e. CYP1A1 and CYP2D6), and can be used in high-throughput (where large numbers of assays can be conducted simultaneously) formats.

Technical Abstract: A real-time fluorescence assay system utilizing a series of 9-N-(alkylamino)acridine derivatives (methyl, ethyl, n-propyl, n-butyl, n-pentyl, and benzyl) that are N-dealkylated to 9-aminoacridine (9AA) is described. The product, 9AA, is ca. 27-fold more fluorescent than the substrates using excitation and emission wavelengths of 405 and 455 nm, respectively. Tests using expressed CYPs 1A1, 1A2, 3A4, 3A5, 1B1, 2C9, 2C19, and 2D6 indicated that N-dealkylase activity is specific for CYP1A1 and CYP2D6. CYP2D6 N-dealkylated methyl, ethyl, n-propyl, and n-butyl substrates while CYP1A1 N-dealkylated these plus the n-pentyl derivative. Activities using 5 mM 9-N-(alkylamino)acridine substrate ranged from 0.1 – 0.9 pmole 9AA/min/pmole CYP. Kinetic constants for CYP1A1 N-dealkylation of the 9-N-(methylamino)- (MAA) and 9-N-(ethylamino)acridine (EAA) were Km 1.09 ± 0.68 and 0.35 ± 0.21 mM and the Vmax 61.9 ± 48.5 and 113.8 ± 8.4 pmol 9AA/min/pmol CYP1A1, respectively. Kinetic constants for CYP2D6 N-dealkylation of MAA and EAA were Km 7.9 ± 5.4 and 3.2 ± 1.6 mM and the Vmax 501 ± 35.4 and 702.7 ± 257 pmol 9AA/min/pmol CYP2D6, respectively. The experimental binding energies (DGbind) were calculated for MAA with CYP1A1 and CYP2D6 to be -8.266 and -7.074 kcal/mole, respectively. The DGbind values for EAA with CYP1A1 and CYP2D6 were -8.950 and -7.618 kcal/mole, respectively. The substrates were suitable for monitoring N-dealkylase activity in microsomal preparations (human, rat, and monkey hepatic preparations) and human hepatocellular carcinoma cell suspensions. Assays were conducted by monitoring reactions either in 96-well microtiter plates using a fluorescence plate reader or in cuvettes using a spectrofluorimeter.