A Real-Time Fluorescence Assay for Measuring N-Dealkylation

A real-time fluorescence assay system using 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 approximately 27-fold more fluorescent than the substrates using excitation and emission wavelengths of 405 and 455 nm, respectively. Tests using expressed CYP1A1, 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, whereas CYP1A1 N -dealkylated these plus the n -pentyl derivative. Activities using 5 μM 9- N -(alkylamino)acridine substrates ranged from 0.1 to 0.9 pmol 9AA/min/pmol P450. Kinetic constants for CYP1A1 N -dealkylation of the 9- N -(methylamino)acridine (MAA) and 9- N -(ethylamino)acridine (EAA) were K m 1.09 ± 0.68 and 0.35 ± 0.21 μM and the V max 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 K m 7.9 ± 5.4 and 3.2 ± 1.6 μM, and V max 501 ± 35.4 and 702.7 ± 257 pmol 9AA/min/pmol CYP2D6, respectively. The experimental binding energies (Δ G bind ) were calculated for MAA with CYP1A1 and CYP2D6 to be –8.266 and –7.074 kcal/mol, respectively. The Δ G bind values for EAA with CYP1A1 and CYP2D6 were –8.950 and –7.618 kcal/mol, 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.