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Vol. 30, Issue 7, 845-852, July 2002
Gentest, a BD Biosciences Company, Woburn, Massachusetts
We have tested a panel of 29 cDNA-expressed rat and human enzymes
with 9 fluorometric substrates to determine the P450 isoform selectivity in the catalysis of the substrates to fluorescent products.
The substrates examined were dibenzyl fluorescein, 7-benzyloxyquinoline (BQ), 3-cyano-7-ethoxycoumarin, 3-cyano-7-methoxycoumarin,
7-methoxy-4-trifluoromethylcoumarin, 3-[2-(N,N-diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcoumarin (AMMC),
3-[2-(N,N-diethyl-N-methylamino)ethyl]-7-methoxy-4-trifluoromethylcoumarin, 7-benzyloxyresorufin, and 7-benzyloxy-4-trifluoromethylcoumarin (BFC). For most substrates, multiple cDNA-expressed cytochrome P450
isoforms were found to catalyze the formation of the fluorescent product. However, among the combinations tested, rat CYP2D2 displayed high selectivity for AMMC demethylation (a substrate selective for
CYP2D6 in human liver microsomes). AMMC demethylation activity was
15-fold lower in microsomes isolated from female Dark Agouti rats, a
model known to have a low abundance of CYP2D2, and apparent KM values were similar for cDNA-expressed
CYP2D2 and male Sprague-Dawley liver microsomes. BFC dealkylation and
BQ dealkylation were selective but not exclusive for human CYP3A4. A
small role for CYP1A2 could be demonstrated. The CYP3A4 selectivity in
hepatic microsomes was supported by studies using chemical and antibody
inhibitors and a correlation analysis within a panel of liver
microsomes from individual donors. BQ demonstrated a higher degree of
selectivity for and higher rates of metabolism by CYP3A than BFC.
However, per unit enzyme the fluorescent signal is lower for BQ than
BFC. AMMC, BQ, and BFC should find uses as enzyme-selective probe substrates.
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