Abstract
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 apparentKM 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.
Footnotes
-
A portion of this work was presented at the 6th International Society for the Study of Xenobiotics meeting; 2001 Oct 7–11; Munich, Germany.
- Abbreviations used are::
- P450
- cytochrome P450
- RLM
- rat liver microsomes
- HLM
- human liver microsomes
- SD
- Sprague-Dawley
- DA
- Dark Agouti
- DBF
- dibenzyl fluorescein
- BQ
- 7-benzyloxyquinoline
- AMMC
- 3-[2-(N,N-diethyl-N-methylamino) ethyl]-7-methoxy-4-methylcoumarin
- MeAMFC
- 3-[2-(N,N-diethyl-N-methylamino) ethyl]-7-methoxy-4-trifluoromethylcoumarin
- BFC
- 7-benzyloxy-4-trifluoromethylcoumarin
- CEC
- 7-ethoxy-3-cyanocoumarin
- CMC
- 7-methoxy-3-cyanocoumarin
- MFC
- 7-methoxy-4-trifluoromethylcoumarin
- BzRes
- 7-benzyloxyresorufin
- E/S
- enzyme-substrate
- MAB
- monoclonal antibody
- Received November 1, 2001.
- Accepted April 11, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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