Abstract
An antipeptide antibody has been produced that recognizes CYP3A4 and exhibits greater than 90–95% inhibition on CYP3A4-mediated reactions [Wang RW and Lu AYH (1997) Drug Metab Dispos25:762–767]. The inhibitory epitope of the 21-amino acid peptide, corresponding to residues 253 to 273 of CYP3A4, has been identified to reside in a 7-amino acid sequence (LEDTQKH: residues 261–267 of CYP3A4). This conclusion was based on the reversal of antibody inhibition of testosterone 6β-hydroxylation when peptides with overlapping sequence in this region were preincubated with the antibody. In immunoblotting analysis, this antibody did not recognize CYP3A5 or CYP3A7 in microsomes prepared from baculovirus-infected cells containing these two expressed isoforms. In addition, the antipeptide antibody did not inhibit testosterone 6β-hydroxylation or midazolam 1′- and 4-hydroxylation in microsomes containing expressed CYP3A5 and CYP3A7. Because the corresponding sequence in CYP3A5 (LNDKQKH) and CYP3A7 (LKETQKH) differs from CYP3A4 by only two amino acids, six peptides with either one or two amino acid changes were used to determine which amino acid is essential for antibody-antigen interaction. Our data indicate that Glu, Asp, and Thr in the 7-amino acid sequence of CYP3A4 are critical determinants of selectivity among CYP3A isoforms.
Footnotes
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Send reprint requests to: Regina W. Wang, Department of Drug Metabolism, RY80-D100, Merck Research Laboratories, P.O. Box 2000, Rahway, NJ 07065. E-mail: regina_wang{at}merck.com.
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Part of this work was presented previously at the Experimental Biology 98 in San Francisco, CA.
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↵1 The abbreviation used is: HPLC, high-pressure liquid chromatography.
- Received June 4, 1998.
- Accepted September 17, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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