Inhibitory anti-CYP3A4 peptide antibody: Mapping of inhibitory epitope andspecificity toward other CYP3A isoforms

An antipeptide antibody has been produced that recognizes CYP3A4 and exhibi ts greater than 90-95% inhibition on CYP3A4-mediated reactions [Wang RW and Lu AYH (1997) Drug Metab Dispos 25:762-767], 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 26 1-267 of CYP3A4), This conclusion was based on the reversal of antibody inh ibition of testosterone 6 beta-hydroxylation when peptides with overlapping sequence in this region were preincubated with the antibody, In immunoblot ting analysis, this antibody did not recognize CYP3A5 or CYP3A7 in microsom es prepared from baculovirus-infected cells containing these two expressed isoforms, In addition, the antipeptide antibody did not inhibit testosteron e 6 beta-hydroxylation or midazolam 1'- and 4-hydroxylation in microsomes c ontaining expressed CYP3A5 and CYP3A7. Because the corresponding sequence i n CYP3A5 (LNDKQKH) and CYP3A7 (LKETQKH) differs from CYP3A4 by only two ami no 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.