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Identification of epitopes on cytochrome P450 3A4/5 recognized by monoclonal antibodies
Bhama Parimoo1, Vladimir M Mishin, Christine M Busch
1Chemical Biology Department, Ernest Mario School of Pharmacy, Rutgers The State University of New Jersey, EOHSI Bldg., 170 Frelinghuysen Road, Piscataway, NJ 08854-8020, USA.
Abstract:
In this study we describe the mapping of epitopes on CYP3A4/5 recognized by a panel of monoclonal antibodies (MAbs). CYP3A4 and CYP3A5 cDNAs were cloned in GST expression vectors and the fusion proteins were subjected to Western blot. Eight MAbs reacted with the full-length GST-3A4 fusion protein as well as baculovirus cDNA-expressed CYP3A4, while six of these reacted with baculovirus cDNA-expressed CYP3A5. Five (MAb 347, 351, 352, 354, and 357) out of 8 MAbs were inhibitory in a metabolic assay using quinine as substrate. MAbs 352, 354, and 357 brought about a moderate inhibition of quinine metabolism (60-70%) while MAb 347 inhibited quinine 3- hydroxylation in human liver microsomes (n=6) by more than 70%. MAb 347 was a potent inhibitor of baculovirus-expressed CYP3A5-catalyzed metabolism of quinine (95%) at