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Antipeptide antibodies against overlapping sequences differentially inhibit human CYP2D6.

Alastair E. Cribb,Cindy E. Nuss,R. Wang

Published 1995 in Drug Metabolism And Disposition

ABSTRACT

Two peptides that correspond to sequences within the major 33-amino acid sequence recognized by human liver-kidney microsomal-1 autoantibodies were used to elicit antibodies in rabbits (four per peptide) against CYP2D6. Peptide 1(DPAQPPRDLTEAFLA) corresponded to amino acids 263-277, and peptide 2 (LLTEHRMTWDPAQPPRDLTE) corresponded to amino acids 254-273 of CYP2D6. The peptide-keyhole limpet hemocyanin conjugates elicited good immune responses against their respective peptides as judged by enzyme-linked immunosorbent assay (titers of 1/10,000 to 1/30,000). The antisera recognized CYP2D6 on Western blots and, to varying extents, inhibited recombinant CYP2D6 and liver microsomal CYP2D6 activity. Immunization with peptide 2 produced antisera with the greatest inhibitory potency. Antiserum from a rabbit (#236) immunized with peptide 2 inhibited up to 95% of dextromethorphan O-demethylase activity in human liver microsomes at the highest concentration tested (40% v/v) but did not significantly inhibit CYP1A2, CYP2C9, CYP2E1, or CYP3A4 marker activities. On Western blot, only a single immunoreactive protein comigrating with recombinant CYP2D6 was recognized. In liver microsomes from a CYP2D6-deficient individual, no proteins were recognized, and the antisera did not cross-react with recombinant CYP1A2, CYP2C9, CYP2E1, or CYP3A4. There was a significant correlation between the quantity of immunoreactive CYP2D6 as determined by immunoblotting with anti-peptide 2 antiserum and dextromethorphan O-demethylation in a panel of 10 human liver microsomes (r = 0.95). These data identify a peptide sequence (peptide 2) that can be used to raise antisera that specifically recognize and inhibit CYP2D6.

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