Selective and potent inhibition of human CYP2C19 activity by a conformationally targeted antipeptide antibody.
A conformationally targeted anti-peptide antibody was produced by immunizing a rabbit with a cyclized peptide corresponding to a loop region of human CYP2C19 (residues 250-261). In an enzyme-linked immunosorbent assay, the antibody bound strongly to recombinant CYP2C19 and poorly to recombinant CYP2C8, CYP2C9, and CYP2C18. In immunoblotting studies, the antibody bound strongly to recombinant CYP2C19 and weakly to recombinant CYP2C8. No binding to recombinant CYP1A2, CYP2C9, CYP2C18, CYP2D6, CYP2E1, and CYP3A4 was detected. In immunoinhibition experiments, the anti-peptide antibody targeted against CYP2C19 potently inhibited (S)-mephenytoin 4'-hydroxylase activity of human hepatic microsomal fraction (>90%). It had no appreciable effect on ethoxyresorufin O-deethylase (CYP1A2), tolbutamide methyl-hydroxylase (CYP2C9), dextromethorphan O-demethylase (CYP2D6), 4-nitrophenol hydroxylase (CYP2E1), or testosterone 6beta-hydroxylase (CYP3A4) activity of human hepatic microsomal fraction. However, large amounts of purified IgG fractions were able to inhibit up to 35% of paclitaxel 6alpha-hydroxylase (CYP2C8) activity. In conclusion, we have demonstrated that an anti-peptide antibody targeted against residues 250 to 261 of human CYP2C19 selectively and potently inhibited CYP2C19 activity of human hepatic microsomal fraction.
Schulz-Utermoehl
T
T
Department of Drug Metabolism, Novo Nordisk A/S, Novo Nordisk Park, Maaloev, Denmark. TiSU@novo.dk
Mountfield
R J
RJ
Madsen
K
K
Jørgensen
P N
PN
Hansen
K T
KT
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D001189:Aryl Hydrocarbon Hydroxylases; D015153:Blotting, Western; D065731:Cytochrome P-450 CYP2C19; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000276:immunology; D010455:Peptides / Q000276:immunology*; D011487:Protein Conformation; D011817:Rabbits; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000276:immunology
D000906:Antibodies; D065607:Cytochrome P-450 Enzyme Inhibitors; D010455:Peptides; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C045793:CYP2C19 protein, human; D065731:Cytochrome P-450 CYP2C19