Affinity and potency of proinhibitory antipeptide antibodies against CYP2D6 is enhanced using cyclic peptides as immunogens.
A series of antipeptide antibodies directed against CYP2D6 were produced by immunizing rabbits with peptides that were sterically unrestrained (linear) or conformationally restricted by cyclization. A variety of sites within the region comprising residues 254 to 290 of CYP2D6 were targeted. In immunoblotting studies, each of the antibodies against the linear and cyclic peptides recognized only a single immunoreactive band of 54 kDa in human liver microsomal fraction and bound to recombinant CYP2D6, but not recombinant CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2E1, or CYP3A4. However, the relative intensity of immunoreactive bands was considerably stronger for those antibodies raised against cyclic peptides. Similarly, in an enzyme-linked immunosorbent assay, antibodies raised against cyclic peptides bound 10 to 100 times more strongly to recombinant CYP2D6 than antibodies raised against the corresponding linear peptides. None of the antibodies raised against linear peptides had any effect on debrisoquine 4-hydroxylase activity of human hepatic microsomal fraction; however, anticyclic peptide antibodies targeted against residues 254 to 273, 261 to 272, and 257 to 268 of CYP2D6 inhibited enzyme activity by a maximum of 60, 75, and 91%, respectively. In contrast, despite binding strongly to CYP2D6, an anticyclic peptide antibody directed against residues 278 to 290 did not inhibit enzyme activity. The epitope of the proinhibitory anticyclic peptide antibody directed against residues 257 to 268 of CYP2D6 included Thr-261 and Trp-262, and indicates a role for these residues in enzyme inhibition. In conclusion, immunization with peptides conformationally restricted by cyclization to mimic loop regions of CYP2D6 resulted in strongly binding antibodies that when targeted appropriately were able to inhibit CYP2D6-catalyzed activity.
Schulz-Utermoehl
T
T
Section on Clinical Pharmacology, Division of Medicine, Imperial College School of Medicine, Hammersmith Campus, London, United Kingdom.
Edwards
R J
RJ
Boobis
A R
AR
D000595:Amino Acid Sequence; D000818:Animals; D019138:Antibodies, Blocking / Q000276:immunology*; D015153:Blotting, Western; D019389:Cytochrome P-450 CYP2D6 / Q000378:metabolism; D065690:Cytochrome P-450 CYP2D6 Inhibitors; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007114:Immunization; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology*; D010456:Peptides, Cyclic / Q000138:chemical synthesis / Q000276:immunology*; D011817:Rabbits / Q000276:immunology; D011994:Recombinant Proteins / Q000378:metabolism
D019138:Antibodies, Blocking; D065690:Cytochrome P-450 CYP2D6 Inhibitors; D010455:Peptides; D010456:Peptides, Cyclic; D011994:Recombinant Proteins; D019389:Cytochrome P-450 CYP2D6