Schulz-Utermoehl T, Edwards R J, Boobis A R
Section on Clinical Pharmacology, Division of Medicine, Imperial College School of Medicine, Hammersmith Campus, London, United Kingdom.
Drug Metab Dispos. 2000 May;28(5):544-51.
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.
通过用空间上无限制（线性）或通过环化进行构象限制的肽免疫兔子，产生了一系列针对CYP2D6的抗肽抗体。在包含CYP2D6第254至290位残基的区域内的多个位点被作为靶点。在免疫印迹研究中，针对线性和环状肽的每种抗体在人肝微粒体组分中仅识别一条54 kDa的免疫反应条带，并与重组CYP2D6结合，但不与重组CYP1A1、CYP1A2、CYP2A6、CYP2B6、CYP2C9、CYP2C19、CYP2E1或CYP3A4结合。然而，针对环状肽产生的那些抗体的免疫反应条带的相对强度明显更强。同样，在酶联免疫吸附测定中，针对环状肽产生的抗体与重组CYP2D6的结合强度比针对相应线性肽产生的抗体强10至100倍。针对线性肽产生的抗体对人肝微粒体组分的去甲丙咪嗪4 - 羟化酶活性均无任何影响；然而，针对CYP2D6第254至273位、261至272位和257至268位残基的抗环状肽抗体分别最多可抑制酶活性60%、75%和91%。相比之下，尽管一种针对第278至290位残基的抗环状肽抗体与CYP2D6强烈结合，但它并未抑制酶活性。针对CYP2D6第257至268位残基的促抑制性抗环状肽抗体的表位包括苏氨酸 - 261和色氨酸 - 262，这表明这些残基在酶抑制中起作用。总之，用通过环化进行构象限制以模拟CYP2D6环区的肽进行免疫可产生强结合抗体，当靶向适当时能够抑制CYP2D6催化的活性。
