Specificity and Structure-Function Studies of Human Drug-Metabolizing Enzymes

目的：确定CYP酶在人体内具有药物特异性和异种特异性的原因。CYP2C亚家族在基因水平上是高度同源的，但在底物特异性上有显著差异。CYP2C9特异性代谢一些抗炎药物，如布洛芬和双氯芬酸。相反，密切相关的CYP2C19特异性代谢其他药物，如抗惊厥药甲苯妥英。本研究的目的是确定参与底物特异性的氨基酸，并解决这些CYP2Cs的结构。结构活性研究将有助于预测哪些药物将被这些多态酶代谢。我们使用布洛芬和双氯芬酸作为CYP2C9的特异性底物，甲苯妥英作为CYP2C19的原型底物。在细菌cDNA表达系统中构建嵌合体和位点定向突变体。底物识别位点（SRS） 3和4（由Gotoh提出用于哺乳动物p450）似乎对特定的CYP2C9底物（如布洛芬和双氯芬酸）具有特异性。SRS 3和SRS 4似乎都是活性或正常表达所必需的。位点定向诱变实验表明，286（N S和289 I N）突变对2C19的布洛芬和双氯芬酸具有很高的特异性活性。然而，反之则不成立。CYP2C19单独的SRS 3和SRS 4不能将甲苯妥英羟化酶赋予CYP2C9。初步实验表明，需要I99H （SRS1）与FG环中的220Ser Pro和220Pro Thr以及2C19的SRS 3和14结合才能将CYP2C9转化为高周转率的甲苯托英羟化酶。因此，CYP2C19特异性似乎需要多个区域，而CYP2C9特异性所需的改变要少得多。-结构活性，cypp, P-450, CYP2C19, CYP2C8, CYP2C9

AIMS: To determine the reason for drug and xenobiotic specificity of the CYP enzymes in humans. The CYP2C subfamily is highly homologous at the gene level, but differ markedly in their substrate specificity. CYP2C9 specifically metabolizes a number of anti-inflammatory drugs such as ibuprofen and diclofenac. In contrast, the closely related CYP2C19 specifically metabolizes other drugs such as the anticonvulsant mephenytoin. The aims of this study are to define the amino acids involved in substrate specificity and solve the structure of these CYP2Cs. Structure activity studies will help predict which drugs will be metabolized by these polymorphic enzymes.ACCOMPLISHMENTS:We used ibuprofen and diclofenac as specific substrates for CYP2C9 and mephenytoin as a prototype substrate for CYP2C19. Chimeras and site directed mutants were constructed in a cDNA expression system in bacteria. Substrate recognition sites (SRS) 3 and 4 (proposed by Gotoh for mammalian P450s) appeared to confer specificity for specific CYP2C9 substrates such as ibuprofen and diclofenac. Both SRS 3 and 4 appeared necessary for activity or proper expression. Site directed mutagenesis experiments showed that mutations at 286(N S and 289 I N) conferred high specific activity toward ibuprofen and diclofenac to 2C19. However, the converse is not true. SRS 3 and 4 of CYP2C19 alone do not confer mephenytoin hydroxylase to CYP2C9. Preliminary experiments suggest that I99H (SRS1) in combination with 220Ser Pro and 220Pro Thr in the FG loop and SRS 3 and 14 of 2C19 are required to convert CYP2C9 to a high turnover mephenytoin hydroxylase. Therefore multiple regions appear to be required for CYP2C19 specificity, but much fewer changes are required for CYP2C9 specificity. - Structure-activity, CYP, P-450, CYP2C19, CYP2C8, CYP2C9