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

人类药物代谢酶的特异性和结构功能研究

• 批准号: 6227941
• 负责人: JOYCE GOLDSTEIN
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6227941
• 关键词: cytochrome P450; drug metabolism; enzyme activity; enzyme structure; enzyme substrate; protein structure function

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

目的：确定人体内细胞色素P450酶的药物和异物专一性的原因。细胞色素P450 2 C亚家族在基因水平上高度同源，但在底物特异性上存在显著差异。CYP2C9能特异性代谢布洛芬、双氯芬酸等多种抗炎药。相比之下，密切相关的CYP2C19专门代谢其他药物，如抗癫痫药美苯妥因。本研究的目的是确定参与底物专一性的氨基酸，并解决这些CYP2Cs的结构问题。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。ACCOMPLISHMENTS：我们使用布洛芬和双氯芬酸作为CYP2C9的特定底物，以美芬妥因作为CYP2C19的原型底物。构建了嵌合体和定点突变体在细菌中的表达系统。底物识别位点(SRS)3和4(由Gotoh提出的哺乳动物P450底物识别位点)似乎对布洛芬和双氯芬酸等特定的CYP2C9底物具有特异性。SRS3和SRS4似乎都是活性或正常表达所必需的。定点突变实验表明，第286位突变(N S和289I N)对布洛芬和双氯芬酸具有较高的2C19特异性。然而，相反的情况并非如此。CYP2C19的SRS 3和4单独不能赋予CYP2C9甲苯妥因羟基酶。初步实验表明，I99H(SRS1)与FG环中的220Ser Pro和220Pro Thr以及2C19的SRS3和14结合是将CYP2C9转化为高周转率的苯妥英羟基酶所必需的。因此，对于CYP2C19的特异性，似乎需要多个区域，但对于CYP2C9的特异性，需要的变化要少得多。-结构活性、细胞色素P-450、细胞色素P450、细胞色素C19、细胞色素C8、细胞色素P450