【作者】 王会娟；

【导师】 陈超；

【作者基本信息】 西北大学 ， 分子微生物学， 2007， 硕士

【摘要】 人细胞色素2C19作为细胞色素P450家族中重要的一员在许多药物的代谢过程中发挥着重要的作用，经研究发现，其基因的多态性是导致个体、种族间的药物代谢差异和药物不良反应的决定因素。本研究利用体外重组技术，克隆及表达CYP2C19野生株及有代表性的10个单核苷酸突变株(它们分别代表在人体内天然存在的多态性等位基因，同时这些突变不包括同义突变，在调控区的突变和引起蛋白缺失的突变)，建立了一个研究CYP2C19多态性酶的平台。通过对这些多态性等位基因进行酶动力学分析和药物抑制高通量筛选，测定酶动力学常数和药物抑制常数，所得数据可为研究多态性等位基因在药物代谢中的作用和在新药开发早期进行新药评价提供重要的信息。通过RT-PCR法从人肝组织中获得CYP2C19野生型的cDNA(1.5Kb)，并将之克隆到酵母表达载体pYES2／CT上，并通过PCR定点突变的方法在野生型CYP2C19基因中引入单核苷酸突变，得到10个SNPs(L17P，E92D，W120R，R132Q，R144H，R150H，P227L，1331V，R410C，和R433W)，通过测序得以证实；将11个重组质粒分别转入已经整合了细胞色素P450氧化还原酶基因(POR)的酿酒酵母中，半乳糖诱导表达目的蛋白，利用SDS-PAGE和Western Blot分析表达产物，结果表明11个菌株均得到表达，所得蛋白约为55Kda，与预期大小相同。大量诱导高表达菌株并制备微粒体蛋白S9部分，将所制备的微粒体用CO还原示差光谱法进行P450含量测定，只有WT、I331V、R150H在450nm处有吸收峰，含量分别为22 pmol／mg、16 pmol／mg、15pmol／mg；其它SNPs只有在420nm处有非常明显的吸收峰。用适量微粒体蛋白与2C19特异性荧光底物CEC(3-cynao-7-ethoxycoumarin)进行酶学反应，所得数据用非线性回归分析法计算出Km值、Vmax值和内在清除率CLint值(Vmax／Km)，结果表明：在10个突变株中，除了R132Q和R433W完全丧失酶活性外，其余有活性的不同的多态性等位基因之间的Km值相差不大，但是Vmax差别很大，其中I331V、R410C与野生株比较接近，其余突变株的Vmax值均有显著降低。将低物浓度固定，与系列浓度的抑制剂共同孵育，用荧光检测技术测定已知抑制剂对重组CYP2C19野生型酶的抑制常数，结果表明：CYP2C19特异性抑制剂反苯环丙胺对重组CYP2C19野生型酶有特异性抑制，IC50约为7.3μM，与相关报道数据接近，而CYP2C9特异性抑制剂磺胺苯吡唑对CYP2C19无抑制。运用荧光高通量检测技术对9种有活性的多态性等位酶和9类20种药物的抑制筛选实验表明：2C19所代谢的底物和已知抑制剂均对其有抑制，8种药物如他汀类药物、噻唑烷二酮类药物等对CYP2C19无抑制；化学结构或治疗作用类似的同类药品对CYP2C19的抑制情况不一；不同基因型被同一种药物抑制程度也不尽相同，甚至差别较大。本研究首次成功构建了11个CYP2C19多态性等位基因的酿酒酵母表达体系，所诱导制备的酵母微粒体蛋白活性良好而稳定，所建立的CYP2C19多态性等位基因体外生化检测系统和药物高通量抑制筛选系统高效、灵敏、简便，从而为下一步研究与CYP2C19有关的药物-药物相互作用奠定基础，同时也有助于临床上的个体化用药、降低不良反应和提高药效。更多还原

【Abstract】 Human cytochrome P450 2C19 plays an important role in the metabolism of drugs and xenobiotics. Pharmacogenetic studies have shown that genetic polymorphisms in this gene are important determinants of inter-individual and inter-ethnic variation in drug metabolism and toxicity. To develop a broadly applicable assay system for studying human CYP2C19 polymorphic enzymes, we cloned and expressed the cDNA of CYP2C19 wild-type(WT) and 10 viarants in budding yeast Saccharomyces cerevisiaee, which was already integreted with CYP450 Oxidoreductase(POR). Using these recombinant enzymes in real-time biochemical assays and high throughput drug screening, the kinetic constants as well as inhibition constants for the test compounds were measured. The results we got can provid valuable information for studying the effects of polymorphic genes on drug metabolism and evluating new drugs in the early phase of drug discovery.The wild-type cDNA of CYP 2C19 was obtained by RT-PCR from human liver tissue and cloned into pYES2/CT vector for galactose-inducible expression in yeast. The cloned cDNA was subsequently used as a template to introduce SNP by site-directed mutagenesis ,the viarants are named I331V, W120R, L17P, E92D, R132Q, R144H, R150H, P227L, R410C, R433W. All the viarants were cloned into the same vector. Transformed yeasts produced large quantities of microsome-bound 2C19 enzymes as determined by Western analysis, a 55 kDa protein was observed.The P450 content was assayed by reduced CO difference spectrum.There was a 450-nm peak observed in 2C19-WT, I331V and R150H , and their content respectively were 22pmol/mg, 16 pmol/mg, 15 pmol/mg; Other SNPs only had a very strong 420-nm peak ,it was a denatured form of CYP2C19. The isolated microsomes were used to measure the kinetic constants of 2C19 enzymes in real-time assays using a fluorogenic substrate CEC. The results showed that all enzymes possess robust activity with the exception of R132Q and R433W, the Km value of 9 catalytically active enzymes showed little difference(no more than two-fold),but the Vmax value were markedly different: I331V and R410C were very close to 2C19-WT, the other viarants had a much lower Vmax .The inhibition of recombinat CYP2C19-WT enzyme by known inhibitor drug was tested by serial titration of drugs in the fluorogenic assays. The results showed that: CYP2C19 was strongly inhibited by its known inhibitor tranylcypromine, IC50 was about 7.3μM, but CYP2C9’known inhibitor Sulfaphenazole can’t inhibit CYP2C19.Used fluorogenic high throughput technique to detect the inhibition of 20 drugs on 9 catalytically active enzymes,the results showed : Except 8 drugs such as statins and Thiozol nediones(TZDs), drugs matabolized by CYP2C19 and its known inhibitors all had specific inhibition on CYP2C19 ; Drugs had similar structure or therapy had different inhibition on CYP2C19; The inhibitory degree of one drug on different genotype varied a lot.For the first time,we successfully constructed the Saccharomyces cerevisiae expressing system for 11 CYP2C19 polymorphic genes; The catalytic activity of yeast microsome protein were robust and stable; Established an in vitro detection system for biochemical analysis and drug high throughput screening of CYP2C19 polymorphic enzymes,which were efficient, sensitive and convenient, This work can guide the further study of CYP2C19 related drug-drug interaction , as well as can aid the future personalized prescription, reducing adverse reaction and enhancing drug efficiency in vivo.更多还原