人细胞色素P4502D64个单核苷酸突变株活性及其与药物相互作用的体外研究

Activities of four strains of human cytochrome P450 2D6 with single nucleotide substitution and their interaction with durgs in vitro

目的探讨单核苷酸替换对人细胞色素P450CYP2D6酶活性及其与药物相互作用的影响。方法体外构建CYP2D6野生株(WT)和G169R、P34S、E410K、V7M等4个单核苷酸突变株的表达载体并在酿酒酵母中诱导表达,裂解并提取各酶的蛋白微粒体,用蛋白质印迹法验证其表达,再分别测定各酶代谢底物丁夫洛尔和3-[2-(N,N二乙基-N-甲铵)乙基]-7-甲氧基-4-甲基香豆素(AMMC)的米氏常数(Km)和最大酶促反应速度(Vmax)。以AMMC为底物对CYP2D6WT和V7M、G169R进行高通量药物抑制实验,所选药物包括已知的2D6抑制剂(奎尼丁、舍曲林)、底物(氯丙嗪、氟西汀、阿米替林)和既非抑制剂又非底物的化合物(酮康唑、曲格列酮),求得不同药物对不同酶的半数抑制浓度(IC50)。结果蛋白质印迹法检测显示各酶均表达良好。CYP2D6WT代谢丁呋洛尔和AMMC的Km值分别为19.22、2.06μmol·L-1,Vmax值分别为154.53、21.60pmol·min-1·mg-1,Vmax/Km值分别为8.04、11.49μl·min-1·mg-1。与CYP2D6WT比较,V7M代谢2种底物的Vmax值均要高得多(P<0.05),G169R和E410K均稍低,而P34S都要低很多(P<0.05);各突变株的Km值也发生了或多或少的改变。药物对CYP2D6WT和V7M和G169R的抑制程度排序均为奎尼丁>氯丙嗪>氟西汀>阿米替林>舍曲林>酮康唑/曲格列酮;药物对V7M和G169R的IC50值与CYP2D6WT的IC50值比值,奎尼丁分别为0.88和0.80,氯丙嗪0.54和0.80,氟西汀0.65和1.02,阿米替林0.85和0.71,舍曲林0.43和0.74。结论CYP2D6部分单核苷酸突变株的酶动力学特征与野生株有明显差异,单核苷酸替换可导致酶对药物抑制的敏感性发生变化。

Objective To study the effects of single nucleotide substitution on the activity of human cytochrome P450 2D6 （CYP2D6） and its interaction with drugs. Methods Yeast expression vectors of prototype （WT）, G169R, P34S, E410K, and V7M strains of CYP2D6 with single nucleotide substitution were constructed in vitro, and the recombinants were expressed in yeast by galactose induction. The proteins were assessed by Western Blot after yeast cells were lysed and the microsomes were prepared. And the bufuralol 1＇-hydroxylation activities and AMMC （3-[2-（N, N-diethyl-N-methylammonium）ethyl]-7-methoxy-4-methylcoumarin） demethylation activities of each enzyme were then determined by measuring the Vmax and Km. At last, high throughput inhibition screening for WT and strains of V7M and G169R were conducted with AMMC as the substrate. The drugs used for the experiment included 2D6 inhibitors, quinidine and sertraline; substrates, chlorpromazine, fluoxetine, and amitriptyline; and ketoconazole and troglitazone, which are neither inhibitor nor substrates. The half-maximal （50%） inhibitory concentration （IC50） for the drugs were calculated. Results As shown by Western Blot, each of the enzyme expressed well. The Km, Vmax, and Vmax/Km of Bufuralol and AMMC for WT were, respectively, 19.22μmol·L^-1, 154.53 pmol·min^-1·mg^-1, and 8.04/μl·min^-1·mg^-1; and 2.06 μmol·L^-1, 21.60 pmol·min^-1·mg^-1, and 11.49 μl·min^-1·mg^-1. The activity （Vmax） of WT was significantly lower than those of the two substrates of V7M, slightly higher than those of G169R and E410K, and significantly higher than that of P34S. The Km of each enzyme strains was changed more or less accordingly. The drugs displayed the following rank order of in vitro potency against CYP2D6 WT and SNP enzymes： quinidine 〉 chlorpromazine 〉 fluoxetine 〉 amitriptyline 〉 sertraline 〉 ketoconazole/troglitazone. The ratios of IC50 （V7M）/IC50（WT） and IC50 （G169）/IC50（WT） for drugs, respectively, were： quinidine （0.88 and 0.80）, chlorpromazine （0.53 and 0.80）, fluoxetine （0.65 and 1.02）, amitriptyline （0.85 and 0.71）, and sertraline （0.43 and 0.74）. Conclusions The kinetic properties of CYP2D6 with single nucleotide substitution are significantly different from that of the prototype enzyme. The enzymes of each SNP showed drug-specific altered susceptibility to inhibition.