The regioselective glucuronidation of morphine by dimerized human UGT2B7, 1A1, 1A9 and their allelic variants  

作　　者：Zi-zhao YANG Li LI Lu WANG Ling-min YUAN Ming-cheng XU Jing-kai GU Hui-di JIANG Lu-shan YU Su ZENG 

机构地区：[1]Institute of Drug Metabolism and Pharmaceutical Analysis, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China [2]Department of Pharmacy, Zhejiang Hospital, Zhejiang Provincial Key Lab of Geriatrics, Hangzhou 310058, China [3]School of Life Sciences, Jilin University, Changchun 130012, China

出　　处：《Acta Pharmacologica Sinica》2017年第8期1184-1194,共11页中国药理学报（英文版）

基　　金：This study was supported by the International：Science ＆ Technology Cooperation Progrcam of China （No 2014DFE30050）; Program for Zhejiang Leading Team of S＆T Innovation Team （No 2011R50014）; Natural Science Foundation of Zhejiang Province （No LQ15H310003）; and Fundamental Research Funds for the Central Universities of China Ministry of Education （No 2016XZZX001-08）.

摘　　要：Uridine diphosphate-glucuronosyltransferase （UGT） 2B7 is expressed mostly in the human liver, lung and kidney and can transfer endogenous glucuronide group into its substrate and impact the pharmacological effects of several drugs such as estriol, AZT and morphine. UGT2B7 and its allelic variants can dimerize with the homologous enzymes UGTIA1 and UGTIA9, as well as their allelic variants, and then change their enzymatic activities in the process of substrate catalysis. The current study was designed to identify this mechanism using morphine as the substrate of UGT2B7. Single-recombinant allozymes, including UGT2B7＊I （wild type）, UGT2B7＊71S （A71S, 211G〉T）, UGT2B7＊2 （H268Y, 802C〉T）, UGT2BT＊5 （D398N, 1192G〉A）, and double-recombinant allozymes formed by the dimerization of UGTIA9＊I （wild type）, UGTIA9＊2 （C3Y, 8G〉A）, UGTIA9＊3 （M33T, 98T〉C）, UGTIA9＊5 （D256N, 766G〉A）, UGTIA1 （wild type） with its splice variant UGTIAlb were established and incubated with morphine in vitro. Each sample was analyzed with HPLC-MS/MS. All enzyme kinetic parameters were then measured and analyzed. From the results, the production ratio of its aberrant metabolism and subsequent metabolites, morphine-3-giucuronide （M3G） and morphine-6-giucuronide （M6G）, changes regioselectively. Double-recombinant allozymes exhibit stronger enzymatic activity catalyzing morphine than the single-recombinant alloyzymes. Compared to UGT2B7＊I, UGT2B7＊2 singles or doubles have lower Km values for M3G and M6G, whereas UGT2B7＊5 allozymes perform opposite effects. The double allozymes of UGTIA9＊2 or UGTIA9＊5 with UGT2B7 tend to produce M6G. Interestingly the majority of single or double allozymes significantly reduce the ratio of M3G to M6G. The UGTIA9＊2-UGT2B7＊I double enzyme has the lowest M3G：M6G ratio, reflecting that more M6G would form in morphine glucuronide metabolism. This study demonstrates that UGT2B7 common SNPs and their dimers with UGTIA1 and UGTIA9 and their alleUridine diphosphate-glucuronosyltransferase （UGT） 2B7 is expressed mostly in the human liver, lung and kidney and can transfer endogenous glucuronide group into its substrate and impact the pharmacological effects of several drugs such as estriol, AZT and morphine. UGT2B7 and its allelic variants can dimerize with the homologous enzymes UGTIA1 and UGTIA9, as well as their allelic variants, and then change their enzymatic activities in the process of substrate catalysis. The current study was designed to identify this mechanism using morphine as the substrate of UGT2B7. Single-recombinant allozymes, including UGT2B7＊I （wild type）, UGT2B7＊71S （A71S, 211G〉T）, UGT2B7＊2 （H268Y, 802C〉T）, UGT2BT＊5 （D398N, 1192G〉A）, and double-recombinant allozymes formed by the dimerization of UGTIA9＊I （wild type）, UGTIA9＊2 （C3Y, 8G〉A）, UGTIA9＊3 （M33T, 98T〉C）, UGTIA9＊5 （D256N, 766G〉A）, UGTIA1 （wild type） with its splice variant UGTIAlb were established and incubated with morphine in vitro. Each sample was analyzed with HPLC-MS/MS. All enzyme kinetic parameters were then measured and analyzed. From the results, the production ratio of its aberrant metabolism and subsequent metabolites, morphine-3-giucuronide （M3G） and morphine-6-giucuronide （M6G）, changes regioselectively. Double-recombinant allozymes exhibit stronger enzymatic activity catalyzing morphine than the single-recombinant alloyzymes. Compared to UGT2B7＊I, UGT2B7＊2 singles or doubles have lower Km values for M3G and M6G, whereas UGT2B7＊5 allozymes perform opposite effects. The double allozymes of UGTIA9＊2 or UGTIA9＊5 with UGT2B7 tend to produce M6G. Interestingly the majority of single or double allozymes significantly reduce the ratio of M3G to M6G. The UGTIA9＊2-UGT2B7＊I double enzyme has the lowest M3G：M6G ratio, reflecting that more M6G would form in morphine glucuronide metabolism. This study demonstrates that UGT2B7 common SNPs and their dimers with UGTIA1 and UGTIA9 and their alle

关 键 词：UGT2B7 single nucleotide polymorphisms DIMERIZATION enzyme kinetics morphine metabolism 

分 类 号：Q538[生物学—生物化学] S512.103[农业科学—作物学]