1药物体外代谢研究III—Phase II酶研究

刘慧鑫Outline•Background & research significance；•Classification & Nomenclature;•Catalytic Mechanisms;•Substrate specificity;•Distribution;•Species differences & examples;•Metabolic stability studies (UGTs)2

Elimination pathways of top 200 drugsWieners et al. (2005) Nature Rev Drug Discov 4:825-833

药物代谢酶

转运蛋白以不变形式清除UGT，ST，甲基转移酶，乙酰转移酶结合代谢

酯酶，酰胺酶，环氧化物水解酶水解代谢CYP，FMO，MAO，Mo-CO，过氧化物酶氧化代谢

可能参与代谢的酶/蛋白清除机制

FMO，黄素单加氧酶；MAO，单胺氧化酶；UGT，UDP-葡糖醛酸转移酶；ST，磺基转移酶；Mo-CO，含钼氧化酶

研究意义Background代谢稳定性(代谢通路及速率）；揭示药物（或毒物）的作用机制(如解毒）；种属差异研究；前药设计与评价；结构改造及新药研发3

Phase II代谢酶Catalyze both detoxificationas well as bioactivationreactionsGenerally considered to be “antioxidant”enzymes when reactions catalyzed lead to detoxification.

partlybecause they remove metabolites capable of causing oxidative stress via redoxcycling

Conjugation reactions also detoxify reactive intermediatesof phase I reactions

Conjugatinreactions requires the presence of a reactive group(R-OH, R-NH2, R-COOH, R-SH, and some others)

实例Conjugation with phase II enzymesDetoxification & Antioxidant

BileUrine4

尿甘二磷酸葡萄糖醛酸转移酶UDP-Glucuronosyltransferases, UGTs

UGTs酶命名TransferasesGlycosyltransferasesHexosyltransferasesGlucuronosyltransferases(UGTs)

EC 2EC 2.4EC 2.4.1EC 2.4.1.17

Full NamesCommon Names

The Enzyme Commission number (EC number) is a numerical classification scheme for enzymes, based on the chemical reactions they catalyze

Accepted name:Glucuronosyltransferase(UGTs)

Reaction: UDP-glucuronate+ acceptor = UDP + acceptor β-D-glucuronoside5

UGTs催化机理There are three steps to form the conjugate:1.Coupling of D-glucose-1-phosphateto UTPto give UDP-glucose2.Oxidation of the primary alcohol yields the coenzyme UDP-glucuronicacid3.Conjugation with the substrateto yield the glucuronide

细胞质细胞膜内质网内腔+ + Signal peptide

UDP-GlcUApH 7.1 （Kim et at., 1998 ）[Mg+] 1mM ( Berg et al., 1995 )

UGTsare a family of membrane-bound enzymes that are concentrated in the endoplasmic reticulum (ER) and nuclear envelope of hepatocytes.

UGTs结构特征

How to remove the latencySonicationDetergent : Toxon-X Brij-58…

Pore-forming peptides: FilipinAlamethicin…6

UGTs酶分类—催化反应类型•O-GlucuronideHydroxyl (alcohol, phenol) Carboxylic acid

•N-Glucuronide

Amine, AmideSulfonamide

•S-Glucuronide

Sulfhydryls(e.g. thiols) •C-Glucuronide

1,3-dicarbonyls

OHAcNH

OH

OPhO

OCONH2

ONH2

OH2NSO2NHNN

OMe

OMeNN

SHCH3

NNO

OPh

Ph

UGTs酶分类-基因相似性The UDP-glucuronosyltransferases(UGTs) are a superfamilywhich comprised of 2 families (UGT1 and UGT2) and 3 subfamilies (UGT1A, UGT2A, and UGT2B)

41%59%

66%

78%85%87%90%94%67%71%93%94%93%89%83%UGT1UGT2UGTBAUGT1A5UGT1A3UGT1A4UGT1A1UGT1A6UGT1A7UGT1A8UGT1A10UGT1A9UGT2B10UGT2B11UGT2B7UGT2B4UGT2B15UGT2B17UGT2A1Fig. Phylogenetictree showing the relationships between the human UGTs.7

UGTs在人体内的分布情况LiverUGT1A1 UGT1A3UGT1A4 UGT1A6UGT1A9 UGT2B4UGT2B7 UGT2B10UGT2B11 UGT2B15

EsophagusUGT 1A7 UGT1A8UGT1A9 UGT1A10UGT2B7 UGT2B10UGT2B15

IntestineUGT1A1 UGT1A4UGT1A8Biliarytissue

UGT1A1 UGT1A3UGT1A4 UGT1A6UGT1A10

StomachUGT1A1 UGT1A3UGT1A6 UGT1A7UGT1A10

KidneyUGT1A9 UGT2B7

BrainUGT1A6 UGT2A1UGT2B7

ColonUGT 1A1 UGT1A3UGT1A4 UGT1A6UGT1A8UGT1A9 UGT1A10UGT2B7

1A1 16%1A3 3%1A4 14%1A6 6%1A9 17%2B4 4%2B7 40%

人体内主要UGTs代谢亚型酶BurchellB. et al. Methods Enzymol. 2005;400:46-57Relative contributions to the glucuronidationof the top 200 drugs prescribed in the United States in 2002 by human UGTs

Relative contributions to the glucuronidationof the top 200 drugs prescribed in the United States in 2002 by human liver UGTs

Williams J.A. et al. DMD 2004; 32:1201-08