尿苷二磷酸葡醛酸转移酶的研究进展

尿苷二磷酸葡醛酸转移酶(UGT)是体内最重要的Ⅱ相代谢酶,它可以参与许多内源性物质如胆红素、甾体激素、甲状腺激素、胆汁酸和脂溶性维生素等的代谢,在许多药物如阿片类药物、镇痛药、非甾体抗炎药和抗惊厥药等的代谢中也发挥着重要的作用。UGT在药物的吸收、分布、代谢和排泄中发挥重要作用。研究UGT特别是其基因多态性及其介导的药物-药物相互作用不仅可以指导临床用药,也可以揭示内源性物质代谢紊乱的机制。本文就UGT的分类、组织分布、对药物吸收的影响、基因多态性及其所介导的药物-药物相互作用进行综述。     

Endogenous substrates for UDP-glucuronosyltransferases

1. Multiple forms of UDP-glucuronosyltransferase (UDPGTs) have been demonstrated in the livers of all mammalian species that have been studied. Rat liver possesses at least eight different isozymes and human liver has at least five different forms which have been identified. 2. Endogenous substrates (e.g., steroids) are helpful in distinguishing UDPGTs as they generally react with only a single form, whereas xenobiotic substrates (e.g., 4-methyl-umbelliferone, p-nitrophenol) react with several forms of the enzyme. 3. Human liver UDPGTs differ in physical properties and substrate specificity from these enzymes obtained from laboratory animals. Hence, it is necessary to study human liver UDPGTs to elucidate substrate specificity and to understand drug-endogenous substrate interaction in humans.     

肝损状态下尿苷二磷酸葡萄糖醛酸转移酶研究进展

肝脏是人体内最重要的代谢器官,包含绝大部分的Ⅰ相和Ⅱ相代谢酶,在内源性和外源性物质的代谢解毒方面起着重要的作用.参与葡萄糖醛酸代谢的尿苷二磷酸葡萄糖醛酸转移酶(UGT)是Ⅱ相代谢中最重要的酶,与常见的CYP450酶相比,研究相对滞后,尤其在肝损伤状态下UGT的相关研究仍处于探索阶段.本文参考最新UGT的研究成果,综述了UGT分子生物学目前研究的概况、肝脏分布及其与肝脏疾病的关系;总结了几种不同诱因的肝损伤病理状态UGT表达和葡萄糖醛酸代谢的变化情况并且深入探讨了相关机制,旨在为药物的肝脏代谢尤其是在肝损状态下葡萄糖醛酸结合消除研究提供一定的参考依据,为肝脏疾病患者的临床合理用药提供指导.     

Isolation and purification of UDP-glucuronosyltransferases

UDPGTs are members of a class of enzymes located in the endoplasmic reticulum and are encoded by a multigene family. These proteins are responsible for the glucuronidation of hundreds of xenobiotics of many chemical classes and many endogenous substances such as steroid hormones, bile acids, and bilirubin. There are a number of UDPGTs which have been identified by purification and characterization studies and a significant number which have been characterized by expression of cDNAs. On the basis of the primary structures elucidated they appear to have marked similarities (5) and are highly conserved. However, key differences in their functional properties appear to depend primarily on differences in amino acid sequences at or about the NH2-terminal area of the protein (5). Many of the UDPGTs have an extraordinarily broad substrate specificity; a few, however, are relatively specific for a given class of substrate (morphine, DT-1 UDPGTs). This places a burden on investigators to clearly identify which substrate and how many UDPGTs will be involved in any analysis of rates of glucuronidation in microsomal preparations. Caution should also be advised for extrapolation of data from hepatic microsomes of experimental animals to human hepatic microsomal preparations because human liver microsomes possess UDPGTs which are qualitatively different and, in certain cases, UDPGTs are present in human liver which are not present in lower animals.     

The UDP-glucuronosyltransferases as oligomeric enzymes

The UDP-glucuronosyltransferases (UGTs) are integral membrane proteins of the endoplasmic reticulum that play important roles in the defense against potentially hazardous xenobiotics. The UGTs also participate in the metabolism and homeostasis of many endogenous compounds, including bilirubin and steroid hormones. Most human UGTs can glucuronidate several substrates the chemical structures of which may vary significantly. Understanding the structural basis for the complex substrate specificity of the UGTs is a major challenge that is hampered by the lack of sufficient structural information on these enzymes. Nevertheless, there is currently a broad interest in the structure and function of the UGTs and here we have focused on their oligomeric state. The question whether or not the UGTs are oligomeric enzymes, either dimeric or tetrameric, was frequently addressed in the past, as well as in recent studies. The current knowledge of protein-protein interactions among the UGTs is limited, however, primarily due to considerable difficulties in purifying individual recombinant UGTs as fully active and mono-dispersed proteins. Such hurdles in studying the oligomeric state of the UGTs prompted researchers to develop less direct approaches for examining the quaternary structure of the UGTs and its functional significance. In this article we have reviewed, sometimes critically, most of the available studies about the oligomeric state of the UGTs. Concluding that the UGTs are oligomeric enzymes, we discuss hetero-oligomerization among UGTs and its possible implications for the structure, function and substrate specificity of the enzymes.     

Carboxyl-glucuronidation of mitiglinide by human UDP-glucuronosyltransferases

Mitiglinide (MGN) is a new potassium channel antagonist for the treatment of type 2 diabetes mellitus. In the present study, a potential metabolic pathway of MGN, via carboxyl-linked glucuronic acid conjugation, was found. MGN carboxyl-glucuronide was isolated from a reaction mixture consisting of MGN and human liver microsomes fortified with UDP-glucuronic acid (UDPGA) and identified by a hydrolysis reaction with beta-glucuronidase and HPLC-MS/MS. Kinetic analysis indicated that MGN from four species had the highest affinity for the rabbit liver microsomal enzyme (K(m)=0.202 mM) and the lowest affinity for the dog liver microsomal enzyme (K(m)=1.164 mM). The metabolic activity (V(max)/K(m)) of MGN to the carboxyl-glucuronidation was in the following order: rabbit>dog>rat>human. With the assessment of MGN glucuronide formation across a panel of recombinant UDP-glucuronosyltransferase (UGT) isoforms (UGT1A3, UGT1A4, UGT1A6, UGT1A9, and UGT2B7), only UGT1A3 and UGT2B7 exhibited high MGN glucuronosyltransferase activity. The K(m) values of MGN glucuronidation in recombinant UGT1A3 and UGT2B7 microsomes were close to those in human liver microsomes. The formation of MGN glucuronidation by human liver microsomes was effectively inhibited by quercetin (substrate for UGT1A3) and diclofenac (substrate for UGT2B7), respectively. The MGN glucuronidation activities in 15 human liver microsomes were significantly correlated with quercetin (r(2)=0.806) and diclofenac glucuronidation activities (r(2)=0.704), respectively. These results demonstrate that UGT1A3 and UGT2B7 are catalytic enzymes in MGN carboxyl-glucuronidation in human liver.     

Structural and functional studies of UDP-glucuronosyltransferases

UDP-Glucuronosyltransferases (UGTs) are glycoproteins localized in the endoplasmic reticulum (ER) which catalyze the conjugation of a broad variety of lipophilic aglycon substrates with glucuronic acid using UDP-glucuronic acid (UDP-GIcUA) as the sugar donor. Glucuronidation is a major factor in the elimination of lipophilic compounds from the body. In this review, current information on the substrate specificities of UGT1A and 2B family isoforms is discussed. Recent findings with regard to UGT structure and topology are presented, including a dynamic topological model of UGTs in the ER. Evidence from experiments on UGT interactions with inhibitors directed at specific amino acids, photoaffinity labeling, and analysis of amino acid alignments suggest that UDP-GIcUA interacts with residues in both the N- and C-terminal domains, whereas aglycon binding sites are localized in the N-terminal domain. The amino acids identified so far as crucial for substrate binding and catalysis are arginine, lysine, histidine, proline, and residues containing carboxylic acid. Site-directed mutagenesis experiments are critical for unambiguous identification of the active-site architecture.     

UDP-glucuronosyltransferases: a family of detoxifying enzymes

Glucuronidation is an important process in the metabolism of xenobiotic and endogenous substances leading to enhancement of excretion of these compounds from the body. A multigene family encodes a number of UDP-glucuronosyltransferase enzymes which catalyse this route of metabolism. Recent advances in biochemical and molecular biological approaches, reviewed here by Thomas Tephly and Brian Burchell, have given new insight into the function and structure of UDP-glucuronosyltransferases. These proteins have surprising similarities and yet appear to be capable of conjugating a remarkable number of different chemicals.     

UDP-glucuronosyltransferases in the metabolic disposition of xenobiotics

UDPGT isoenzymes are products of multiple gene families as demonstrated by sequence analysis of purified proteins and by molecular cloning experiments. These isoenzymes are relatively specific for endogenous substrates but have broad substrate specificities for xenobiotic substrates. They are important metabolic enzymes capable of converting exogenous and endogenous substances to more hydrophilic metabolites. Each species has its own pattern of UDPGTs and it is not possible at this time to extrapolate information directly from one species to another.     

Vertebrate UDP-glucuronosyltransferases: functional and evolutionary aspects

UDP-glucuronosyltransferases (UGTs) represent major phase II drug metabolizing enzymes. They are part of a rapidly growing, sequence similarly based superfamily of UDP-glycosyltransferases, including a number of enzymes, which presumably are functionally unrelated to UGTs. The present commentary discusses evolutionary aspects of the large glycosyltransferase superfamily emphasizing functionally related members which share roles in detoxication and elimination of endo- and xenobiotics. The discussion starts with the two human UGT families and polymorphism frequencies in different populations. These families probably evolved in vertebrates as a result of the struggle against toxic phytoalexins at the hepatogastrointestinal barrier. Co-regulation of some UGTs with other drug metabolizing enzymes may also have evolved in the course of 'animal-plant warfare'. Related UDP-glucosyltransferases evolved in insects. Even in plants and bacteria UDP-glucosyltransferases have been characterized which may be functionally related.     

Inhibition of aflatoxins on UDP-glucuronosyltransferases (UGTs)

Aflatoxins have been recognized as the most harmful mycotoxins leading to various toxic effects. The present study aims to determine the inhibition behavior of aflatoxins on the activity of the important phase II metabolizing enzymes, UDP-glucuronosyltransferases (UGTs), based on in vitro incubation system of recombinant human UGTs-catalyzed glucuronidation of 4-methylumbelliferone (4-MU). 100 μM AFB1 and AFG1 exhibited extensive inhibition towards UGT isoforms especially UGT1A7 and UGT1A8, with the inhibition ratios to be 71.38%, 72.95% and 72.79% for AFB1 to UGT1A7, AFB1 to UGT1A8 and AFG1 to UGT1A8, respectively. Molecular docking results showed that hydrogen bonds and hydrophobic contacts of the particular structure consisting of double furan ring with double bond contributed to the interaction of aflatoxins and UGTs. Kinetics analysis, including inhibition types and kinetics parameters (K_i), and in vitro-in vivo extrapolation (IVIVE) indicated that there might be a medium possibility of inhibition on UGTs by aflatoxins in vivo. In conclusion, the present study indicated that aflatoxins could possibly disturb endogenous metabolism by inhibiting the activity of UGTs so as to exhibit toxic effects.     

Induction of rat hepatic UDP-glucuronosyltransferases by dietary ethoxyquin

Summary Dietary administration of 0.5% ethoxyquin markedly enhanced rat hepatic UDP-glucuronosyltransferase activities. Both 3-methylcholanthrene- and phenobarbital-inducible glucuronidation reactions were stimulated by the antioxidant. In contrast, phenobarbital-inducible bilirubin glucuronidation was not affected by ethoxyquin.     

Everolimus-inhibited multiple isoforms of UDP-glucuronosyltransferases (UGTs)

1.?Everolimus is an inhibitor of mammalian target of rapamycin (mTOR) and has been clinically utilized to prevent the rejection of organ transplants. This study aims to determine the inhibition of everolimus on the activity of phase-II drug-metabolizing enzymes UDP-glucuronosyltransferases (UGTs).

2.?The results showed that 100 μM of everolimus exerted more than 80% inhibition toward UGT1A1, UGT-1A3 and UGT-2B7. UGT1A3 and UGT2B7 were selected to elucidate the inhibition mechanism, and in silico docking showed that hydrogen bonds and hydrophobic interactions mainly contributed to the strong binding of everolimus toward the activity cavity of UGT1A3 and UGT2B7. Inhibition kinetic-type analysis using Lineweaver–Burk plot showed competitive inhibition toward all these UGT isoforms. The inhibition kinetic parameters (K_i) were calculated to be 2.3, 0.07 and 4.4 μM for the inhibition of everolimus toward UGT1A1, UGT-1A3 and UGT-2B7, respectively.

3.?In vitro–in vivo extrapolation (IVIVE) showed that [I]/K_i value was calculated to be 0.004, 0.14 and 0.002 for UGT1A1, UGT-1A3 and UGT-2B7, respectively. Therefore, high DDI potential existed between everolimus and clinical drugs mainly undergoing UGT1A3-catalyzed glucuronidation.     

Isolation and purification of two human liver UDP-glucuronosyltransferases

Two UDP-glucuronosyltransferases (EC 2.4.1.17) were purified from human liver microsomes. Human liver microsomes were solubilized with Emulgen 911 and the UDP-glucuronosyltransferases were separated and purified by chromatofocusing and UDP-hexanolamine Sepharose 4B affinity chromatography. One isoenzyme eluted with an apparent pl of 7.4, displayed a subunit molecular weight of 53,000 after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and catalyzed the glucuronidation of p-nitrophenol, 4-methylumbelliferone, alpha-naphthylamine, and estriol, but not that of 4-aminobiphenyl. A second isoenzyme eluted with an apparent pl of 6.2, displayed a subunit molecular weight of 54,000 after SDS-PAGE, and catalyzed the glucuronidation of p-nitrophenol, 4-methylumbelliferone, alpha-naphthylamine, and 4-aminobiphenyl, but not that of estriol. Neither of the purified human liver UDP-glucuronosyltransferases employed estrone, beta-estradiol, testosterone, androsterone, or 5 alpha-androstane-3 alpha,17 beta-diol as substrate. These enzymes displayed apparent Km values in the same order of magnitude for a given substrate. In general, high concentrations of phosphatidylcholine were required for reconstitution of maximal glucuronidation activity. This report documents the existence of multiple UDP-glucuronosyltransferases in human liver.     

Glucuronidation and the UDP-glucuronosyltransferases in health and disease.

This article is an updated report of a symposium held at the June 2000 annual meeting of the American Society for Pharmacology and Experimental Therapeutics in Boston. The symposium was sponsored by the ASPET Divisions for Drug Metabolism and Molecular Pharmacology. The report covers research from the authors' laboratories on the structure and regulation of UDP-glucuronosyltransferase (UGT) genes, glucuronidation of xenobiotics and endobiotics, the toxicological relevance of UGTs, the role of UGT polymorphisms in cancer susceptibility, and gene therapy for UGT deficiencies.     

尿苷二磷酸葡萄糖醛酸转移酶的调控及其介导的中药-药物相互作用研究进展

尿苷二磷酸葡萄糖醛酸转移酶(UGT)催化的葡萄糖醛酸化反应是的是Ⅱ相代谢中重要的代谢反应之一,对于维持内源性化合物如胆红素、胆汁酸的动态平衡和药物、致癌物等外源性化合物的处置过程起着至关重要的作用。尿苷二磷酸葡萄糖醛酸转移酶的表达和酶活性受多维机制的调控。深入研究其调控网络以及尿苷二磷酸葡萄糖醛酸转移酶介导的相关中药-药物相互作用,对于临床更安全、有效的使用中药提供了指导。因此,本文总结了尿苷二磷酸葡萄糖醛酸转移酶的转录前、转录水平、翻译后修饰等分子调节机制,以及由尿苷二磷酸葡萄糖醛酸转移酶介导的中药-药物相互作用相关的研究进展。