The crystal structure of human UDP-glucuronosyltransferase 2B7 C-terminal end is the first mammalian UGT target to be revealed: the significance for human UGTs from both the 1A and 2B families

Human UDP-glucuronosyltransferases (EC 2.4.1.17) (UGTs) are major phase II metabolism enzymes that detoxify a multitude of endo- and xenobiotics through the covalent addition of a glucuronic acid moiety. UGTs are promiscuous enzymes that regulate the levels of numerous important endobiotics in a range of tissues, and inactivate most therapeutic compounds in concert with phase I enzymes. In spite of the importance of these enzymes, we have only a limited understanding of the molecular mechanisms governing their substrate specificity and catalytic activity. Until recently, no three-dimensional structural information was available for any mammalian UGT. The 1.8-Å resolution apo crystal structure of the UDP-glucuronic acid binding domain of human UGT2B7 (2B7CT) is the only structure of a mammalian UGT target determined to date. In this review, we summarize what has been learned about human UGT function from the analysis of this and other related glycosyltransferase (GT) crystal structures

Ethylenediamine functionalized-single-walled nanotube (f-SWNT)-assisted in vitro delivery of the oncogene suppressor p53 gene to breast cancer MCF-7 cells

A gene delivery concept based on ethylenediamine-functionalized single-walled carbon nanotubes (f-SWCNTs) using the oncogene suppressor p53 gene as a model gene was successfully tested in vitro in MCF-7 breast cancer cells. The f-SWCNTs-p53 complexes were introduced into the cell medium at a concentration of 20 μg mL−1 and cells were exposed for 24, 48, and 72 hours. Standard ethidium bromide and acridine orange assays were used to detect apoptotic cells and indicated that a significantly larger percentage of the cells (approx 40%) were dead after 72 hours of exposure to f-SWCNTs-p53 as compared to the control cells, which were exposed to only p53 or f-SWCNTs, respectively. To further support the uptake and expression of the genes within the cells, green fluorescent protein-tagged p53, attached to the f-SWCNTs was added to the medium and the complex was observed to be strongly expressed in the cells. Moreover, caspase 3 activity was found to be highly enhanced in cells incubated with the f-SWCNTs-p53 complex, indicating strongly induced apoptosis. This system could be the foundation for novel gene delivery platforms based on the unique structural and morphological properties of multi-functional nanomaterials

Crystal Structure of the Cofactor-Binding Domain of the Human Phase II Drug-Metabolism Enzyme UDP-Glucuronosyltransferase 2B7

Human UDP-glucuronosyltransferases (UGT) are the dominant phase II conjugative drug metabolism enzymes that also play a central role in the processing of a range of endobiotic compounds. UGTs catalyze the covalent addition of glucuronic acid sugar moieties to a host of therapeutics and environmental toxins, as well as to a variety of endogenous steroids and other signaling molecules. We report the 1.8 Å resolution apo crystal structure of the UDP-glucuronic acid binding domain of human UGT isoform 2B7 (UGT2B7), which catalyzes the conjugative elimination of opioid, antiviral, and anticancer drugs. This is the first crystal structure of any region of a mammalian UGT drug metabolism enzyme. Designed UGT2B7 mutants at residues predicted to interact with the UDP-glucuronic acid cofactor exhibited significantly impaired catalytic activity, with maximum effects observed for amino acids closest to the glucuronic acid sugar transferred to the acceptor molecule. Homology modeling of UGT2B7 with related plant flavonoid glucosyltransferases indicate that human UGTs share a common catalytic mechanism. Point mutations at predicted catalytic residues in UGT2B7 abrogated activity, strongly suggesting that human UGTs also utilize a serine hydrolase-like catalytic mechanism to facilitate glucuronic acid transfer

Analysis of R- and S-hydroxywarfarin glucuronidation catalyzed by human liver microsomes and recombinant UDP-glucuronosyltransferases

Coumadin (R-, S-warfarin) is a challenging drug to accurately dose, both initially and for maintenance, because of its narrow therapeutic range and wide interpatient variability and is typically administered as a racemic (Rac) mixture, which complicates the biotransformation pathways. The goal of the current work was to identify the human UDP-glucuronosyltransferases (UGTs) involved in the glucuronidation of the separated R- and S-enantiomers of 6-, 7-, and 8-hydroxywarfarin and the possible interactions between these enantiomers. The kinetic and inhibition constants for human recombinant 1A family UGTs toward these separated enantiomers have been assessed using high-performance liquid chromatography (HPLC)-UV-visible analysis, and product confirmations have been made using HPLC-mass spectrometry/mass spectrometry. We found that separated R- and S-enantiomers of 6-, 7-, and 8-hydroxywarfarin demonstrate significantly different glucuronidation kinetics and can be mutually inhibitory. In some cases significant substrate inhibition was observed, as shown by K(m), V(max), and K(i), comparisons. In particular, UGT1A1 and extrahepatic UGT1A10 have significantly higher capacities than other isoforms for S-7-hydroxywarfarin and R-7-hydroxywarfarin glucuronidation, respectively. Activity data generated using a set of well characterized human liver microsomes supported the recombinant enzyme data, suggesting an important (although not exclusive) role for UGT1A1 in glucuronidation of the main warfarin metabolites, including Rac-6- and 7-hydroxywarfarin and their R- and S-enantiomers in the liver. This is the first demonstration that the R- and S-enantiomers of hydroxywarfarins are glucuronidated, with significantly different enzymatic affinity and capacity, and supports the importance of UGT1A1 as the major hepatic isoform involved

Characterization of Human Hepatic and Extrahepatic UDP-Glucuronosyltransferase Enzymes Involved in the Metabolism of Classic Cannabinoids

Tetrahydrocannabinol (Δ9-THC), the primary psychoactive ingredient in marijuana, is subject to cytochrome P450 oxidation and subsequent UDP-glucuronosyltransferase (UGT)-dependent glucuronidation. Many studies have shown that CYP2C9 and CYP3A4 are the primary enzymes responsible for these cytochrome P450-dependent oxidations, but little work has been done to characterize phase II metabolic pathways. In this study, we test the hypothesis that there are specific human UGTs responsible for classic cannabinoid metabolism. The activities of 12 human recombinant UGTs toward classic cannabinoids [cannabinol (CBN), cannabidiol (CBD), (–)-Δ8-THC, (–)-Δ9-THC, (±)-11-hydroxy-Δ9-THC (THC-OH), and (–)-11-nor-9-carboxy-Δ9-THC (THC-COOH)] were evaluated using high-performance liquid chromatography-tandem mass spectrometry and labeling assays. Despite activity by UGT1A1, 1A3, 1A8, 1A9, 1A10, and 2B7 toward CBN, CBD, THC-OH, and THC-COOH, only selected UGTs demonstrate sufficient activity for further characterization of steady-state kinetics. CBN was the most recognized substrate as evidenced by activities from hepatic UGT1A9 and extrahepatic UGT1A7, UGT1A8, and UGT1A10. These results may reflect the introduction of an aromatic ring to Δ9-THC, leading to favorable π stacking with phenylalanines in the UGT active site. Likewise, oxidation of Δ9-THC to THC-OH results in UGT1A9 and UGT1A10 activity toward the cannabinoid. Further oxidation to THC-COOH surprisingly leads to a loss in metabolism by UGT1A9 and UGT1A10, while creating a substrate recognized by UGT1A1 and UGT1A3. The resulting glucuronide of THC-COOH is the main metabolite found in urine, and thus these hepatic enzymes play a critical role in the metabolic clearance of cannabinoids. Taken together, glucuronidation of cannabinoids depends on upstream processing including enzymes such as CYP2C9 and CYP3A4