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1.
J Agric Food Chem ; 67(42): 11650-11656, 2019 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-31554401

RESUMO

Occurring in hops (Humulus lupulus) and beer as a racemic mixture, (2R,2S)-8-prenylnaringenin (8-PN) is a potent phytoestrogen in hop dietary supplements used by women as alternatives to conventional hormone therapy. With a half-life exceeding 20 h, 8-PN is excreted primarily as 8-PN-7-O-glucuronide or 8-PN-4'-O-glucuronide. Human liver microsomes and 11 recombinant human UDP-glucuronosyltransferases (UGTs) were used to catalyze the formation of the two oxygen-linked glucuronides of purified (2R)-8-PN and (2S)-8-PN, which were subsequently identified using mass spectrometry and nuclear magnetic resonance spectroscopy. Formation of (2R)- and (2S)-8-PN-7-O-glucuronides predominated over the 8-PN-4'-O-glucuronides except for intestinal UGT1A10, which formed more (2S)-8-PN-4'-O-glucuronide. (2R)-8-PN was a better substrate for all 11 UGTs except for UGT1A1, which formed more of both (2S)-8-PN glucuronides than (2R)-8-PN glucuronides. Although several UGTs conjugated both enantiomers of 8-PN, some conjugated just one enantiomer, suggesting that human phenotypic variation might affect the routes of metabolism of this chiral estrogenic constituent of hops.


Assuntos
Flavanonas/química , Glucuronídeos/química , Glucuronosiltransferase/química , Extratos Vegetais/química , Biocatálise , Flavanonas/metabolismo , Glucuronídeos/metabolismo , Glucuronosiltransferase/metabolismo , Humanos , Humulus/química , Humulus/metabolismo , Espectrometria de Massas , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Extratos Vegetais/metabolismo , Estereoisomerismo
2.
Environ Pollut ; 254(Pt B): 113093, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31472452

RESUMO

Per- and polyfluoroalkyl substances (PFASs) are a large group of chemicals and can be detected in environmental and human samples all over the world. Toxicity of existing and emerging PFASs will be a long-term source of concern. This study aimed to investigate structure-dependent inhibitory effects of 14 PFASs towards the activity of 11 UDP-glucuronosyltransferase (UGT) isoforms. In vitro UGTs-catalyzed glucuronidation of 4-methylumbelliferone (4-MU) was employed to determine the inhibition of PFASs towards different UGT isoforms. All the PFASs showed <75% of inhibition or stimulation effects on UGT1A3, UGT1A7, UGT1A9, UGT2B4, UGT2B7 and UGT2B17. However, PFASs showed broad inhibition on the activity of UGT1A1 and UGT1A8. The activity of UGT1A1 was inhibited by 98.8%, 98%, 79.9%, 77.1%, and 76.9% at 100 µmoL/L of perfluorodecanoic acid (PFDA), perfluorooctanesulfonic acid potassium salt (PFOS), perfluorotetradecanoic acid (PFTA), perfluorooctanoic acid (PFOA) and perfluorododecanoic acid (PFDoA), respectively. UGT1A8 was inhibited by 97.6%, 94.8%, 86.3%, 83.4% and 77.1% by PFDA, PFTA, perfluorooctadecanoic acid (PFOcDA), PFDoA and PFOS, respectively. Additionally, PFDA significantly inhibited UGT1A6 and UGT1A10 by 96.8% and 91.6%, respectively. PFDoA inhibited the activity of UGT2B15 by 88.2%. PFDA and PFOS exhibited competitive inhibition towards UGT1A1, and PFDA and PFTA showed competitive inhibition towards UGT1A8. The inhibition kinetic parameter (Ki) were 3.15, 1.73, 13.15 and 20.21 µmoL/L for PFDA-1A1, PFOS-1A1, PFDA-1A8 and PFTA-1A8, respectively. The values were calculated to be 0.3 µmoL/L and 1.3 µmoL/L for the in vivo inhibition of PFDA towards UGT1A1-and UGT1A8-catalyzed metabolism of substances, and 0.2 µmoL/L and 2.0 µmoL/L for the inhibition of PFOS towards UGT1A1 and the inhibition of PFTA towards UGT1A8, respectively. Molecular docking indicated that hydrogen bonds and hydrophobic interactions contributed to the interaction between PFASs and UGT isoforms. In conclusion, exposure to PFASs might inhibit the activity of UGTs to disturb metabolism of endogenous compounds and xenobiotics. The structure-related effects of PFASs on UGTs would be very important for risk assessment of PFASs.


Assuntos
Fluorcarbonetos/química , Glucuronosiltransferase/química , Simulação de Acoplamento Molecular , Simulação por Computador , Humanos , Isoformas de Proteínas/química
3.
Int J Mol Sci ; 20(15)2019 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-31349586

RESUMO

Uridine diphosphate (UDP)-glycosyltransferases (UGTs) are major phase II detoxification enzymes involved in glycosylation of lipophilic endobiotics and xenobiotics, including phytoalexins. Nicotine, one of the most abundant secondary plant metabolites in tobacco, is highly toxic to herbivorous insects. Plant-herbivore competition is the major impetus for the evolution of large superfamilies of UGTs and other detoxification enzymes. However, UGT functions in green peach aphid (Myzus persicae) adaptation are unknown. In this study, we show that UGT inhibitors (sulfinpyrazone and 5-nitrouracil) significantly increased nicotine toxicity in M. persicae nicotianae, suggesting that UGTs may be involved in nicotine tolerance. In total, 101 UGT transcripts identified in the M. persicae genome/transcriptome were renamed according to the UGT Nomenclature Committee guidelines and grouped into 11 families, UGT329, UGT330, UGT339, UGT341-UGT345, and UGT348-UGT350, with UGT344 containing the most (57). Ten UGTs (UGT330A3, UGT339A2, UGT341A6, UGT342B3, UGT343C3, UGT344D5, UGT344D8, UGT348A3, UGT349A3, and UGT350A3) were highly expressed in M. persicae nicotianae compared to M. persicae sensu stricto. Knockdown of four UGTs (UGT330A3, UGT344D5, UGT348A3, and UGT349A3) significantly increased M. persicae nicotianae sensitivity to nicotine, suggesting that UGT expression in this subspecies may be associated with nicotine tolerance and thus host adaptation. This study reveals possible UGTs relevant to nicotine adaptation in tobacco-consuming M. persicae nicotianae, and the findings will facilitate further validation of the roles of these UGTs in nicotine tolerance.


Assuntos
Adaptação Biológica , Afídeos/fisiologia , Glucuronosiltransferase/metabolismo , Nicotina/metabolismo , Sequência de Aminoácidos , Animais , Afídeos/classificação , Afídeos/efeitos dos fármacos , Sequência Conservada , Resistência a Medicamentos/genética , Expressão Gênica , Técnicas de Silenciamento de Genes , Glucuronosiltransferase/antagonistas & inibidores , Glucuronosiltransferase/química , Glucuronosiltransferase/genética , Família Multigênica , Nicotina/farmacologia , Filogenia , Domínios Proteicos
4.
PLoS One ; 14(5): e0217695, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31150474

RESUMO

Demethoxycurcumin (DMC) is a safe and natural food-coloring additive, as well as an agent with several therapeutic properties. However, extensive glucuronidation in vivo has resulted in its poor bioavailability. In this study, we aimed to investigate the formation of DMC-O-glucuronides by uridine 5'-diphospho-glucuronosyltransferase 1A1 (UGT1A1) and its transport by breast cancer resistance protein (BCRP) and multidrug resistance-associated proteins (MRPs) in HeLa cells stably transfected with UGT1A1 (named HeLa1A1 cells). The chemical inhibitors Ko143 (a selective BCRP inhibitor) and MK571 (a pan-MRP inhibitor) both induced an obvious decrease in the excretion rate of DMC-O-glucuronides and a significant increase in intracellular DMC-O-glucuronide concentrations. Furthermore, BCRP knock-down resulted in a marked reduction in the level of excreted DMC-O-glucuronides (maximal 55.6%), whereas MRP1 and MRP4 silencing significantly decreased the levels of excreted DMC-O-glucuronides (a maximum of 42.9% for MRP1 and a maximum of 29.9% for MRP3), respectively. In contrast, neither the levels of excreted DMC-O-glucuronides nor the accumulation of DMC-O-glucuronides were significantly altered in the MRP4 knock-down HeLa cells. The BCRP, MRP1 and MRP3 transporters were identified as the most important contributors to the excretion of DMC-O-glucuronides. These results may significantly contribute to improving our understanding of mechanisms underlying the cellular disposition of DMC via UGT-mediated metabolism.


Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Diarileptanoides/farmacologia , Glucuronosiltransferase/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas de Neoplasias/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Disponibilidade Biológica , Diarileptanoides/química , Dicetopiperazinas/farmacologia , Corantes de Alimentos/química , Corantes de Alimentos/farmacologia , Inativação Gênica , Glucuronídeos/biossíntese , Glucuronídeos/genética , Glucuronosiltransferase/química , Células HeLa , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Proteínas de Neoplasias/antagonistas & inibidores , Propionatos/farmacologia , Transporte Proteico/genética , Quinolinas/farmacologia , Transfecção
5.
Hum Mol Genet ; 28(12): 2062-2077, 2019 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-31163085

RESUMO

Glycosylation is a common post-translational modification of proteins. Glycosylation is associated with a number of human diseases. Defining genetic factors altering glycosylation may provide a basis for novel approaches to diagnostic and pharmaceutical applications. Here we report a genome-wide association study of the human blood plasma N-glycome composition in up to 3811 people measured by Ultra Performance Liquid Chromatography (UPLC) technology. Starting with the 36 original traits measured by UPLC, we computed an additional 77 derived traits leading to a total of 113 glycan traits. We studied associations between these traits and genetic polymorphisms located on human autosomes. We discovered and replicated 12 loci. This allowed us to demonstrate an overlap in genetic control between total plasma protein and IgG glycosylation. The majority of revealed loci contained genes that encode enzymes directly involved in glycosylation (FUT3/FUT6, FUT8, B3GAT1, ST6GAL1, B4GALT1, ST3GAL4, MGAT3 and MGAT5) and a known regulator of plasma protein fucosylation (HNF1A). However, we also found loci that could possibly reflect other more complex aspects of glycosylation process. Functional genomic annotation suggested the role of several genes including DERL3, CHCHD10, TMEM121, IGH and IKZF1. The hypotheses we generated may serve as a starting point for further functional studies in this research area.


Assuntos
Fucosiltransferases/genética , Glicosiltransferases/genética , Polissacarídeos/sangue , Cromatografia Líquida de Alta Pressão , Estudos de Coortes , Fucosiltransferases/sangue , Fucosiltransferases/química , Estudo de Associação Genômica Ampla , Glucuronosiltransferase/sangue , Glucuronosiltransferase/química , Glicosilação , Fator 1-alfa Nuclear de Hepatócito/sangue , Fator 1-alfa Nuclear de Hepatócito/química , Humanos , Imunoglobulina G/metabolismo , Proteínas de Membrana/metabolismo , Polimorfismo Genético , Locos de Características Quantitativas
6.
J Agric Food Chem ; 67(22): 6275-6284, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31083910

RESUMO

Many O-glucuronides exhibiting various pharmacological activities have been found in nature and in drug metabolism. The glucuronidation of bioactive natural products or drugs to generate glucuronides with better activity and druggability is important in drug discovery and research. In this study, by using two uridine diphosphate (UDP)-dependent glucuronosyltransferases (GATs, UGT88D4 and UGT88D7) from plants, we developed two glucuronidation approaches, pure enzyme catalysis in vitro and recombinant whole-cell catalysis in vivo, to efficiently synthesize bioactive O-glucuronides by the glucuronidation of natural products. In total, 14 O-glucuronides with different structures, including flavonoids, anthraquinones, coumarins, and lignans, were obtained, 7 of which were new compounds. Furthermore, one of the biosynthesized O-glucuronides, kaempferol-7- O-ß-d-glucuronide (3a), potently inhibited protein tyrosine phosphatase (PTP) 1B with an IC50 value of 8.02 × 10-6 M. Some of the biosynthesized O-glucuronides also exhibited significant antioxidant activities.


Assuntos
Antirrhinum/enzimologia , Glucuronídeos/química , Glucuronosiltransferase/química , Perilla frutescens/enzimologia , Proteínas de Plantas/química , Antirrhinum/genética , Biocatálise , Estabilidade Enzimática , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Cinética , Estrutura Molecular , Perilla frutescens/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Especificidade por Substrato
7.
Mol Pharmacol ; 96(2): 128-137, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31127008

RESUMO

Drug transporters play a crucial role in pharmacokinetics. One subfamily of transporters with proven clinical relevance are the OATP1B transporters. Recently we identified a new member of the OATP1B family named OATP1B3-1B7 (LST-3TM12). This functional transporter is encoded by SLCO1B3 and SLCO1B7 OATP1B3-1B7 is expressed in hepatocytes and is located in the membrane of the smooth endoplasmic reticulum (SER). One aim of this study was to test whether OATP1B3-1B7 interacts with commercial drugs. First, we screened a selection of OATP1B substrates for inhibition of OATP1B3-1B7-mediated transport of dehydroepiandrosterone sulfate and identified several inhibitors. One such inhibitor was ezetimibe, which not only inhibited OATP1B3-1B7 but is also a substrate, as its cellular content was significantly increased in cells heterologously expressing the transporter. In humans, ezetimibe is extensively metabolized by hepatic and intestinal uridine-5'-diphospho-glucuronosyltransferases (UGTs), the catalytic site of which is located within the SER lumen. After verification of OATP1B3-1B7 expression in the small intestine, we determined in microsomes whether SER access can be modulated by inhibitors of OATP1B3-1B7. We were able to show that these compounds significantly reduced accumulation in small intestinal and hepatic microsomes, which influenced the rate of ezetimibe ß-D-glucuronide formation as determined in microsomes treated with bromsulphthalein. Notably, this molecule not only inhibits the herein reported transporter but also other transport systems. In conclusion, we report that multiple drugs interact with OATP1B3-1B7; for ezetimibe, we were able to show that SER access and metabolism is significantly reduced by bromsulphthalein, which is an inhibitor of OATP1B3-1B7. SIGNIFICANCE STATEMENT: OATP1B3-1B3 (LST-3TM12) is a transporter that has yet to be fully characterized. We provide valuable insight into the interaction potential of this transporter with several marketed drugs. Ezetimibe, which interacted with OATP1B3-1B7, is highly metabolized by uridine-5'-diphospho-glucuronosyltransferases (UGTs), whose catalytic site is located within the smooth endoplasmic reticulum (SER) lumen. Through microsomal assays with ezetimibe and the transport inhibitor bromsulphthalein we investigated the interdependence of SER access and the glucuronidation rate of ezetimibe. These findings led us to the hypothesis that access or exit of drugs to the SER is orchestrated by SER transporters such as OATP1B3-1B7.


Assuntos
Retículo Endoplasmático Liso/química , Ezetimiba/farmacocinética , Transportadores de Ânions Orgânicos/metabolismo , Proteínas Carreadoras de Solutos/metabolismo , Sulfobromoftaleína/farmacologia , Transporte Biológico , Domínio Catalítico , Glucuronosiltransferase/química , Células HeLa , Humanos , Intestino Delgado/metabolismo , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo
8.
Molecules ; 24(8)2019 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-31013570

RESUMO

GL-V9 is a prominent derivative of wogonin with a wide therapeutic spectrum and potent anti-tumor activity. The metabolism characteristics of GL-V9 remain unclear. This study aimed to clarify the metabolic pathway of GL-V9 and investigate the generation of its glucuronidation metabolites in vitro and in vivo. HPLC-UV-TripleTOF was used to identify metabolites. The main metabolite that we found was chemically synthesized and the synthetic metabolite was utilized as standard substance for the subsequent metabolism studies of GL-V9, including enzyme kinetics in liver microsomes of five different species and reaction phenotyping metabolism using 12 recombinant human UDP-glucuronosyltransferase (UGT) isoforms. Results indicated that the glucuronidation reaction occurred at C5-OH group, and 5-O-glucuronide GL-V9 is the only glucuronide metabolite and major phase II metabolite of GL-V9. Among 12 recombinant human UGTs, rUGT1A9 showed the strongest catalytic capacity for the glucuronidation reaction of GL-V9. rUGT1A7 and rUGT1A8 were also involved in the glucuronidation metabolism. Km of rUGT1A7-1A9 was 3.25 ± 0.29, 13.92 ± 1.05, and 4.72 ± 0.28 µM, respectively. In conclusion, 5-O-glucuronide GL-V9 is the dominant phase II metabolite of GL-V9 in vivo and in vitro, whose formation rate and efficiency are closely related to isoform-specific metabolism profiles and the distribution of UGTs in different tissues of different species.


Assuntos
Flavanonas , Glucuronídeos/química , Glucuronosiltransferase/química , Microssomos Hepáticos/enzimologia , Animais , Flavanonas/química , Flavanonas/farmacocinética , Glucuronídeos/farmacocinética , Glucuronosiltransferase/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie
9.
J Anal Toxicol ; 43(7): 536-542, 2019 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-30927356

RESUMO

Desomorphine, the principal opioid in Krokodil, has an analgesic potency approximately ten-times that of morphine. Similar to other opioids, during phase II metabolism it undergoes conjugation with glucuronic acid to form desomorphine-glucuronide. Although hydrolysis of conjugated species is sometimes required prior to analysis, desomorphine-glucuronide has not been fully investigated. In this study, six hydrolysis procedures were optimized and evaluated. Deconjugation efficiencies using chemical and enzymatic hydrolysis were evaluated and stability in aqueous solution was assessed. Acid hydrolysis was compared with five ß-glucuronidase sources (BGTurbo™, IMCSzyme™, Escherichia coli, Helix pomatia and Patella vulgata). At optimal conditions, each hydrolysis method produced complete hydrolysis (≥96%). However, under simulated challenging conditions, P. vulgata was the most efficient ß-glucuronidase for the hydrolysis of desomorphine-glucuronide. Both BGTurbo™ and IMCSzyme™ offered fast hydrolysis with no need for sample cleanup prior to liquid chromatography-quadrupole/time of flight-mass spectrometry (LC-Q/TOF-MS) analysis. Hydrolysates using E. coli, H. pomatia and P. vulgata underwent additional sample treatment using ß-Gone™ cartridges. Additionally, the stability of free and conjugated drug was evaluated at elevated temperature (60°C) in aqueous solutions between pH 4 and 10. No degradation was observed for either desomorphine or desomorphine-glucuronide under any of the conditions tested.


Assuntos
Codeína/análogos & derivados , Glucuronidase/química , Glucuronídeos/química , Glucuronosiltransferase/química , Animais , Codeína/química , Estabilidade de Medicamentos , Escherichia coli/enzimologia , Gastrópodes/enzimologia , Humanos , Hidrólise , Estrutura Molecular , Proteínas Recombinantes/química
10.
Drug Test Anal ; 11(1): 45-50, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29996009

RESUMO

Multiple new psychoactive substances (NPS) are released into the recreational drug market each year. One NPS drug class that has become more common in recent years is that of the benzodiazepines (designer benzodiazepines, DBZ). Several metabolism studies have been performed to improve their bioanalytical detection via the best target. These studies have shown the presence of parent glucuronides and, as polymorphisms have been noted for the catalyzing enzymes (UDP-glucuronyltransferases) responsible for glucuronide conjugation reactions, it is important to keep this in mind when interpreting DBZ cases in clinical and/or forensic toxicology. Therefore, the aim of this study was to determine the UDP-glucuronyltransferases (UGTs) responsible for parent compound conjugation of nine DBZ to facilitate interpretation of related cases. Clonazolam, deschloroetizolam, etizolam, flubromazolam, flunitrazolam, metizolam, nifoxipam, nitrazolam, and pyrazolam were incubated with pooled human liver microsomes (pHLM) or 13 different human UGTs. The samples were analyzed using liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS). Glucuronide conjugates of flunitrazolam and nifoxipam were only detected in pHLM, suggesting that these reactions are performed by dimer complexes of several UGTs or complexes between UGTs and other metabolizing enzymes contained in pHLM. Nitrazolam or pyrazolam glucuronides were not detected. Glucuronidation of clonazolam, deschloroetizolam, etizolam, flubromazolam, and metizolam was catalyzed exclusively by UGT1A4. The conjugation of the majority of the DBZ was performed by the UGT isoform 1A4 for which polymorphisms have been described. This underlines the importance of taking glucuronidation polymorphism into consideration when interpreting intoxication cases.


Assuntos
Benzodiazepinas/metabolismo , Drogas Desenhadas/metabolismo , Glucuronídeos/metabolismo , Glucuronosiltransferase/metabolismo , Espectrometria de Massas em Tandem/métodos , Animais , Benzodiazepinas/análise , Benzodiazepinas/química , Cromatografia Líquida/métodos , Drogas Desenhadas/análise , Drogas Desenhadas/química , Glucuronídeos/química , Glucuronosiltransferase/química , Humanos , Insetos , Microssomos/metabolismo , Detecção do Abuso de Substâncias/métodos
11.
Xenobiotica ; 49(10): 1158-1163, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30484368

RESUMO

Parthenolide (PTL) and micheliolide (MCL) are sesquiterpene lactones with similar structures, and both of them have been reported to exhibit multiple biochemical and pharmacological activities. This study aims to investigate the inhibition of these two compounds on the activity of UDP-glucuronosyltransferases (UGTs). In vitro incubation mixture for recombinant UGTs-catalyzed glucuronidation metabolism of 4-methylumbelliferone (4-MU) was utilized to investigate the inhibition potential. Inhibition kinetics (including inhibition type and parameters) were determined, and in silico docking was employed to elucidate the inhibition difference between PTL and MCL on UGT1A1. MCL showed no inhibition toward all the UGT isoforms, and PTL showed strong inhibition toward UGT1A1. The half-maximal inhibitory concentration (IC50) of PTL on the activity of UGT1A1 was determined to be 64.4 µM. Inhibition kinetics determination showed that PTL exerted noncompetitive inhibition toward UGT1A1, and the inhibition kinetic constant (Ki) was determined to be 12.1 µM. In silico docking method has been employed to show that hydrogen bonds between PTL and the activity cavity of UGT1A1 contributed to the stronger inhibition of PTL on the activity of UGT1A1 than MCL. In conclusion, PTL can more easily induce drug-drug interaction (DDI) with clinical drugs mainly undergoing UGT1A1-catalyzed glucuronidation.


Assuntos
Inibidores Enzimáticos , Glucuronosiltransferase/antagonistas & inibidores , Glucuronosiltransferase/química , Sesquiterpenos de Guaiano , Sesquiterpenos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Humanos , Cinética , Sesquiterpenos/química , Sesquiterpenos/farmacocinética , Sesquiterpenos/farmacologia , Sesquiterpenos de Guaiano/química , Sesquiterpenos de Guaiano/farmacocinética , Sesquiterpenos de Guaiano/farmacologia
12.
Int J Biol Macromol ; 126: 653-661, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30594625

RESUMO

Flavonoids are widely distributed phytochemicals in vegetables, fruits and medicinal plants. Recent studies demonstrate that some natural flavonoids are potent inhibitors of the human UDP-glucuronosyltransferase 1A1 (UGT1A1), a key enzyme in detoxification of endogenous harmful compounds such as bilirubin. In this study, the inhibitory effects of 56 natural and synthetic flavonoids on UGT1A1 were assayed, while the structure-inhibition relationships of flavonoids as UGT1A1 inhibitors were investigated. The results demonstrated that the C-3 and C-7 hydroxyl groups on the flavone skeleton would enhance UGT1A1 inhibition, while flavonoid glycosides displayed weaker inhibitory effects than their corresponding aglycones. Further investigation on inhibition kinetics of two strong flavonoid-type UGT1A1 inhibitors, acacetin and kaempferol, yielded interesting results. Both flavonoids were competitive inhibitors against UGT1A1-mediated NHPN-O-glucuronidation, but were mixed and competitive inhibitors toward UGT1A1-mediated NCHN-O-glucuronidation, respectively. Furthermore, docking simulations showed that the binding areas of NHPN, kaempferol and acacetin on UGT1A1 were highly overlapping, and convergence with the binding area of bilirubin within UGT1A1. In summary, detailed structure-inhibition relationships of flavonoids as UGT1A1 inhibitors were investigated carefully and the findings shed new light on the interactions between flavonoids and UGT1A1, and will contribute considerably to the development of flavonoid-type drugs without strong UGT1A1 inhibition.


Assuntos
Flavonoides/farmacologia , Glucuronosiltransferase/antagonistas & inibidores , Domínio Catalítico , Flavonas/química , Flavonas/farmacologia , Flavonoides/química , Corantes Fluorescentes/metabolismo , Glucuronosiltransferase/química , Glucuronosiltransferase/metabolismo , Humanos , Concentração Inibidora 50 , Quempferóis/química , Quempferóis/farmacologia , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Simulação de Acoplamento Molecular , Especificidade por Substrato/efeitos dos fármacos
13.
Molecules ; 23(10)2018 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-30347696

RESUMO

The co-use of conventional drug and herbal medicines may lead to herb-drug interaction via modulation of drug-metabolizing enzymes (DMEs) by herbal constituents. UDP-glucuronosyltransferases (UGTs) catalyzing glucuronidation are the major metabolic enzymes of Phase II DMEs. The in vitro inhibitory effect of several herbal constituents on one of the most important UGT isoforms, UGT2B7, in human liver microsomes (HLM) and rat liver microsomes (RLM) was investigated. Zidovudine (ZDV) was used as the probe substrate to determine UGT2B7 activity. The intrinsic clearance (Vmax/Km) of ZDV in HLM is 1.65 µL/mg/min which is ten times greater than in RLM, which is 0.16 µL/mg/min. Andrographolide, kaempferol-3-rutinoside, mitragynine and zerumbone inhibited ZDV glucuronidation in HLM with IC50 values of 6.18 ± 1.27, 18.56 ± 8.62, 8.11 ± 4.48 and 4.57 ± 0.23 µM, respectively, hence, herb-drug interactions are possible if andrographolide, kaempferol-3-rutinoside, mitragynine and zerumbone are taken together with drugs that are highly metabolized by UGT2B7. Meanwhile, only mitragynine and zerumbone inhibited ZDV glucuronidation in RLM with IC50 values of 51.20 ± 5.95 µM and 8.14 ± 2.12 µM, respectively, indicating a difference between the human and rat microsomal model so caution must be exercised when extrapolating inhibitory metabolic data from rats to humans.


Assuntos
Glucuronosiltransferase/antagonistas & inibidores , Interações Ervas-Drogas , Microssomos Hepáticos/efeitos dos fármacos , Zidovudina/administração & dosagem , Animais , Diterpenos/administração & dosagem , Glucuronídeos/antagonistas & inibidores , Glucuronosiltransferase/química , Glucuronosiltransferase/isolamento & purificação , Glucuronosiltransferase/metabolismo , Medicina Herbária , Humanos , Microssomos Hepáticos/enzimologia , Ratos , Alcaloides de Triptamina e Secologanina/administração & dosagem , Sesquiterpenos/administração & dosagem , Zidovudina/antagonistas & inibidores , Zidovudina/química
14.
J Biol Chem ; 293(49): 19047-19063, 2018 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-30327429

RESUMO

Homogalacturonan (HG) is a pectic glycan in the plant cell wall that contributes to plant growth and development and cell wall structure and function, and interacts with other glycans and proteoglycans in the wall. HG is synthesized by the galacturonosyltransferase (GAUT) gene family. Two members of this family, GAUT1 and GAUT7, form a heteromeric enzyme complex in Arabidopsis thaliana Here, we established a heterologous GAUT expression system in HEK293 cells and show that co-expression of recombinant GAUT1 with GAUT7 results in the production of a soluble GAUT1:GAUT7 complex that catalyzes elongation of HG products in vitro The reaction rates, progress curves, and product distributions exhibited major differences dependent upon small changes in the degree of polymerization (DP) of the oligosaccharide acceptor. GAUT1:GAUT7 displayed >45-fold increased catalytic efficiency with DP11 acceptors relative to DP7 acceptors. Although GAUT1:GAUT7 synthesized high-molecular-weight polymeric HG (>100 kDa) in a substrate concentration-dependent manner typical of distributive (nonprocessive) glycosyltransferases with DP11 acceptors, reactions primed with short-chain acceptors resulted in a bimodal product distribution of glycan products that has previously been reported as evidence for a processive model of GT elongation. As an alternative to the processive glycosyltransfer model, a two-phase distributive elongation model is proposed in which a slow phase, which includes the de novo initiation of HG and elongation of short-chain acceptors, is distinguished from a phase of rapid elongation of intermediate- and long-chain acceptors. Upon reaching a critical chain length of DP11, GAUT1:GAUT7 elongates HG to high-molecular-weight products.


Assuntos
Proteínas de Arabidopsis/metabolismo , Glucuronosiltransferase/metabolismo , Pectinas/biossíntese , Arabidopsis/enzimologia , Proteínas de Arabidopsis/química , Glucuronosiltransferase/química , Células HEK293 , Humanos , Modelos Biológicos , Estrutura Molecular , Pectinas/química , Eletricidade Estática , Especificidade por Substrato , Açúcares de Uridina Difosfato/metabolismo
15.
Drug Metab Dispos ; 46(10): 1446-1452, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30111624

RESUMO

UDP-glucuronosyltransferase (UGT), as an integral membrane protein localized in the endoplasmic reticulum, has the ability to detoxify potentially hazardous xenobiotic substances. Most UGTs are expressed in liver, but UGT1A10 has proven to be an extrahepatic enzyme considerably expressed throughout the gastrointestinal tract. Earlier studies indicated that different UGT isoforms could exist in higher-order homo-oligomers or at least dimers within the membrane, but the formation of intermolecular disulfide bridges between UGT molecules was not often observed. In this study, we expressed recombinant human UGT1A10 in human embryonic kidney (HEK)293 and Chinese hamster ovary (CHO) cells to examine its oligomeric states and characterize its enzymatic activities against two therapeutically interesting substrates, morphine and entacapone, including determination of the catalytic rate constant (kcat) values for the first time. It was observed that a majority of the UGT1A10 protein expressed in HEK293 cells existed in covalently crosslinked higher-order oligomers via formation of intermolecular disulfide bonds, whereas formation of the intermolecular disulfide bonds was not observed in the UGT1A10 protein expressed in CHO cells. Owing to the formation of the covalently crosslinked higher-order oligomers, the UGT1A10 protein expressed in HEK293 cells had much lower catalytic activity (particularly the catalytic rate constant kcat) against both morphine and entacapone, compared with the UGT1A10 protein form expressed in CHO cells against the same substrates.


Assuntos
Glucuronosiltransferase/química , Proteínas Recombinantes/química , Animais , Células CHO , Catálise , Catecóis/metabolismo , Cricetulus , Ativação Enzimática , Glucuronosiltransferase/metabolismo , Células HEK293 , Humanos , Nitrilos/metabolismo , Multimerização Proteica , Proteínas Recombinantes/metabolismo
16.
J Asian Nat Prod Res ; 20(7): 605-614, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29989425

RESUMO

Glucuronidation is an important and popular metabolic reaction in vivo of drugs. The further evaluation of biological activity and toxicity of glucuronides is necessary in the course of the drug research and development. However, the synthesis of glucuronides is limited by the lack of efficient approach. Herein, we have developed a new glucuronide synthesis method using plant uridine diphosphate-dependent glucuronosyltransferases (UGTs), UGT88D4, UGT88D7, and EpGT8, enabling the convenient preparation for corresponding O-glucuronide metabolites (1a, 2a, 3a, and 3b) in milligram scale of two neurological active agents, IMM-H004 (1) and FLZ (2). Their structures were characterized by spectroscopic data analyses.


Assuntos
Glucuronídeos/síntese química , Glucuronosiltransferase/metabolismo , Plantas/enzimologia , Clonagem Molecular , Glucuronosiltransferase/química , Glucuronosiltransferase/genética , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Metais/química , Estrutura Molecular , Espectrometria de Massas por Ionização por Electrospray , Temperatura Ambiente
17.
Biomol NMR Assign ; 12(2): 315-318, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29934866

RESUMO

A major component of phase II drug metabolism is the covalent addition of glucuronic acid to metabolites and xenobiotics. This activity is carried out by UDP-glucuronosyltransferases (UGT) which bind the UDP-glucuronic acid donor and catalyze the covalent addition of glucuronic acid sugar moieties onto a wide variety of substrates. UGTs play important roles in drug detoxification and were recently shown to act in an inducible form of multi-drug resistance in cancer patients. Despite their biological importance, structural understanding of these enzymes is limited. The C-terminal domain is identical for all UGT1A family members and required for binding to UDP-glucuronic acid as well as involved in contacts with substrates. Here, we report the backbone assignments for the C-terminal domain of UGT1A. These assignments are a critical tool for the development of a deeper biochemical understanding of substrate specificity and enzymatic activity.


Assuntos
Glucuronosiltransferase/química , Ressonância Magnética Nuclear Biomolecular , Glucuronosiltransferase/metabolismo , Humanos , Domínios Proteicos , Especificidade por Substrato
18.
Chemosphere ; 206: 9-16, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29723751

RESUMO

Chlorophenols (CPs) are important pollutants extensively utilized in industry, agriculture and forestry. The present study aims to determine the inhibition of CPs on the activity of the important phase II drug-metabolizing enzymes (DMEs) UDP-glucuronosyltransferases (UGTs). 100 µM of fourteen CPs were used for preliminary screening using in vitro incubation. Furthermore, half inhibition concentration (IC50) and inhibition kinetics were determined for CPs with significant inhibition towards UGT isoforms. In silico docking was used to explain the inhibition difference among CPs. Multiple UGT isoforms were inhibited by CPs. In silico docking showed that higher free binding energy due to hydrophobic interactions of 2.4-Dichlorophenol (2.4-DCP) or 4-Chloro-3-methylphenol (4C3MP) with UGT1A9 contributed to stronger inhibition potential of 2.4-Dichlorophenol (2.4-DCP) or 4-Chloro-3-methylphenol (4C3MP) towards UGT1A9 than 4-CP. Pentachlorophenol (PCP) was chosen as the representative CPs to determine the IC50 value towards UGT1A6, UGT1A9 and UGT2B7. IC50 was calculated to be 0.33 µM, 0.24 µM and 31.35 µM for the inhibition of PCP towards UGT1A6, UGT1A9 and UGT2B7. PCP was demonstrated to show competitive inhibition towards UGT1A6, UGT1A9 and UGT2B7, and the inhibition kinetic parameters (Ki) was calculated to be 0.18 µM, 0.01 µM and 5.37 µM for the inhibition of PCP towards UGT1A6, UGT1A9 and UGT2B7. All these information will be beneficial for elucidating the risk of CPs exposure from a new perspective.


Assuntos
Clorofenóis/química , Glucuronosiltransferase/química , Glucuronosiltransferase/antagonistas & inibidores , Humanos , Fatores de Risco
19.
FEBS Lett ; 592(11): 1837-1846, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29710432

RESUMO

Uridine diphosphate-glucuronosyltransferases (UGTs) are the most important phase II enzymes in human drug metabolism. Using permeabilized recombinant fission yeast cells (enzyme bags), we demonstrate that UGT1A5 can catalyze an N-glucuronidation reaction. We characterized two new polymorphic UGT1A5 variants: a common ninefold mutant (UGT1A5*8) with double-fold activity and a much rarer sixfold mutant (UGT1A5*9), which has the same activity as the wild-type. Molecular modeling studies indicate that the minor effects of all mutations, except for Gly259Arg, are due to their distance to the substrate binding site. Extensive molecular dynamics simulations revealed that the Gly259Arg mutation stabilizes helix Q through a newly formed hydrogen bonding network, which places the cofactor in a much more favorable geometry in UGT1A5*8 as compared to the wild-type.


Assuntos
Glucuronosiltransferase/química , Simulação de Dinâmica Molecular , Mutação de Sentido Incorreto , Substituição de Aminoácidos , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Humanos , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo
20.
Nat Biotechnol ; 36(3): 249-257, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29431741

RESUMO

Cell walls in crops and trees have been engineered for production of biofuels and commodity chemicals, but engineered varieties often fail multi-year field trials and are not commercialized. We engineered reduced expression of a pectin biosynthesis gene (Galacturonosyltransferase 4, GAUT4) in switchgrass and poplar, and find that this improves biomass yields and sugar release from biomass processing. Both traits were maintained in a 3-year field trial of GAUT4-knockdown switchgrass, with up to sevenfold increased saccharification and ethanol production and sixfold increased biomass yield compared with control plants. We show that GAUT4 is an α-1,4-galacturonosyltransferase that synthesizes homogalacturonan (HG). Downregulation of GAUT4 reduces HG and rhamnogalacturonan II (RGII), reduces wall calcium and boron, and increases extractability of cell wall sugars. Decreased recalcitrance in biomass processing and increased growth are likely due to reduced HG and RGII cross-linking in the cell wall.


Assuntos
Biocombustíveis , Parede Celular/genética , Glucuronosiltransferase/genética , Pectinas/biossíntese , Biomassa , Boro/metabolismo , Cálcio/metabolismo , Parede Celular/enzimologia , Parede Celular/metabolismo , Produtos Agrícolas , Glucuronosiltransferase/química , Panicum/enzimologia , Panicum/genética , Pectinas/genética , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Populus/enzimologia , Populus/genética , Açúcares/metabolismo
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