Human UDP-glucuronosyltransferase 1As catalyze aristolochic acid D O-glucuronidation to form a lesser nephrotoxic glucuronide.

ETHNOPHARMACOLOGICAL RELEVANCE: Aristolochic acids (AAs) are naturally occurring nitro phenanthrene carboxylic acids primarily found in plants of the Aristolochiaceae family. Aristolochic acid D (AAD) is a major constituent in the roots and rhizomes of the Chinese herb Xixin (the roots and rhizomes of Asarum heterotropoides F. Schmidt), which is a key material for preparing a suite of marketed Chinese medicines. Structurally, AAD is nearly identical to the nephrotoxic aristolochic acid I (AAI), with an additional phenolic group at the C-6 site. Although the nephrotoxicity and metabolic pathways of AAI have been well-investigated, the metabolic pathway(s) of AAD in humans and the influence of AAD metabolism on its nephrotoxicity has not been investigated yet. AIM OF THE STUDY: To identify the major metabolites of AAD in human tissues and to characterize AAD O-glucuronidation kinetics in different enzyme sources, as well as to explore the influence of AAD O-glucuronidation on its nephrotoxicity. MATERIALS AND METHODS: The O-glucuronide of AAD was biosynthesized and its chemical structure was fully characterized by both 1H-NMR and 13C-NMR. Reaction phenotyping assays, chemical inhibition assays, and enzyme kinetics analyses were conducted to assess the crucial enzymes involved in AAD O-glucuronidation in humans. Docking simulations were performed to mimic the catalytic conformations of AAD in human UDP-glucuronosyltransferases (UGTs), while the predicted binding energies and distances between the deprotonated C-6 phenolic group of AAD and the glucuronyl moiety of UDPGA in each tested human UGT isoenzyme were measured. The mitochondrial membrane potentials (MMP) and reactive oxygen species (ROS) levels in HK-2 cells treated with either AAI, or AAD, or AAD O-glucuronide were tested, to elucidate the impact of O-glucuronidation on the nephrotoxicity of AAD. RESULTS: AAD could be rapidly metabolized in human liver and intestinal microsomes (HLM and HIM, respectively) to form a mono-glucuronide, which was purified and fully characterized as AAD-6-O-ß-D-glucuronide (AADG) by NMR. UGT1A1 was the predominant enzyme responsible for AAD-6-O-glucuronidation, while UGT1A9 contributed to a lesser extent. AAD-6-O-glucuronidation in HLM, HIM, UGT1A1 and UGT1A9 followed Michaelis-Menten kinetics, with the Km values of 4.27 µM, 9.05 µM, 3.87 µM, and 7.00 µM, respectively. Docking simulations suggested that AAD was accessible to the catalytic cavity of UGT1A1 or UGT1A9 and formed catalytic conformations. Further investigations showed that both AAI and AAD could trigger the elevated intracellular ROS levels and induce mitochondrial dysfunction and in HK-2 cells, but AADG was hardly to trigger ROS accumulation and mitochondrial dysfunction. CONCLUSION: Collectively, UGT1A-catalyzed AAD 6-O-glucuronidation represents a crucial detoxification pathway of this naturally occurring AAI analogs in humans, which is very different from that of AAI.

Fluorescence-Based High-Throughput Assays for Investigating Cytochrome P450 Enzyme-Mediated Drug-Drug Interactions.

The cytochrome P450 enzymes (CYPs), a group of heme-containing enzymes, catalyze oxidative metabolism of a wide range of drugs and xenobiotics, as well as different endogenous molecules. Strong inhibition of human CYPs is the most common cause of clinically associated pharmacokinetic drug-drug/herb-drug interactions (DDIs/HDIs), which may result in serious adverse drug reactions, even toxicity. Accurate and rapid assessing of the inhibition potentials on CYP activities for therapeutic agents is crucial for the prediction of clinically relevant DDIs/HDIs. Over the past few decades, significant efforts have been invested into developing optical substrates for the human CYPs, generating a variety of powerful tools for high-throughput assays to detect CYP activities in biologic specimens and for screening of CYP inhibitors. This minireview focuses on recent advances in optical substrates developments for human CYPs, as well as their applications in screening CYP inhibitors and DDIs/HDIs studies. The examples for rational design and optimization of highly specific optical substrates for the target CYP enzyme, as well as applications in investigating CYP-mediated DDIs, are illustrated. Finally, the challenges and future perspectives in this field are proposed. Collectively, this review summarizes the reported optical-based biochemical assays for highly efficient CYP activities detection, which strongly facilitated the discovery of CYP inhibitors and the investigations on CYP-mediated DDIs. SIGNIFICANCE STATEMENT: Optical substrates for cytochrome P450 enzymes (CYPs) have emerged as powerful tools for the construction of high-throughput assays for screening of CYP inhibitors. This mini-review covers the advances and challenges in the development of highly specific optical substrates for sensing human CYP isoenzymes, as well as their applications in constructing fluorescence-based high-throughput assays for investigating CYP-mediated drug-drug interactions.

Optical substrates for drug-metabolizing enzymes: Recent advances and future perspectives.

Drug-metabolizing enzymes (DMEs), a diverse group of enzymes responsible for the metabolic elimination of drugs and other xenobiotics, have been recognized as the critical determinants to drug safety and efficacy. Deciphering and understanding the key roles of individual DMEs in drug metabolism and toxicity, as well as characterizing the interactions of central DMEs with xenobiotics require reliable, practical and highly specific tools for sensing the activities of these enzymes in biological systems. In the last few decades, the scientists have developed a variety of optical substrates for sensing human DMEs, parts of them have been successfully used for studying target enzyme(s) in tissue preparations and living systems. Herein, molecular design principals and recent advances in the development and applications of optical substrates for human DMEs have been reviewed systematically. Furthermore, the challenges and future perspectives in this field are also highlighted. The presented information offers a group of practical approaches and imaging tools for sensing DMEs activities in complex biological systems, which strongly facilitates high-throughput screening the modulators of target DMEs and studies on drug/herb‒drug interactions, as well as promotes the fundamental researches for exploring the relevance of DMEs to human diseases and drug treatment outcomes.

A broad-spectrum substrate for the human UDP-glucuronosyltransferases and its use for investigating glucuronidation inhibitors.

Strong inhibition of the human UDP-glucuronosyltransferase enzymes (UGTs) may lead to undesirable effects, including hyperbilirubinaemia and drug/herb-drug interactions. Currently, there is no good way to examine the inhibitory effects and specificities of compounds toward all the important human UGTs, side-by-side and under identical conditions. Herein, we report a new, broad-spectrum substrate for human UGTs and its uses in screening and characterizing of UGT inhibitors. Following screening a variety of phenolic compound(s), we have found that methylophiopogonanone A (MOA) can be readily O-glucuronidated by all tested human UGTs, including the typical N-glucuronidating enzymes UGT1A4 and UGT2B10. MOA-O-glucuronidation yielded a single mono-O-glucuronide that was biosynthesized and purified for structural characterization and for constructing an LC-UV based MOA-O-glucuronidation activity assay, which was then used for investigating MOA-O-glucuronidation kinetics in recombinant human UGTs. The derived Km values were crucial for selecting the most suitable assay conditions for assessing inhibitory potentials and specificity of test compound(s). Furthermore, the inhibitory effects and specificities of four known UGT inhibitors were reinvestigated by using MOA as the substrate for all tested UGTs. Collectively, MOA is a broad-spectrum substrate for the human UGTs, which offers a new and practical tool for assessing inhibitory effects and specificities of UGT inhibitors.

A fluorescence-based microplate assay for high-throughput screening and evaluation of human UGT inhibitors.

Human UDP-glucuronosyltransferase enzymes (hUGTs), one of the most important classes of conjugative enzymes, are responsible for the glucuronidation and detoxification of a variety of endogenous substances and xenobiotics. Inhibition of hUGTs may cause undesirable effects or adverse drug-drug interactions (DDI) via modulating the glucuronidation rates of endogenous toxins or the drugs that are primarily conjugated by the inhibited hUGTs. Herein, to screen hUGTs inhibitors in a more efficient way, a novel fluorescence-based microplate assay has been developed by utilizing a fluorogenic substrate. Following screening of series of 4-hydroxy-1,8-naphthalimide derivatives, we found that 4-HN-335 is a particularly good substrate for a panel of hUGTs. Under physiological conditions, 4-HN-335 can be readily O-glucuronidated by ten hUGTs, such reactions generate a single O-glucuronide with a high quantum yield (Ф = 0.79) and bring remarkable changes in fluorescence emission. Subsequently, a fluorescence-based microplate assay is developed to simultaneously measure the inhibitory effects of selected compound(s) on ten hUGTs. The newly developed fluorescence-based microplate assay is time- and cost-saving, easy to manage and can be adapted for 96-well microplate format with the Z-factor of 0.92. We further demonstrate the utility of the fluorescence-based assay for high-throughput screening of two compound libraries, resulting in the identification of several potent UGT inhibitors, including natural products and FDA-approved drugs. Collectively, this study reports a novel fluorescence-based microplate assay for simultaneously sensing the residual activities of ten hUGTs, which strongly facilitates the identification and characterization of UGT inhibitors from drugs or herbal constituents and the investigations on UGT-mediated DDI.

An ultra-sensitive and easy-to-use assay for sensing human UGT1A1 activities in biological systems.

The human UDP-glucuronosyltransferase 1A1 (UGT1A1), one of the most essential conjugative enzymes, is responsible for the metabolism and detoxification of bilirubin and other endogenous substances, as well as many different xenobiotic compounds. Deciphering UGT1A1 relevance to human diseases and characterizing the effects of small molecules on the activities of UGT1A1 requires reliable tools for probing the function of this key enzyme in complex biological matrices. Herein, an easy-to-use assay for highly-selective and sensitive monitoring of UGT1A1 activities in various biological matrices, using liquid chromatography with fluorescence detection (LC-FD), has been developed and validated. The newly developed LC-FD based assay has been confirmed in terms of sensitivity, specificity, precision, quantitative linear range and stability. One of its main advantages is lowering the limits of detection and quantification by about 100-fold in comparison to the previous assay that used the same probe substrate, enabling reliable quantification of lower amounts of active enzyme than any other method. The precision test demonstrated that both intra- and inter-day variations for this assay were less than 5.5%. Furthermore, the newly developed assay has also been successfully used to screen and characterize the regulatory effects of small molecules on the expression level of UGT1A1 in living cells. Overall, an easy-to-use LC-FD based assay has been developed for ultra-sensitive UGT1A1 activities measurements in various biological systems, providing an inexpensive and practical approach for exploring the role of UGT1A1 in human diseases, interactions with xenobiotics, and characterization modulatory effects of small molecules on this conjugative enzyme.

Human efflux transport of testosterone, epitestosterone and other androgen glucuronides.

Several drug-metabolizing enzymes are known to control androgen homeostasis in humans. UDP-glucuronosyltransferases convert androgens to glucuronide conjugates in the liver and intestine, which enables subsequent elimination of these conjugated androgens via urine. The most important androgen is testosterone, while others are the testosterone metabolites androsterone and etiocholanolone, and the testosterone precursor dehydroepiandrosterone. Epitestosterone is another endogenous androgen, which is included as a crucial marker in urine doping tests. Since glucuronide conjugates are hydrophilic, efflux transporters mediate their excretion from tissues. In this study, we employed the membrane vesicle assay to identify the efflux transporters for glucuronides of androsterone, dehydroepiandrosterone, epitestosterone, etiocholanolone and testosterone. The human hepatic and intestinal transporters MRP2 (ABCC2), MRP3 (ABCC3), MRP4 (ABCC4), BCRP (ABCG2) and MDR1 (ABCB1) were studied in vitro. Of these transporters, only MRP2 and MRP3 transported the androgen glucuronides investigated. In kinetic analyses, MRP3 transported glucuronides of androsterone, epitestosterone and etiocholanolone at low Km values, between 0.4 and 4 µM, while the Km values for glucuronides of testosterone and dehydroepiandrosterone were 14 and 51 µM, respectively. MRP2 transported the glucuronides at lower affinity, as indicated by Km values over 100 µM. Interestingly, the MRP2-mediated transport of androsterone and epitestosterone glucuronides was best described by sigmoidal kinetics. The inability of BCRP to transport any of the androgen glucuronides investigated is drastically different from its highly active transport of several estrogen conjugates. Our results explain the transporter-mediated disposition of androgen glucuronides in humans, and shed light on differences between the human efflux transporters MRP2, MRP3, MRP4, BCRP and MDR1.

In vitro glucuronidation of 7-hydroxycoumarin derivatives in intestine and liver microsomes of Beagle dogs.

Beagle dog is a standard animal model for evaluating nonclinical pharmacokinetics of new drug candidates. Glucuronidation in intestine and liver is an important first-pass drug metabolic pathway, especially for phenolic compounds. This study evaluated the glucuronidation characteristics of several 7-hydroxycoumarin derivatives in beagle dog's intestine and liver in vitro. To this end, glucuronidation rates of 7-hydroxycoumarin (compound 1), 7-hydroxy-4-trifluoromethylcoumarin (2), 6-methoxy-7-hydroxycoumarin (3), 7-hydroxy-3-(4-tolyl)coumarin (4), 3-(4-fluorophenyl)coumarin (5), 7-hydroxy-3-(4-hydroxyphenyl)coumarin (6), 7-hydroxy-3-(4-methoxyphenyl)coumarin (7), and 7-hydroxy-3-(1H-1,2,4-tirazole)coumarin (8) were determined in dog's intestine and liver microsomes, as well as recombinant dog UGT1A enzymes. The glucuronidation rates of 1, 2 and 3 were 3-10 times higher in liver than in small intestine microsomes, whereas glucuronidation rates of 5, 6, 7 and 8 were similar in microsomes from both tissues. In the colon, glucuronidation of 1 and 2 was 3-5 times faster than in small intestine. dUGT1A11 glucuronidated efficiently all the substrates and was more efficient catalyst for 8 than any other dUGT1A. Other active enzymes were dUGT1A2 that glucuronidated efficiently 2, 3, 4, 5, 6 and 7, while dUGT1A10 glucuronidated efficiently 1, 2, 3, 4, 5 and 7. Kinetic analyses revealed that the compounds' Km values varied between 1.1 (dUGT1A10 and 2) and 250 µM (dUGT1A7 and 4). The results further strengthen the concept that dog intestine has high capacity for glucuronidation, and that different dUGT1As mediate glucuronidation with distinct substrates selectivity in dog and human.

Neobavaisoflavone Induces Bilirubin Metabolizing Enzyme UGT1A1 via PPARα and PPARγ.

UDP-glucuronosyltransferase 1A1 (UGT1A1) is an essential enzyme in mammals that is responsible for detoxification and metabolic clearance of the endogenous toxin bilirubin and a variety of xenobiotics, including some crucial therapeutic drugs. Discovery of potent and safe UGT1A1 inducers will provide an alternative therapy for ameliorating hyperbilirubinaemia and drug-induced hepatoxicity. This study aims to find efficacious UGT1A1 inducer(s) from natural flavonoids, and to reveal the mechanism involved in up-regulating of this key conjugative enzyme by the flavonoid(s) with strong UGT1A1 induction activity. Among all the tested flavonoids, neobavaisoflavone (NBIF) displayed the most potent UGT1A1 induction activity, while its inductive effects were confirmed by both western blot and glucuronidation activity assays. A panel of nuclear receptor reporter assays demonstrated that NBIF activated PPARα and PPARγ in a dose-dependent manner. Meanwhile, we also found that NBIF could up-regulate the expression of PPARα and PPARγ in hepatic cells, suggesting that the induction of UGT1A1 by NBIF was mainly mediated by PPARs. In silico simulations showed that NBIF could stably bind on pocket II of PPARα and PPARγ. Collectively, our results demonstrated that NBIF is a natural inducer of UGT1A1, while this agent induced UGT1A1 mainly via activating and up-regulating PPARα and PPARγ. These findings suggested that NBIF can be used as a promising lead compound for the development of more efficacious UGT1A1 inducers to treat hyperbilirubinaemia and UGT1A1-associated drug toxicities.

Characterization of human UGT2A3 expression using a prepared specific antibody against UGT2A3.

UDP-Glucuronosyltransferase (UGT) 2A3 belongs to a UGT superfamily of phase II drug-metabolizing enzymes that catalyzes the glucuronidation of many endobiotics and xenobiotics. Previous studies have demonstrated that UGT2A3 is expressed in the human liver, small intestine, and kidney at the mRNA level; however, its protein expression has not been determined. Evaluation of the protein expression of UGT2A3 would be useful to determine its role at the tissue level. In this study, we prepared a specific antibody against human UGT2A3 and evaluated the relative expression of UGT2A3 in the human liver, small intestine, and kidney. Western blot analysis indicated that this antibody is specific to UGT2A3 because it did not cross-react with other human UGT isoforms or rodent UGTs. UGT2A3 expression in the human small intestine was higher than that in the liver and kidney. Via treatment with endoglycosidase, it was clearly demonstrated that UGT2A3 was N-glycosylated. UGT2A3 protein levels were significantly correlated with UGT2A3 mRNA levels in a panel of 28 human liver samples (r = 0.64, p < 0.001). In conclusion, we successfully prepared a specific antibody against UGT2A3. This antibody would be useful to evaluate the physiological, pharmacological, and toxicological roles of UGT2A3 in human tissues.

Efflux transport of nicotine, cotinine and trans-3'-hydroxycotinine glucuronides by human hepatic transporters.

Nicotine is the addiction causing alkaloid in tobacco, and it is used in smoking cessation therapies. Although the metabolic pathways of nicotine are well known and mainly occur in the liver, the transport of nicotine and its metabolites is poorly characterized. The highly hydrophilic nature and urinary excretion of nicotine glucuronide metabolites indicate that hepatic basolateral efflux transporters mediate their excretion. We aimed here to find the transporters responsible for the hepatic excretion of nicotine, cotinine and trans-3'-hydroxycotinine (OH-cotinine) glucuronides. To this end, we tested their transport by multidrug resistance-associated proteins 1 (MRP1, ABCC1) and MRP3-6 (ABCC3-6), which are located on the basolateral membranes of hepatocytes, as well as MRP2 (ABCC2), breast cancer resistance protein (BCRP, ABCG2) and multidrug resistance protein 1 (MDR1, P-gp, ABCB1) that are expressed in the apical membranes of these cells. ATP-dependent transport of these glucuronides was evaluated in inside-out membrane vesicles expressing the transporter of interest. In addition, potential interactions of both the glucuronides and parent compounds with selected transporters were tested by inhibition assays. Considerable ATP-dependent transport was observed only for OH-cotinine glucuronide by MRP3. The kinetics of this transport activity was characterized, resulting in an estimated Km value of 895 µmol/L. No significant transport was found for nicotine or cotinine glucuronides by any of the tested transporters at either 5 or 50 µmol/L substrate concentration. Furthermore, neither nicotine, cotinine nor OH-cotinine inhibited MRP2-4, BCRP or MDR1. In this study, we directly examined, for the first time, efflux transport of the three hydrophilic nicotine glucuronide metabolites by the major human hepatic efflux transporters. Despite multiple transporters studied here, our results indicate that an unknown transporter may be responsible for the hepatic excretion of nicotine and cotinine glucuronides.

Inhibition of human carboxylesterases by magnolol: Kinetic analyses and mechanism.

Magnolol, the most abundant bioactive constituent of the Chinese herb Magnolia officinalis, has been found with multiple biological activities, including anti-oxidative, anti-inflammatory and enzyme-regulatory activities. In this study, the inhibitory effects and inhibition mechanism of magnolol on human carboxylesterases (hCEs), the key enzymes responsible for the hydrolytic metabolism of a variety of endogenous esters as well as ester-bearing drugs, have been well-investigated. The results demonstrate that magnolol strongly inhibits hCE1-mediated hydrolysis of various substrates, whereas the inhibition of hCE2 by magnolol is substrate-dependent, ranging from strong to moderate. Inhibition of intracellular hCE1 and hCE2 by magnolol was also investigated in living HepG2 cells, and the results showed that magnolol could strongly inhibit intracellular hCE1, while the inhibition of intracellular hCE2 was weak. Inhibition kinetic analyses and docking simulations revealed that magnolol inhibited both hCE1 and hCE2 in a mixed manner, which could be partially attributed to its binding at two distinct ligand-binding sites in each carboxylesterase, including the catalytic cavity and the regulatory domain. In addition, the potential risk of the metabolic interactions of magnolol via hCE1 inhibition was predicted on the basis of a series of available pharmacokinetic data and the inhibition constants. All these findings are very helpful in deciphering the metabolic interactions between magnolol and hCEs, and also very useful for avoiding deleterious interactions via inhibition of hCEs.

Recent progress and challenges in screening and characterization of UGT1A1 inhibitors.

Uridine-diphosphate glucuronosyltransferase 1A1 (UGT1A1) is an important conjugative enzyme in mammals that is responsible for the conjugation and detoxification of both endogenous and xenobiotic compounds. Strong inhibition of UGT1A1 may trigger adverse drug/herb-drug interactions, or result in metabolic disorders of endobiotic metabolism. Therefore, both the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) have recommended assaying the inhibitory potential of drugs under development on the human UGT1A1 prior to approval. This review focuses on the significance, progress and challenges in discovery and characterization of UGT1A1 inhibitors. Recent advances in the development of UGT1A1 probes and their application for screening UGT1A1 inhibitors are summarized and discussed in this review for the first time. Furthermore, a long list of UGT1A1 inhibitors, including information on their inhibition potency, inhibition mode, and affinity, has been prepared and analyzed. Challenges and future directions in this field are highlighted in the final section. The information and knowledge that are presented in this review provide guidance for rational use of drugs/herbs in order to avoid the occurrence of adverse effects via UGT1A1 inhibition, as well as presenting methods for rapid screening and characterization of UGT1A1 inhibitors and for facilitating investigations on UGT1A1-ligand interactions.

Data on biosynthesis of BPAF glucuronide, enzyme kinetics of BPAF glucuronidation, and molecular modeling.

Bisphenol AF (BPAF) is in the body mainly metabolized to the corresponding bisphenol AF glucuronide (BPAF-G). While BPAF-G is not commercially available, enzyme-assisted synthesis of BPAF-G using the human recombinant enzyme UGT2A1, purification of BPAF-G by solid phase extraction and semi-preparative HPLC and chemical characterization of BPAF-G by NMR and LC-MS/MS were performed and are described here. Furthermore, BPAF glucuronidation kinetics with the UGT enzymes that showed the highest glucuronidation activity in previous studies (i.e hepatic UGTs 1A3, 2B7, and 2B17, intestinal UGT 1A10 and UGT 2A1 that is present in airways) was performed and data is presented. Hepatic enzymes exhibited high affinities toward BPAF, while extrahepatic UGTs 2A1 and 1A10 showed the high vmax values (3.3 and 3.0 nmol/min/mg, respectively). To understand molecular interactions of BPA, BPAF and BPAF-G with ligand biding sites of several nuclear receptors, molecular modeling was performed and data on the binding modes of BPAF, BPA, and BPAF-G in the ligand-binding sites of nuclear receptors are presented. This article is related to "Endocrine activities and adipogenic effects of bisphenol AF and its main metabolite" (Skledar et al., 2019).

Metabolism of Scoparone in Experimental Animals and Humans.

Scoparone, a major constituent of the Chinese herbal medicine Yin Chen Hao, expresses beneficial effects in experimental models of various diseases. The intrinsic doses and effects of scoparone are dependent on its metabolism, both in humans and animals. We evaluated in detail the metabolism of scoparone in human, mouse, rat, pig, dog, and rabbit liver microsomes in vitro and in humans in vivo. Oxidation of scoparone to isoscopoletin via 6-O-demethylation was the major metabolic pathway in liver microsomes from humans, mouse, rat, pig and dog, whereas 7-O-demethylation to scopoletin was the main reaction in rabbit. The scoparone oxidation rates in liver microsomes were 0.8 - 1.2 µmol/(min*g protein) in mouse, pig, and rabbit, 0.2 - 0.4 µmol/(min*g protein) in man and dog, and less than 0.1 µmol/(min*g) in rat. In liver microsomes of all species, isoscopoletin was oxidized to 3-[4-methoxy-ρ-(3, 6)-benzoquinone]-2-propenoate and esculetin, which was formed also in the oxidation of scopoletin. Human CYP2A13 exhibited the highest rate of isoscopoletin and scopoletin oxidation, followed by CYP1A1 and CYP1A2. Glucuronidation of isoscopoletin and scopoletin was catalyzed by the human UGT1A1, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, and UGT2B17. Dog was most similar to man in scoparone metabolism. Isoscopoletin glucuronide and sulfate conjugates were the major scoparone in vivo metabolites in humans, and they were completely excreted within 24 h in urine. Scoparone and its metabolites did not activate key nuclear receptors regulating CYP and UGT enzymes. These results outline comprehensively the metabolic pathways of scoparone in man and key preclinical animal species.

Inhibition of UGT1A1 by natural and synthetic flavonoids.

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.

Endocrine activities and adipogenic effects of bisphenol AF and its main metabolite.

Bisphenol AF (BPAF) is a fluorinated analog of bisphenol A (BPA), and it is a more potent estrogen receptor (ER) agonist. BPAF is mainly metabolized to BPAF-glucuronide (BPAF-G), which has been reported to lack ER agonist activity and is believed to be biologically inactive. The main goal of the current study was to examine the influence of the metabolism of BPAF via glucuronidation on its ER activity and adipogenesis. Also, as metabolites can have different biological activities, the effects of BPAF-G on other nuclear receptors were evaluated. First, in-vitro BPAF glucuronidation was investigated using recombinant human enzymes. Specific reporter-gene assays were used to determine BPAF and BPAF-G effects on estrogen, androgen, glucocorticoid, and thyroid receptor pathways, and on PXR, FXR, and PPARγ pathways. Their effects on lipid accumulation and differentiation were determined in murine 3T3L1 preadipocytes using Nile Red, with mRNA expression analysis of the adipogenic markers adiponectin, Fabp4, Cebpα, and PPARγ. BPAF showed strong agonistic activity for hERα and moderate antagonistic activities for androgen and thyroid receptors, and for PXR. BPAF-G was antagonistic for PXR and PPARγ. BPAF (0.1 µM) and BPAF-G (1.0 µM) induced lipid accumulation and increased expression of key adipogenic markers in murine preadipocytes. BPAF-G is therefore not an inactive metabolite of BPAF. Further toxicological and epidemiological investigations of BPAF effects on human health are warranted, to provide better understanding of the metabolic end-elimination of BPAF.

A Novel Pathogenic UGT1A1 Variant in a Sudanese Child with Type 1 Crigler-Najjar Syndrome.

Uridine diphosphate glucuronosyltransferases (UGTs) are key enzymes responsible for the body's ability to process a variety of endogenous and exogenous compounds. Significant gains in understanding UGT function have come from the analysis of variants seen in patients. We cared for a Sudanese child who showed clinical features of type 1 Crigler-Najjar syndrome (CN-1), namely severe unconjugated hyperbilirubinemia leading to liver transplantation. CN-1 is an autosomal recessive disorder caused by damaging mutations in the gene for UGT1A1, the hepatic enzyme responsible for bilirubin conjugation in humans. Clinical genetic testing was unable to identify a known pathogenic UGT1A1 mutation in this child. Instead, a novel homozygous variant resulting in an in-frame deletion, p.Val275del, was noted. Sanger sequencing demonstrated that this variant segregated with the disease phenotype in this family. We further performed functional testing using recombinantly expressed UGT1A1 with and without the patient variant, demonstrating that p.Val275del results in a complete lack of glucuronidation activity, a hallmark of CN-1. Sequence analysis of this region shows a high degree of conservation across all known catalytically active human UGTs, further suggesting that it plays a key role in the enzymatic function of UGTs. Finally, we note that the patient's ethnicity likely played a role in his variant being previously undescribed and advocate for greater diversity and inclusion in genomic medicine.

Molecular Docking-Based Design and Development of a Highly Selective Probe Substrate for UDP-glucuronosyltransferase 1A10.

Intestinal and hepatic glucuronidation by the UDP-glucuronosyltransferases (UGTs) greatly affect the bioavailability of phenolic compounds. UGT1A10 catalyzes glucuronidation reactions in the intestine, but not in the liver. Here, our aim was to develop selective, fluorescent substrates to easily elucidate UGT1A10 function. To this end, homology models were constructed and used to design new substrates, and subsequently, six novel C3-substituted (4-fluorophenyl, 4-hydroxyphenyl, 4-methoxyphenyl, 4-(dimethylamino)phenyl, 4-methylphenyl, or triazole) 7-hydroxycoumarin derivatives were synthesized from inexpensive starting materials. All tested compounds could be glucuronidated to nonfluorescent glucuronides by UGT1A10, four of them highly selectively by this enzyme. A new UGT1A10 mutant, 1A10-H210M, was prepared on the basis of the newly constructed model. Glucuronidation kinetics of the new compounds, in both wild-type and mutant UGT1A10 enzymes, revealed variable effects of the mutation. All six new C3-substituted 7-hydroxycoumarins were glucuronidated faster by human intestine than by liver microsomes, supporting the results obtained with recombinant UGTs. The most selective 4-(dimethylamino)phenyl and triazole C3-substituted 7-hydroxycoumarins could be very useful substrates in studying the function and expression of the human UGT1A10.