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Uridine-disphosphate glucuronosyltransferase 1A9 (UGT1A9), an important detoxification and inactivation enzyme for toxicants, regulates the exposure level of environmental pollutants in the human body and induces various toxicological consequences. However, an effective tool for high-throughput monitoring of UGT1A9 function under exposure to environmental pollutants is still lacking. In this study, 1,3-dichloro-7-hydroxy-9,9-dimethylacridin-2(9H)-one (DDAO) was found to exhibit excellent specificity and high affinity towards human UGT1A9. Remarkable changes in absorption and fluorescence signals after reacting with UGT1A9 were observed, due to the intramolecular charge transfer (ICT) mechanism. Importantly, DDAO was successfully applied to monitor the biological functions of UGT1A9 in response to environmental pollutant exposure not only in microsome samples, but also in living cells by using a high-throughput screening method. Meanwhile, the identified pollutants that disturb UGT1A9 functions were found to significantly influence the exposure level and retention time of bisphenol S/bisphenol A in living cells. Furthermore, the molecular mechanism underlying the inhibition of UGT1A9 by these pollutant-derived disruptors was elucidated by molecular docking and molecular dynamics simulations. Collectively, a fluorescent probe to characterize the responses of UGT1A9 towards environmental pollutants was developed, which was beneficial for elucidating the health hazards of environmental pollutants from a new perspective., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This study aimed to characterize the glucuronidation pathway of licochalcone A (LCA) in human liver microsomes (HLM). HLM incubation systems were employed to catalyze the formation of LCA glucuronide. The glucuronidation activity of commercially recombinant UDP-glucuronosyltransferase (UGT) isoforms toward LCA was screened. Kinetic analysis was used to identify the UGT isoforms involved in the glucuronidation of LCA in HLM. LCA could be metabolized to two monoglucuronides in HLM, including a major monoglucuronide, namely, 4-O-glucuronide, and a minor monoglucuronide, namely, 4'-O-glucuronide. Species-dependent differences were observed among the glucuronidation profiles of LCA in liver microsomes from different species. UGT1A1, UGT1A3, UGT1A7, UGT1A8, UGT1A9, UGT1A10 and UGT2B7 participated in the formation of 4-O-glucuronide, with UGT1A9 exhibiting the highest catalytic activity in this biotransformation. Only UGT1A1 and UGT1A3 were involved in the formation of 4'-O-glucuronide, exhibiting similar reaction rates. Kinetic analysis demonstrated that UGT1A9 was the major contributor to LCA-4-O-glucuronidation, while UGT1A1 played important roles in the formation of both LCA-4-O- and 4'-O-glucuronide. UGT1A9 was the major contributor to the formation of LCA-4-O-glucuronide, while UGT1A1 played important roles in both LCA-4-O- and 4'-O-glucuronidation.

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., (Copyright © 2018. Published by Elsevier B.V.)

UGTs play crucial roles in the metabolism and detoxification of both endogenous and xenobiotic compounds. The key roles of UGTs in human health have garnered great interest in the design and development of specific probes for human UGTs. However, in contrast to other human enzymes, the probe substrates for human UGTs are rarely reported, owing to the highly overlapping substrate specificities of UGTs and the lack of the integrated crystal structures of UGTs. Over the past decades, many efforts are made to develop specific probe substrates for UGTs and use them in both basic research and drug discovery. This review focuses on recent progress in the development of probe substrates for UGTs and their biomedical applications. A long list of chemical probes for UGTs, including non-fluorescent and fluorescent probes along with their structural information and kinetic parameters, are prepared and analyzed. Additionally, challenges and future directions in this field are highlighted in the final section. All information and knowledge presented in this review provide practical tools/methods for measuring UGT activities in complex biological samples, which will be very helpful for rapid screening and characterization of UGT modulators, and for exploring the relevance of UGT enzymes to human diseases., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Amentoflavone (AMF), an abundant natural biflavonoid found in many medicinal plants, displays various beneficial effects including anti-inflammatory, anti-oxidative and anti-cancer. Despite the extensive studies on pharmacological activities, the toxicity or undesirable effects of AMF are rarely reported. In this study, the inhibitory effects of AMF on human UDP-glucuronosyltransferases (UGTs) were carefully investigated. AMF displayed strong inhibition towards most of human UGTs including UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4 and 2B17, with the IC 50 values ranging from 0.12 μM to 16.81 μM. Inhibition constants (K i ) of AMF against various human UGTs varied from 0.29 μM to 11.51 μM. Further investigation demonstrated that AMF was a noncompetitive inhibitor of UGT1A1 mediated NCHN-O-glucuronidation but functioned as a competitive inhibitor of UGT1A1 mediated 4-MU-O-glucuronidation. In addition, AMF was a competitive inhibitor of UGT1A4 mediated TFP-N-glucuronidation in both UGT1A4 and human liver microsomes, while functioned as a competitive inhibitor of UGT1A9 mediated propofol or 4-MU-O-glucuronidation. These findings demonstrated that AMF was a strong and broad-spectrum natural inhibitor of most human UGTs, which might bring potential risks of herb-drug interactions (HDIs) via UGT inhibition. Additionally, this study provided novel insights into the underlying mechanism of AMF-associated toxicity from the perspective of UGT inhibition., (Copyright © 2018 Elsevier B.V. All rights reserved.)

1. Xanthotoxol is a furanocoumarin that possesses many pharmacological activities and in this study its in vitro glucuronidation was studied. 2. Xanthotoxol can be rapidly metabolized to a mono-glucuronide in both human intestine microsomes (HIM) and human liver microsomes (HLM); the structure of the metabolite was confirmed by NMR spectroscopy. 3. Reaction phenotyping with 12 commercial recombinant human UGTs, as well as with the Helsinki laboratory UGT1A10 that carry a C-terminal His-tag (UGT1A10-H), revealed that UGT1A10-H catalyzes xanthotoxol glucuronidation at the highest rate, followed by UGT1A8. The other enzymes, namely UGT1A3, UGT1A1, UGT1A6, UGT1A10 (commercial), and UGT2B7 displayed moderate-to-low reaction rates. 4. In kinetic analyses, HIM exhibited much higher affinity for xanthotoxol, along with high V max and mild substrate inhibition, whereas the kinetics in HLM was biphasic. UGT1A1 (high K m value), UGT1A10-H (low K m value), and UGT1A8 exhibited mild substrate inhibition. 5. Considering the above findings and the current knowledge on UGTs expression in HIM, it is likely that UGT1A10 is mainly responsible for xanthotoxol glucuronidation in the human small intestine, with some contribution from UGT1A1. In the liver, this reaction is mainly catalyzed by UGT1A1 and UGT2B7. 6. Glucuronidation appears to be the major metabolic pathway of xanthotoxol in human.

This study aimed to develop a practical and high-affinity fluorescent probe for uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1), a key conjugative enzyme responsible for the elimination and detoxification of many potentially harmful compounds. Several substrates derived from N-butyl-4-phenyl-1,8-naphthalimide were designed and synthesized on the basis of the substrate preference of UGT1A1 and the principle of photoinduced electron transfer (PET). Following the preliminary screening, substrate 2 was found with a high specificity and high affinity toward UGT1A1, while such biotransformation brought remarkable changes in fluorescence emission. Both inhibition kinetic analyses and molecular docking simulations demonstrated that 2 could bind on UGT1A1 at the same ligand-binding site as bilirubin. Furthermore, this newly developed probe was successfully used for sensing UGT1A1 activities and the high-throughput screening of UGT1A1 modulators in complex biological samples. In conclusion, a practical and high-affinity fluorescent probe for UGT1A1 was designed and well-characterized, which could serve as a good surrogate for bilirubin to investigate UGT1A1-ligand interactions.

Tolcapone and entacapone are two potent catechol-O-methyltransferase (COMT) inhibitors with a similar skeleton and displaying similar pharmacological activities. However, entacapone is a very safe drug used widely in the treatment of Parkinson's disease, while tolcapone is only in limited use for Parkinson's patients and needs careful monitoring of hepatic functions due to hepatotoxicity. This study aims to investigate and compare the inhibitory effects of entacapone and tolcapone on human UDP-glucosyltransferases (UGTs), as well as to evaluate the potential risks from the view of drug-drug interactions (DDI). The results demonstrated that both tolcapone and entacapone exhibited inhibitory effects on UGT1A1, UGT1A7, UGT1A9 and UGT1A10. In contrast to entacapone, tolcapone exhibited more potent inhibitory effects on UGT1A1, UGT1A7, and UGT1A10, while their inhibitory potentials against UGT1A9 were comparable. It is noteworthy that the inhibition constants (Ki) of tolcapone and entacapone against bilirubin-O-glucuronidation in human liver microsomes (HLM) are determined as 0.68μM and 30.82μM, respectively, which means that the inhibition potency of tolcapone on UGT1A1 mediated bilirubin-O-glucuronidation in HLM is much higher than that of entacapone. Furthermore, the potential risks of tolcapone or entacapone via inhibition of human UGT1A1 were quantitatively predicted by the ratio of the areas under the plasma drug concentration-time curve (AUC). The results indicate that tolcapone may result in significant increase in AUC of bilirubin or the drugs primarily metabolized by UGT1A1, while entacapone is unlikely to cause a significant DDI through inhibition of UGT1A1., (Copyright © 2016 Elsevier Inc. All rights reserved.)

As an edible traditional Chinese herb, Fructus psoraleae (FP) has been widely used in Asia for the treatment of vitiligo, bone fracture and osteoporosis. Several cases on markedly elevated bilirubin and acute liver injury following administration of FP and its related proprietary medicine have been reported, but the mechanism in FP-associated toxicity has not been well investigated yet. This study aimed to investigate the inhibitory effects of FP extract and its major constituents against human UDP-glucuronosyltransferase 1A1 (UGT1A1), the key enzyme responsible for metabolic elimination of bilirubin. To this end, N-(3-carboxy propyl)-4-hydroxy-1,8-naphthalimide (NCHN), a newly developed specific fluorescent probe for UGT1A1, was used to evaluate the inhibitory effects of FP extract or its fractions in human liver microsomes (HLM), while LC-UV fingerprint and UGT1A1 inhibition profile were combined to identity and characterize the naturally occurring inhibitors of UGT1A1 in FP. Our results demonstrated that both the extract of FP and five major components of FP displayed evident inhibitory effects on UGT1A1 in HLM. Among these five identified naturally occurring inhibitors, bavachin and corylifol A were found to be strong inhibitors of UGT1A1 with the inhibition kinetic parameters (Ki) values lower than 1 μM, while neobavaisoflavone, isobavachalcone, and bavachinin displayed moderate inhibitory effects against UGT1A1 in HLM, with the Ki values ranging from 1.61 to 9.86μM. These findings suggested that FP contains natural compounds with potent inhibitory effects against human UGT1A1, which may be one of the important reasons for triggering FP-associated toxicity, including elevated bilirubin levels and liver injury., (Copyright © 2015 Elsevier Inc. All rights reserved.)

This study aimed to develop a practical ratiometric fluorescent probe for highly selective and sensitive detection of human UDP-glucuronosyltransferase 1A1 (UGT1A1), one of the most important phase II enzymes. 4-Hydroxy-1,8-naphthalimide (HN) was selected as the fluorophore for this study because it possesses intramolecular charge transfer (ICT) feature and displays outstanding optical properties. A series of N-substituted derivatives with various hydrophobic, acidic and basic groups were designed and synthesized to evaluate the selectivity of HN derivatives toward UGT1A1. Our results demonstrated that the introduction of an acidic group to HN could significantly improve the selectivity of UGT1A1. Among the synthesized fluorescent probes, NCHN (N-3-carboxy propyl-4-hydroxy-1,8-naphthalimide) displayed the best combination of selectivity, sensitivity and ratiometric fluorescence response following UGT1A1-catalyzed glucuronidation. UGT1A1-catalyzed NCHN-4-O-glucuronidation generated a single fluorescent product with a high quantum yield (Φ=0.688) and brought remarkable changes in both color and fluorescence in comparison with the parental substrate. The newly developed probe has been successfully applied for sensitive measurements of UGT1A1 activities in human liver preparations, as well as for rapid screening of UGT1A1 modulators, using variable enzyme sources. Furthermore, its potential applications for live imaging of endogenous UGT1A1in cells have also been demonstrated., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Bavachinin (BCI), a major bioactive compound in Chinese herbal Psoralea corylifolia, possesses a wide range of biological activities. In this study, the glucuronidation pathway of BCI was characterized for the first time, by using pooled human liver microsomes (HLM), pooled human intestine microsomes (HIM) and recombinant human UDP-glucosyltransferases (UGTs). One mono-glucuronide was detected in HLM in the presence of uridine-diphosphate glucuronic acid (UDPGA), and it was biosynthesized and well-characterized as BCI-4'-O-glucuronide (BCIG). Reaction phenotyping assay showed that UGT1A1, UGT1A3 and UGT1A8 were involved in BCI-4'-O-glucuronidation, while UGT1A1 and UGT1A8 displayed the higher catalytic ability among all tested UGT isoforms. Kinetic analysis demonstrated that BCI-4'-O-glucuronidation in both HLM and UGT1A1 followed sigmoidal kinetic behaviors and displayed much close Km values (12.4 μM in HLM & 9.7 μM in UGT1A1). Both chemical inhibition assays and correlation analysis demonstrated that UGT1A1 displayed a predominant role in BCI-4'-O-glucuronidation in HLM. Both HIM and UGT1A8 exhibited substrate inhibition at high concentrations, and Km values of HIM and UGT1A8 were 3.6 and 2.3 μM, respectively. Similar catalytic efficiencies were observed for HIM (199.3 μL/min/mg) and UGT1A8 (216.2 μL/min/mg). These findings suggested that UGT1A1 and UGT1A8 were the primary isoforms involved in BCI-4'-O-glucuronidation in HLM, and HIM, respectively., (Copyright © 2015 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Fraxetin, a major constituent of the traditional medicine plant Fraxinus rhynchophylla Hance (Oleaceae), has been found to possess multiple bioactivities. However, the metabolic pathway(s) of fraxetin in human tissues has not been reported yet. This study aimed to characterize the glucuronidation pathway(s) of fraxetin in human tissues. Fraxetin could be metabolized to two glucuronides in human liver microsomes (HLMs). These two glucuronides were biosynthesized and characterized as 7-O-glucuronide (7-O-G) and 8-O-glucuronide (8-O-G). UGT1A1, -1A6, -1A7, -1A8, -1A9 and -1A10 participated in the formation of 7-O-G, while the formation of 8-O-G was catalyzed selectively by UGT1A6 and UGT1A9. UGT1A9 showed the highest catalytic activities in the formation of 7-O-G and 8-O-G. Both kinetic characterization and inhibition assays demonstrated that UGT1A9 played important roles in fraxetin glucuronidations in HLMs, especially in the formation of the major metabolite 8-O-G. Furthermore, the intrinsic clearance of fraxetin in both human liver microsomes and UGT1A9 was greater than that of 7,8-dihydroxylcoumarin, revealing that the addition of a C-6 methoxy group led to the higher metabolic clearance. In summary, the glucuronidation pathways of fraxetin in human liver microsomes were well-characterized, and UGT1A9 was the major isoform responsible for the glucuronidations of fraxetin.

UDP-glucuronosyltransferase 1A1 (UGT1A1) plays a key role in detoxification of many potentially harmful compounds and drugs. UGT1A1 inhibition may bring risks of drug–drug interactions (DDIs), hyperbilirubinemia and drug-induced liver injury. This study aimed to investigate and compare the inhibitory effects of icotinib and erlotinib against UGT1A1, as well as to evaluate their potential DDI risks via UGT1A1 inhibition. The results demonstrated that both icotinib and erlotinib are UGT1A1 inhibitors, but the inhibitory effect of icotinib on UGT1A1 is weaker than that of erlotinib. The IC 50 values of icotinib and erlotinib against UGT1A1-mediated NCHN- O -glucuronidation in human liver microsomes (HLMs) were 5.15 and 0.68 μmol/L, respectively. Inhibition kinetic analyses demonstrated that both icotinib and erlotinib were non-competitive inhibitors against UGT1A1-mediated glucuronidation of NCHN in HLMs, with the K i values of 8.55 and 1.23 μmol/L, respectively. Furthermore, their potential DDI risks via UGT1A1 inhibition were quantitatively predicted by the ratio of the areas under the concentration–time curve (AUC) of NCHN. These findings are helpful for the medicinal chemists to design and develop next generation tyrosine kinase inhibitors with improved safety, as well as to guide reasonable applications of icotinib and erlotinib in clinic, especially for avoiding their potential DDI risks via UGT1A1 inhibition. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • A new fluorescence-based microplate assay is developed for screening and evaluation of UGT inhibitors. • 4-HN-335 is a fluorogenic co-substrate for a panel of human UGTs with O -glucuronidation activity. • The newly developed assay is time- and cost-saving, and capable for 96-well microplate format. • The newly developed assay strongly facilitates the investigations on UGT-mediated drug-drug interactions. [ABSTRACT FROM AUTHOR]