Type 2 diabetes mellitus (T2DM) is characterized by high blood glucose levels and has emerged as an increasing public health burden worldwide. α-Glucosidases from both host and gut microbiota played important roles in digestion of carbohydrate to monosaccharides. Dual inhibition against host and gut microbial α-glucosidases hold great potentials in regulating glucose absorption of T2DM patients. This study aimed to discover a dual inhibitor of both host α-glucosidase (α-Glu) and gut microbial glucosidase (SusB) derived from Bacteroides based on bioassay-guided screening. The results demonstrated that the ethanol extract of Galla chinensis exhibited superior dual inhibition on both α-glucosidases and its major constituent 1,2,3,4,6-O-pentagalloylglucose (PGG) could remarkably inhibit α-Glu and SusB with IC50 values of 0.21 ± 0.01 μM and 13.23 ± 1.00 μM, respectively, while the IC50 values of the positive control (acarbose) on α-Glu and SusB were 430.40 ± 19.56 μM and 0.68 ± 0.05 μM, respectively. Inhibition kinetic analysis revealed that PGG functioned as a non-competitive inhibitor against α-Glu with Ki values of 0.27 ± 0.13 μM, and it acted as competitive inhibitor towards SusB with Ki values of 4.50 ± 0.64 μM. Docking simulations demonstrated that PGG could generate hydrogen bonds with allosteric sites in α-Glu and catalytic residues in SusB, respectively. The binding energies of PGG with α-Glu and SusB were - 7.5 kcal/mol and - 9.3 kcal/mol, while them for acarbose were - 8.2 kcal/mol and - 7.3 kcal/mol, respectively. These findings revealed the potential mechanism of PGG for diabetic therapy through retarding the absorption of carbohydrates.

Mycotoxins, a category of fungal secondary metabolites, frequently contaminate food products and pose a severe threat to human health. Aspergillus, a genus of fungi, is capable of producing mycotoxins, with aflatoxins (AFs) and ochratoxins being its principal types. Aspergillus mycotoxins can contaminate a wide range of crops and their derivatives, such as maize, wheat, rice, minor cereals, and peanuts, thereby threatening food and feed safety. In the paper, the related biosynthesis genes and multifaceted biosynthesis pathways of these mycotoxins are first discussed in detail, and elucidated several global regulators, including growth conditions, oxidative stress, and cell signal. Furthermore, how global shifts in temperature and water availability, driven by climate change (including rising temperatures, increased heavy rainfall frequency, prolonged droughts, and elevated carbon dioxide levels), are key determinants of Aspergillus proliferation and mycotoxin production are explored. Finally, to safeguard animal and human health from the detrimental impacts of Aspergillus mycotoxins, the effective and convenient analytical techniques and management strategies for the detection and prevention of contamination are analyzed. Overall, this review provides effective detection techniques and promising solutions to the global contamination of food with Aspergillus mycotoxins, which is of great significance to ensuring food security and protecting people's lives and health.

Gut microbial Loop-1 β-glucuronidases (gmGUS) played an important role in irinotecan-induced gastrointestinal toxicity by regulating the level of its active metabolite SN38 through enterohepatic recirculation. gmGUS inhibition has emerged as a promising approach to relieve its dose-limiting intestinal toxicity and improve its medication efficacy. This study aims to investigate the inhibitory effects and mechanisms of Platycladi cacumen and its main constituent hinokiflavone against four different types of Loop-1 gmGUS (EeGUS, SaGUS, CpGUS and EcGUS). Our results showed that the ethanol extract of Platycladi cacumen displayed strong broad-spectrum inhibition against four gmGUS, and hinokiflavone could potently inhibit EeGUS, SaGUS, CpGUS and EcGUS with IC50 values of 0.09 ± 0.01 μM, 0.44 ± 0.01 μM, 0.20 ± 0.01 μM and 0.69 ± 0.10 μM, respectively. Inhibition kinetic analyses demonstrated that hinokiflavone acted as a strong competitive inhibitor of EeGUS with Ki value of 0.13 μM, while it displayed non-competitive inhibition against SaGUS, CpGUS and EcGUS, with the Ki values of 0.43 μM, 0.33 μM and 0.76 μM, respectively. Docking simulations revealed that hinokiflavone could tightly bind with Tyr-485 and Glu-516 in catalytic sites of EeGUS, as well it created strong interactions with amino acids in loop structures of SaGUS (Asn-362), CpGUS (Phe-363, Met-364, Ala-365 and Arg-375) and EcGUS (Leu-361) to interfere the substrate entry into the catalytic pocket. Collectively, these results confirmed that hinokiflavone from Platycladi cacumen is a potent naturally occurring inhibitor of gmGUS with broad efficiency, suggesting hinokiflavone will be helpful for alleviating intestinal toxicity in irinotecan therapy.

Gut microbial ll-glucuronidases (gmll-GUS) played crucial roles in regulating a variety of endogenous substances and xenobiotics on the circulating level, thus had been recognized as key modulators of drug toxicity and human diseases. Inhibition or inactivation of gmll-GUS enzymes has become a promising therapeutic strategy to alleviate drug-induced intestinal toxicity. Herein, the Rhodiola crenulata extract (RCE) was found with potent and broadspectrum inhibition on multiple gmll-GUS enzymes. Subsequently, the anti-gmll-GUS activities of the major constituents in RCE were tested and the results showed that 1,2,3,4,6-penta-O-galloyl-ll-D-glucopyranose (PGG) acted as a strong and broad-spectrum inhibitor on multiple gmll-GUS (including EcGUS, CpGUS, SaGUS, and EeGUS). Inhibition kinetic assays demonstrated that PGG effectively inhibited four gmll-GUS in a noncompetitive manner, with the Ki values ranging from 0.12 mu M to 1.29 mu M. Docking simulations showed that PGG could tightly bound to the non-catalytic sites of various gmll-GUS, mainly via hydrogen bonding and aromatic interactions. It was also found that PGG could strongly inhibit the total gmll-GUS activity in mice feces, with the IC50 value of 1.24 mu M. Collectively, our findings revealed that RCE and its constituent PGG could strongly inhibit multiple gmll-GUS enzymes, suggesting that RCE and PGG could be used for alleviating gmll-GUS associated enterotoxicity.

Gut microbial Loop-1 (3-glucuronidases (gmGUS) played an important role in irinotecan-induced gastrointestinal toxicity by regulating the level of its active metabolite SN38 through enterohepatic recirculation. gmGUS inhibition has emerged as a promising approach to relieve its dose-limiting intestinal toxicity and improve its medication efficacy. This study aims to investigate the inhibitory effects and mechanisms of Platycladi cacumen and its main constituent hinokiflavone against four different types of Loop-1 gmGUS (EeGUS, Ee GUS, Sa GUS, Cp GUS and Ec GUS). Our results showed that the ethanol extract of Platycladi cacumen displayed strong broad-spectrum inhibition against four gmGUS, and hinokiflavone could potently inhibit Ee GUS, Sa GUS, Cp GUS and Ec GUS with IC50 50 values of 0.09 f 0.01 mu M, 0.44 f 0.01 mu M, 0.20 f 0.01 mu M and 0.69 f 0.10 mu M, respectively. Inhibition kinetic analyses demonstrated that hinokiflavone acted as a strong competitive inhibitor of Ee GUS with Ki i value of 0.13 mu M, while it displayed non-competitive inhibition against Sa GUS, Cp GUS and Ec GUS, with the Ki i values of 0.43 mu M, 0.33 mu M and 0.76 mu M, respectively. Docking simulations revealed that hinokiflavone could tightly bind with Tyr-485 and Glu-516 in catalytic sites of E eGUS, as well it created strong interactions with amino acids in loop structures of Sa GUS (Asn-362), Cp GUS (Phe-363, Met-364, Ala-365 and Arg-375) and Ec GUS (Leu-361) to interfere the substrate entry into the catalytic pocket. Collectively, these results confirmed that hinokiflavone from Platycladi cacumen is a potent naturally occurring inhibitor of gmGUS with broad efficiency, suggesting hinokiflavone will be helpful for alleviating intestinal toxicity in irinotecan therapy.

Obesity has been recognized as a key risk factor for multiple metabolic disorders, including diabetes, cardiovascular diseases and many types of cancer. Herbal medicines have been frequently used for preventing and treating obesity in many countries, but in most cases, the key anti-obesity constituents in herbs and their anti-obesity mechanisms are poorly understood. This study demonstrated a case study for uncovering the anti-obesity constituents in an anti-obesity herbal medicine (Ginkgo biloba extract) and deciphering their synergistic effects via targeting human pancreatic lipase (hPL). Following screening the anti-hPL effects of eighty herbal medicines, Ginkgo biloba extract (GBE50) was found with the most potent anti-hPL activity. Global chemical profiling of herbal constituents coupling with hPL inhibition assay revealed that the bioflavonoids and several flavonoids in GBE50 were key anti-hPL constituents. Among all tested thirty-eight constituents, sciadopitysin, bilobetin, quercetin, isoginkgetin, and ginkgetin showed potent anti-hPL effects (IC50 values <2.5 μM). Inhibition kinetic analyses suggested that sciadopitysin, bilobetin, quercetin, isoginkgetin, and ginkgetin acted as non-competitive inhibitors of hPL, with the Ki values were <2 μM. Docking simulations revealed that four bioflavonoids (sciadopitysin, bilobetin, isoginkgetin, and ginkgetin) could tightly bind on hPL at cavity 2, which it is different from the binding cavity of quercetin on hPL. Further investigations demonstrated that the combinations of quercetin and one bioflavonoid-type hPL inhibitor (sciadopitysin or bilobetin) showed synergistic anti-hPL effects, suggesting that the multi-components in GBE50 may generate more potent anti-hPL effect. Collectively, our findings uncovered the anti-obesity constituents in GBE50, and explored their anti-hPL mechanisms as well as synergistic effects at molecular levels, which will be very helpful for further understanding the anti-obesity mechanisms of Ginkgo biloba.

Coumarin-based fluorogenic probes have emerged as preferred imaging tools to real-time and noninvasively monitor target analyte(s) in biological systems, due to their inherent merits of tunable optical properties, facile synthesis, excellent cell-membrane permeability, and favorable biocompatibility. Compared to natural coumarins, coumarin-based near-infrared probes (CBNPs) display improved signal-to-noise ratio and higher spatial imaging resolution. Over the past few decades, multifunctional coumarin scaffolds have been used to construct practical CBNPs for dynamically tracking cellular microenvironments, metal ions, small molecules, and biomacromolecules of interest, as well as for drug screening and disease diagnosis. Herein, the advances in the development of CBNPs from molecular engineering to biomedical applications in the last decade are reviewed. Firstly, the structure-fluorescence relationships (SFRs) of coumarin derivatives, along with diverse structural types, as well as sensing mechanisms of diverse high-performance CBNPs, are well-summarized. Next, a wide array of state-of-the-art CBNPs with various recognition fragments and sensing mechanisms, together with their biomedical applications in native living systems, are illustrated. Meanwhile, the applications of CBNPs for sensing or visualizing disease biomarkers, drug screening, assessing drug efficacy in vivo, and photothermal/photodynamic therapy are also covered. Finally, the existing challenges and future perspectives in this field are highlighted. Collectively, the information and knowledge presented here provide valuable strategies and practical cases for constructing multifunctional CBNPs, which will be very helpful for researchers to devise more versatile CBNPs tools for both fundamental and applied studies.(c) 2023 Elsevier B.V. All rights reserved.

382 Background: Q-1802 is a humanized bispecific antibody that targets both the tumor -specific antigen CLDN18.2 and the immune checkpoint PD-L1. Q-1802 monotherapy dose-escalation study has been completed in China and dose-expansion study has enrolled 17 advanced subjects with advanced Gastric adenocarcinoma, pancreatic cancer, biliary tract cancer. Methods: This is a FIH, phase 1a/1b, multicenter, open label, single arm, dose escalation and dose expansion study of Q-1802, administered intravenously to 20-30 adult patients with resistant/refractory advanced or metastatic solid tumors who had failed standard therapies (NCT04856150). It aimed to evaluate the safety, tolerability, PK/PD profiles and preliminary efficacy of Q-1802 monotherapy. In the dose escalation phase (phase 1a), an accelerated titration followed by a 3+3 design was used to assess the safety and tolerability of Q-1802 (dose range 0.1 mg/kg to 20 mg/kg); and determine the maximum tolerated dose (MTD). Q-1802 was administered in a dose limiting toxicity (DLT) observation period followed by a Q2w treatment schedule. In the dose expansion phase (Phase 1b), one to two dose groups will be selected to enroll 9-23 Pts, including 3 negative CLDN18.2 expression and 6 positive CLDN18.2 expression in each group. Q-1802 was administered Q2w on a 14-day treatment cycle. Results: As of September 20, 2022, a total of 29 patients (median age 57.0 y; most patients received ≥3 prior regimens) were enrolled (12 Pts in phase 1a with DLT evaluation and 17 Pts in Phase 1b). The most common tumor types were GI cancers. There were no DLTs up to 20 mg/kg of Q-1802, inclusive. Two dose groups:10mg/kg and 20mg/kg were extended in Phase 1b study. Treatment-emergent adverse events were mostly grade 1-2 .The most common treatment related adverse events were Gastrointestinal AEs (89.7%, 26/29), including nausea 18/29 (62.1%), vomiting 18/29 (62.1%), abdominal pain 8/29 (27.6%), Gastroesophageal reflux disease 7/29 (24.1%). Grade 3 treatment-related AEs occurred 7/29 ( 24.1%), and gastrointestinal disease including nausea, vomiting has a higher percentage of 10.3% (3/29). Only one case has a grade 4 AE, hyponatremia, with a long term use of diuretics history. No death reported due to study related treatment. irAEs happened in 7 subjects, including abnormal thyroid function, fatigue, rash, arthritis. Most of irAEs were grade1-2. Among the 9 GIsubjects in the dose-expansion phase with CLDN18.2 positive expression who had measurable lesions and had received at least one post treatment tumor assessments, 2ptsachieved partial response and 4 achieved stable disease as the best overall response per RECIST1.1. Conclusions: Interim data from the present phase 1 study, demonstrate that Q-1802 has excellent preliminary safety, tolerability and preliminary anti-tumor activity up to doses of 20 mg/kg. The dose extension is still ongoing. Clinical trial information: NCT04856150 .

Mycobacterium tuberculosis (M. tb) is an intracellular pathogen persisting in phagosomes that has the ability to escape host immune surveillance causing tuberculosis (TB). Lipoarabinomannan (LAM), as a glycolipid, is one of the complex outermost components of the mycobacterial cell envelope and plays a critical role in modulating host responses during M. tb infection. Different species within the Mycobacterium genus exhibit distinct LAM structures and elicit diverse innate immune responses. However, little is known about the mechanisms. In this study, we first constructed a LAM-truncated mutant with fewer arabinofuranose (Araf) residues named M. sm-ΔM_6387 (Mycobacterium smegmatis arabinosyltransferase EmbC gene knockout strain). It exhibited some prominent cell wall defects, including tardiness of mycobacterial migration, loss of acid-fast staining, and increased cell wall permeability. Within alveolar epithelial cells (A549) infected by M. sm-ΔM_6387, the uptake rate was lower, phagosomes with bacterial degradation appeared, and microtubule-associated protein light chain 3 (LC3) recruitment was enhanced compared to wild type Mycobacterium smegmatis (M. smegmatis). We further confirmed that the variability in the removal capability of M. sm-ΔM_6387 resulted from host cell responses rather than the changes in the mycobacterial cell envelope. Moreover, we found that M. sm-ΔM_6387 or its glycolipid extracts significantly induced expression changes in some genes related to innate immune responses, including Toll-like receptor 2 (TLR2), class A scavenger receptor (SR-A), Rubicon, LC3, tumor necrosis factor alpha (TNF-α), Bcl-2, and Bax. Therefore, our studies suggest that nonpathogenic M. smegmatis can deposit LC3 on phagosomal membranes, and the decrease in the quantity of Araf residues for LAM molecules not only impacts mycobacterial cell wall integrity but also enhances host defense responses against the intracellular pathogens and decreases phagocytosis of host cells.

Gut bacterial nitroreductases play an important role in reduction of various nitroaromatic compounds to the corresponding N-nitroso compounds, hydroxylamines or aromatic amines, most of which are carcinogenic and mutagenic agents. Inhibition of gut nitroreductases has been recognized as an attractive approach for reducing mutagen metabolites in the colon, so as to prevent colon diseases. In this study, the inhibitory effects of 55 herbal medicines against Escherichia coli(E. coli) nitroreductase (EcNfsA) were examined. Compared with other herbal extracts, Syzygium aromaticum extract showed superior inhibitory potency toward EcNfsA mediated nitrofurazone reduction. Then, the inhibitory effects of 22 major constituents in Syzygium aromaticum against EcNfsA were evaluted. Compared with other tested natural compounds, ellagic acid, corilagin, betulinic acid, oleanic acid, ursolic acid, urolithin M5 and isorhamnetin were found with strong to moderate inhibitory effect against EcNfsA, with IC50 values ranging from 0.67 to 28.98 mol·L-1. Furthermore, the inhibition kinetic analysis and docking simulation demonstrated that ellagic acid and betulinic acid potently inhibited EcNfsA (Ki < 2 μmol·L -1) in a competitively inhibitory manner, which created strong interactions with the catalytic triad of EcNfsA. In summary, our findings provide new scientific basis for explaining the anti-mutagenic activity of Syzygium aromaticum, where some newly identified EcNfsA inhibitors can be used for developing novel agents to reduce the toxicity induced by bacterial nitroreductase.

Although herbal medicines (HMs) are widely used in the prevention and treatment of obesity and obesity-associated disorders, the key constituents exhibiting anti-obesity activity and their molecular mechanisms are poorly understood. Recently, we assessed the inhibitory potentials of several HMs against human pancreatic lipase (hPL, a key therapeutic target for human obesity), among which the root-extract of Rhodiola crenulata (ERC) showed the most potent anti-hPL activity. In this study, we adopted an integrated strategy, involving bioactivity-guided fractionation techniques, chemical profiling, and biochemical assays, to identify the key anti-hPL constituents in ERC. Nine ERC fractions (retention time = 12.5-35 min), obtained using reverse-phase liquid chromatography, showed strong anti-hPL activity, while the major constituents in these bioactive fractions were subsequently identified using liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS/MS). Among the identified ERC constituents, 1,2,3,4,6-penta-O-galloyl-β-d-glucopyranose (PGG) and catechin gallate (CG) showed the most potent anti-hPL activity, with pIC50 values of 7.59 ± 0.03 and 7.68 ± 0.23, respectively. Further investigations revealed that PGG and CG potently inhibited hPL in a non-competitive manner, with inhibition constant (Ki) values of 0.012 and 0.082 μM, respectively. Collectively, our integrative analyses enabled us to efficiently identify and characterize the key anti-obesity constituents in ERC, as well as to elucidate their anti-hPL mechanisms. These findings provide convincing evidence in support of the anti-obesity and lipid-lowering properties of ERC.

Gut bacterial nitroreductases are found to be heavily related with the intestinal toxicity of nitroaromatic com-pounds in food or medicine, which can be converted into mutagenic and enterotoxic nitroso or N-hydroxyl in-termediates. Thus, inhibiting the gut microbe-encoded nitroreductases has become an attractive method to reduce the mutagen metabolites in colon and prevent intestinal diseases. In this study, the inhibitory effects of sixteen constituents in Cortex Mori Radicis on two kinds of gut bacterial nitroreductases (EcNfsA and EcNfsB) were evaluated with nitrofurazone (NFZ) as substrate and NADPH as electron donor. The results clearly demonstrated that four flavonoids including kuwanon G, kuwanon A, sanggenol A and kuwanon C showed dual inhibition on both EcNfsA and EcNfsB mediated NFZ reduction; morusin, morin, and sanggenone C were strong inhibitors towards EcNfsA; kuwanon H and kuwanon E exhibited effective inhibition on EcNfsB. Further inhi-bition kinetic analysis and molecular docking simulations displayed that all inhibitors above suppressed both EcNfsA and EcNfsB activities in competitive manners, except non-competitive inhibition of morin on EcNfsA and non-competitive inhibition of kuwanon C on EcNfsB, respectively. Taking together, these findings revealed that most flavonoids in Cortex Mori Radicis presented effective inhibition on gut microbial nitroreductases, suggesting that Cortex Mori Radicis might be a promising candidate for ameliorating nitroreductases mediated intestinal mutagenicity.

Pancreatic lipase (PL) inhibitor therapy has been validated as an efficacious way for preventing and treating obesity and overweight. In the past few decades, porcine PL (pPL) is widely used as the enzyme source for screening the PL inhibitors, which generates a wide range of pPL inhibitors. By contrast, the efficacious inhibitors against human PL (hPL) are rarely reported. This study aims to discover the naturally occurring hPL inhibitors from edible herbal medicines (HMs) and to characterize the inhibitory mechanisms of the newly identified hPL inhibitors. Following the screening of the inhibition potentials of more than 100 HMs against hPL, Ampelopsis grossedentata extract (AGE) displayed the most potent hPL inhibition activity. After that, the major constituents in AGE were identified and purified, while their anti-hPL effects were assayed in vitro. The results clearly showed that two abundant constituents in AGE (dihydromyricetin and iso-dihydromyricetin) were moderate hPL inhibitors, while myricetin and quercetin were strong hPL inhibitors [half-maximal inhibitory concentration (IC50) values were around 1.5 μM]. Inhibition kinetic analyses demonstrated that myricetin and quercetin potently inhibited hPL-catalyzed near-infrared fluorogenic substrate of human pancreatic lipase (DDAO-ol) hydrolysis in a non-competitive inhibition manner, with Ki values of 2.04 and 2.33 μM, respectively. Molecular dynamics simulations indicated that myricetin and quercetin could stably bind on an allosteric site of hPL. Collectively, this study reveals the key anti-obesity constituents in AGE and elucidates their inhibitory mechanisms against hPL, which offers convincing evidence to support the anti-obesity and lipid-lowering effects of this edible herb.

Bile salt hydrolases (BSHs) play crucial roles in the deconjugation of conjugated bile acids and therefore are key targets for modulating bile acid metabolism. This study aimed to identify efficacious BSH inhibitors from a natural compound library and to characterize their inhibitory mechanisms. The inhibitory potential of more than 100 natural compounds against BSH produced by Lactobacillus salivarius (lsBSH) was assayed, and several chalcones with strong or moderate lsBSH inhibitory activity were identified. Of all tested chalcones, licochalcone C and isobavachalcone showed the most potent lsBSH inhibitory activity (IC 50 < 1 μM). Inhibition kinetic analyses demonstrated that both licochalcone C and isobavachalcone reversibly inhibited lsBSH-catalyzed CA-AMCA hydrolysis via a mixed manner. Docking simulations suggested that they bind lsBSH at two distinct sites mainly via hydrogen bonding and hydrophobic interactions. Additionally, licochalcone C and isobavachalcone were found to inhibit various BSHs and decrease the total BSH activity in mouse feces, thus suggesting that these agents are broad-spectrum BSH inhibitors. Collectively, our findings revealed that licochalcone C and isobavachalcone are naturally occurring inhibitors of BSH, which may serve as promising lead compounds in the development of more efficacious BSH inhibitors for modulating bile acid metabolism.

Bile salt hydrolases (BSHs), a group of cysteine-hydrolases produced by gut microbes, play a crucial role in the hydrolysis of glycine- or taurine-conjugated bile acids and have been validated as key targets to modulate bile acid metabolism. This study aims to discover one or more efficacious inhibitors against a BSH produced by Lactobacillus salivarius (lsBSH) from natural products and to characterize the mechanism of the newly identified BSH inhibitor(s). Following screening of the inhibition potentials of more than 100 natural compounds against lsBSH, amentoflavone (AMF), a naturally occurring biflavone isolated from various medicinal plants, was discovered to be an efficacious BSH inhibitor (IC50 = 0.34 μM). Further investigation showed that AMF could strongly inhibit the lsBSH-catalyzed hydrolytic reaction in living gut microbes. Inhibition kinetic analyses demonstrated that AMF reversibly inhibited the lsBSH-catalyzed hydrolytic reaction in a mixed-inhibition manner, with an apparent Ki value of 0.65 μM. Fluorescence quenching assays suggested that AMF could quench the fluorescence of lsBSH via a static quenching procedure. Docking simulations suggested that AMF could be fitted into lsBSH at two distinct ligand-binding sites, mainly via hydrophobic interactions and hydrogen bonding, which explained well the mixed inhibition mode of this agent. Animal tests showed that the hydrolytic activities of BSHs in mice feces could be significantly blocked by AMF. In summary, this study reports that AMF is a strong, naturally occurring inhibitor of lsBSH, which offers a promising lead compound to develop novel agents for modulating bile acid metabolism in the host via targeting BSHs.

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.

The worldwide prevalence of nonalcoholic fatty liver disease (NAFLD) is increasing, and this metabolic disorder has been recognized as a severe threat to human health. A variety of chemical drugs have been approved for treating NAFLD, however, they always has serious side effects. Chinese herbal medicines (CHMs) have been widely used for preventing and treating a range of metabolic diseases with satisfactory safety and effective performance in clinical treatment of NAFLD. Recent studies indicated that imbanlance of the intestinal microbiota was closely associated with the occurrence and development of NAFLD, thus, the intestinal microbiota has been recognized as a promising target for treatment of NAFLD. In recent decades, a variety of CHMs have been reported to effectively prevent or treat NAFLD by modulating intestinal microbiota to further interfer the gut-liver axis. In this review, recent advances in CHMs for the treatment of NAFLD via rebuilding the intestinal microecology were systematically reviewed. The key roles of CHMs in the regulation of gut microbiota and the gut-liver axis along with their mechanisms (such as modulating intestinal permeability, reducing the inflammatory response, protecting liver cells, improving lipid metabolism, and modulating nuclear receptors), were well summarized. All the knowledge and information presented here will be very helpful for researchers to better understand the applications and mechanisms of CHMs for treatment of NAFLD.

Gut bacterial β-glucuronidases (GUS) play an important role in deconjugation of various O-glucuronides, which are tightly linked with the drug-induced intestinal toxicity. Increasing evidence has indicated that inhibition of bacterial GUS could alleviate GUS-associated intestinal toxicity, but the potent and broad-spectrum inhibitors against multiple bacterial GUS have been rarely reported. This study aimed to find potent and broad-spectrum GUS inhibitors from Ginkgo biloba. It was found that amentoflavone displayed relatively strong inhibition on three GUS including CpGUS, SpasGUS and EcGUS. Further investigations demonstrated that amentoflavone could inhibit GUS-mediated PNPG hydrolysis in a dose-dependent manner with IC50 values of 2.36 μM, 2.88 μM and 3.43 μM for CpGUS, SpasGUS and EcGUS, respectively. Inhibition kinetic studies showed that amentoflavone functioned as a non-competitive inhibitor against all tested GUS with Ki values of less than 2 μM. Docking simulations indicated that amentoflavone could tightly bind on allosteric sites of three GUS mainly via hydrogen bonding interactions, and the number of hydroxyl groups of amentoflavone played crucial roles in these interactions. Collectively, our findings suggested that amentoflavone was a potent broad-spectrum inhibitor against bacterial GUS, which can be used as a promising lead compound for developing novel agents to alleviate GUS-associated intestinal toxicity.