Β-glucuronidase Inhibitor Research Articles

Dissecting molecular mechanisms underlying the inhibition of β-glucuronidase by alkaloids from Hibiscus trionum: Integrating in vitro and in silico perspectives

β-Glucuronidase, a crucial enzyme in drug metabolism and detoxification, represents a promising target for therapeutic intervention due to its potential to modulate drug pharmacokinetics and enhance therapeutic efficacy. Herein, we assessed the inhibitory potential of phytochemicals from Hibiscus trionum against β-glucuronidase. Grossamide and grossamide K emerged as the most potent β-glucuronidase inhibitors with IC50 values of 0.73 ± 0.03 and 1.24 ± 0.03 μM, respectively. The investigated alkaloids effectively inhibited β-glucuronidase-catalyzed PNPG hydrolysis through a noncompetitive inhibition mode, whereas steppogenin displayed a mixed inhibition mechanism. Molecular docking analyses highlighted grossamide and grossamide K as inhibitors with the lowest binding free energy, all compounds successfully docked into the same main binding site occupied by the reference drug Epigallocatechin gallate (EGCG). We explored the interaction dynamics of isolated compounds with β-glucuronidase through a 200 ns molecular dynamics (MD) simulation. Analysis of various MD parameters revealed that grossamide and grossamide K maintained stable trajectories and demonstrated significant energy stabilization upon binding to β-glucuronidase. Additionally, these compounds exhibited the lowest average interaction energies with the target enzyme. The MM/PBSA calculations further supported these findings, showing the lowest binding free energies for grossamide and grossamide K. These computational results are consistent with experimental data, suggesting that grossamide and grossamide K could be potent inhibitors of β-glucuronidase.

Investigation of the inhibitory activity of triterpenoids isolated from Actinidia polygama stems against β-glucuronidase via enzyme kinetics, molecular docking, and molecular dynamics analyses

Actinidia polygama, well-known for its traditional medicinal properties, has garnered significant attention due to its distinctive chemical composition. In this study, we isolated and structurally elucidated 11 known compounds, including a monoterpene glycoside (1), eight triterpenoids (2–9), and two lignan glycosides (10 and 11), from A. polygama stems. Notably, the inhibitory activity of these compounds on β-glucuronidase activity was evaluated for the first time. Our results show that 2α,3α,24-trihydroxyurs-12-en-28-oic acid (4), strongly inhibited β-glucuronidase (IC50 = 14.36 ± 0.42 μM) to a degree much higher than that of the positive control (d-saccharic acid 1,4-lactone, DSA; IC50 = 16.30 ± 0.21 μM). Furthermore, the results of an enzyme kinetics analysis indicate that compound 4 acts as a non-competitive inhibitor of β-glucuronidase. Finally, the findings from molecular docking and molecular dynamics simulations confirmed the inhibition mode of compound 4, its interaction with the allosteric site of β-glucuronidase, and the stability of the complex in the bound state.

In vitro and in vivo studies on the metabolism and pharmacokinetics of the selective gut microbial β-glucuronidase targeting compound Inh 1

In vitro studies using rat, mouse, and human microsomes and hepatocytes on the bacterial β-glucuronidase inhibitor 1-((6,8-dimethyl-2-oxo-1,2-dihydroquinolin-3-yl)methyl)-3-(4-ethoxyphenyl)-1-(2-hydroxyethyl)thiourea) (Inh 1) revealed extensive metabolism in all species. The intrinsic clearances of Inh 1 in human, mouse, and rat hepatic microsomes were 30.9, 67.8, and 201 µL/min/mg, respectively. For intact hepatocytes intrinsic clearances of 21.6, 96.0, and 129 µL/min/106 cells were seen for human, mouse and rat, respectively. The metabolism of Inh 1 involved an uncommon desulphurisation reaction in addition to oxidation, deethylation, and conjugation reactions at multiple sites. Six metabolites were detected in microsomal incubations in human and rat, and seven for the mouse. With hepatocytes, 18 metabolites were characterised, 9 for human, and 11 for mouse and rat. Following IV administration to mice (3 mg/kg), plasma concentrations of Inh 1 exhibited a monophasic decline with a terminal elimination half-life of 0.91 h and low systemic clearance (11.8% of liver blood flow). After PO dosing to mice (3 mg/kg), peak observed Inh 1 concentrations of 495 ng/mL were measured 0.5 h post dose, declining to under 10 ng/mL at 8 h post dose. The absolute oral bioavailability of Inh 1 in the mouse was ca. 26%.

Glucuronic acid confers colonization advantage to enteric pathogens

Glucuronidation is a detoxification process to eliminate endo- and xeno-biotics and neurotransmitters from the host circulation. Glucuronosyltransferase binds these compounds to glucuronic acid (GlcA), deactivating them and allowing their elimination through the gastrointestinal (GI) tract. However, the microbiota produces β-glucuronidases that release GlcA and reactivate these compounds. Enteric pathogens such as enterohemorrhagic Escherichia coli (EHEC) and Citrobacter rodentium sense and utilize galacturonic acid (GalA), an isomer of GlcA, to outcompete the microbiota promoting gut colonization. However, the role of GlcA in pathogen colonization has not been explored. Here, we show that treatment of mice with a microbial β-glucuronidase inhibitor (GUSi) decreased C. rodentium's colonization of the GI tract, without modulating bacterial virulence or host inflammation. Metagenomic studies indicated that GUSi did not change the composition of the intestinal microbiota in these animals. GlcA confers an advantage for pathogen expansion through its utilization as a carbon source. Congruently mutants unable to catabolize GlcA depict lower GI colonization compared to wild type and are not sensitive to GUSi. Germfree mice colonized with a commensal E. coli deficient for β-glucuronidase production led to a decrease of C. rodentium tissue colonization, compared to animals monocolonized with an E. coli proficient for production of this enzyme. GlcA is not sensed as a signal and doesn't activate virulence expression but is used as a metabolite. Because pathogens can use GlcA to promote their colonization, inhibitors of microbial β-glucuronidases could be a unique therapeutic against enteric infections without disturbing the host or microbiota physiology.

Hybrid molecules of thiadiazole-based benzothioate and benzenesulfonothioate: synthesis, structural analysis, and evaluation as potential inhibitors of thymidine phosphorylase and β-glucuronidase through in vitro and in silico approaches

The thiadiazole-based benzothioate (3a-f) and benzenesulfonothioate (5a-f) hybrid derivatives were designed and synthesized in a single step reaction. All the synthesized compounds structures were confirmed through spectroscopic techniques including 1H NMR, 13C NMR and HRMS (ESI). Then, the synthesized compounds were subjected to in vitro thymidine phosphorylase and beta-glucuronidase inhibition profile. All the newly synthesized moieties (3a-f) & (5a-f) were found potent and showed moderately to good inhibition profile. Among benzothioate analogues (3a-f), the analogues 3d and 3c were identified to be active inhibitors of thymidine phosphorylase and beta-glucuronidase enzymes having IC50 values of 2.11 ± 0.31 μM & 3.28 ± 0.20 μM (against thymidine phosphorylase) and 2.50 ± 0.20 μM & 3.71 ± 0.10 μM (against beta-glucuronidase) respectively. However, among benzenesulfonothioate (5a-f) analogues, analogues 5a and 5d were found to be significantly active, even more than standard drugs with IC50 values of 2.23 ± 0.13 μM & 3.10 ± 0.17 μM (against thymidine phosphorylase) and 3.12 ± 0.20 μM & 5.60 ± 0.34 μM respectively. The results were compared to standard drugs such as D-saccharic acid with IC50 values of 10.10 ± 0.10 µM (for benzothioate series) & 8.50 ± 0.10 µM (for benzenesulfonothioate series) and 7-Deazaxanthine having IC50 values of 12.20 ± 0.10 µM (for benzothioate series) & 9.20 ± 0.30 µM (benzenesulfonothioate series) respectively. From structure-activity (SAR) analysis, it was confirmed that any variation found in inhibitory activities of both thymidine phosphorylase beta-glucuronidase enzymes was due to different substitution pattern of substituent(s) at variable position of aryl ring. Moreover, a good protein-ligand interaction (PLI) subjected to molecular docking study against shown by the most active analogues against corresponding target with key binding interactions with least binding energies. These findings reveal that thiadiazole-containing benzothioate (3a-f) and benzenesulfonothioate (5a-f) act as thymidine phosphorylase beta-glucuronidase inhibitors to develop novel therapeutics agents for the purpose of drug discovery.

Effect of Saccharolactone on CYP-mediated Metabolism of Xenobiotics.

Saccharolactone is used as a β-glucuronidase inhibitor in in vitro microsomal and recombinant uridine diphosphoglucuronosyl transferases (rUGTs) incubations to enhance glucuronide pathway and, thereby, formation of glucuronide metabolites. We investigated its effect on CYP mediated metabolism of drugs (compound-174, phenacetin and quinidine) using human liver microsomes (HLM) supplemented with Phase-1 and Phase-2 co-factors. Compounds were incubated in HLM supplemented with co-factors to assess Phase-1 (NADPH) and Phase-2 (NADPH, alamethicin, saccharolactone and UDPGA) metabolism. CYP phenotype assay for compound-174 was conducted in HLM (± 1-ABT) and human recombinant CYP isoforms. CYP inhibition profile of saccharolactone was also generated in HLM. The metabolism of compound-174, phenacetin and quinidine in HLM significantly decreased in reactions containing additional components like alamethicin, saccharolactone and UDPGA and indicated that the addition of saccharolactone inhibited the metabolism. Phenacetin and quinidine are known substrates of CYP1A2 and CYP3A4 isoforms. The metabolism of compound- 174 was significantly inhibited in the presence of 1-ABT in HLM, and CYP3A4 and CYP2C8 isoforms were found to be the predominant isoforms responsible for its metabolism. Further evaluation of CYP inhibition in HLM indicated saccharolactone to be a strong inhibitor of CYP1A2, 2D6, 3A4 and 2C8 isoforms with IC50 values of less than 4 mM. The findings indicated that saccharolactone being a strong inhibitor of CYP1A2, 2D6, 3A4 and 2C8 isoforms (IC50 < 4 mM), resulted in significant inhibition of the metabolism of compound-174, phenacetin and quinidine in HLM and caution should be exercised in using it with proper titration of the concentrations.

Novel benzoxazole-based thiosemicarbazide derivatives as new inhibitors of urease and β-Glucuronidase: Synthesis, in vivo anti-nematodal activity and ADMET prediction along with in silico study

Here, we discuss the synthesis of thiosemicarbazide derivatives based on benzoxazole. These compounds were obtained via sequence of reactions. The targeted products wereconfirmed using a number of spectroscopic methods, including NMR (1H and 13C) and EI-MS. After spectral confirmation all the synthesized compounds were evaluated for urease and β-Glucuronidase inhibitory activity in order to explore their biological significances in the presence of standard drug thiourea (IC50 = 21.86 ± 0.40) and D-saccharic acid 1,4-lactone (IC50 value 22.00 ± 1.10 µM) respectively. Among the evaluated series, compounds 14 and 15 (1.10 and 0.01 and 2.20 and 0.60) were shown to have slightly greater potential than standard drugs. Anti-nematodal activity was also employed to explore the cytotoxic nature of synthesized analogs. In order to establish the binding relationship with enzyme active sites, molecular docking experiments were done and directions for compound modification based on SAR features were addressed. In addition, ADMET prediction study also investigated to found drug like properties of the potential analogs.

Synthesis of Uronic Acid 1-Azasugars as Putative Inhibitors of α-Iduronidase, β-Glucuronidase and Heparanase.

1-Azasugar analogues of l-iduronic acid (l-IdoA) and d-glucuronic acid (d-GlcA) and their corresponding enantiomers have been synthesized as potential pharmacological chaperones for mucopolysaccharidosis I (MPS I), a lysosomal storage disease caused by mutations in the gene encoding α-iduronidase (IDUA). The compounds were efficiently synthesized in nine or ten steps from d- or l-arabinose, and the structures were confirmed by X-ray crystallographic analysis of key intermediates. All compounds were inactive against IDUA, although l-IdoA-configured 8 moderately inhibited β-glucuronidase (β-GLU). The d-GlcA-configured 9 was a potent inhibitor of β-GLU and a moderate inhibitor of the endo-β-glucuronidase heparanase. Co-crystallization of 9 with heparanase revealed that the endocyclic nitrogen of 9 forms close interactions with both the catalytic acid and catalytic nucleophile.

Synthesis of 2-Aminopyrimidine Derivatives and Their Evaluation as β-Glucuronidase Inhibitors: In Vitro and In Silico Studies.

Currently the discovery and development of potent β-glucuronidase inhibitors is an active area of research due to the observation that increased activity of this enzyme is associated with many pathological conditions, such as colon cancer, renal diseases, and infections of the urinary tract. In this study, twenty-seven 2-aminopyrimidine derivatives 1-27 were synthesized by fusion of 2-amino-4,6-dichloropyrimidine with a variety of amines in the presence of triethylamine without using any solvent and catalyst, in good to excellent yields. All synthesized compounds were characterized by EI-MS, HREI-MS and NMR spectroscopy. Compounds 1-27 were then evaluated for their β-glucuronidase inhibitory activity, and among them, compound 24 (IC50 = 2.8 ± 0.10 µM) showed an activity much superior to standard D-saccharic acid 1,4-lactone (IC50 = 45.75 ± 2.16 µM). To predict the binding mode of the substrate and β-glucuronidase, in silico study was performed. Conclusively, this study has identified a potent β-glucuronidase inhibitor that deserves to be further studied for the development of pharmaceutical products.

Evaluation of herbal ingredients used in an ethno-polyherbal formulation for treating menorrhagia and dysmenorrhea in Bangladesh

BackgroundGlobally, gynecological complications such as menorrhagia and dysmenorrhea lack attention. A female folk medicine practitioner (FMP) residing in the Thakurgaon district of Bangladesh, uses a phytomedicine that has explicitly been prescribed for many years to treat abnormal uterine bleeding accompanying severe lower abdominal pain. ObjectiveWe aim to corroborate original ethno-phytomedicine data that was prescribed for menorrhagia and dysmenorrhea treatment, by studying the evidence-based therapeutic efficacy of medicinal plants and the potential role of gut microbiota in relevant phytopharmacology. MethodsFirst, we documented the process of the phytomedicine preparation by visiting the folk practitioner with her Prior Informed Consent and completed the plant's taxonomic identification from Bangladesh National Herbarium. This was followed by the study of relevant literature available on PubMed and Google Scholar databases to elucidate the scientific efficacy of the herbal ingredients used in the phytomedicine preparation. ResultsThe plant species Phyllanthus reticulatus, Piper nigrum, and Acacia catechu was used as ingredients in the phytomedicine preparation, which has been corroborated by phytochemical and pharmacological data, as well as previous ethnopharmacological uses. The accumulated information aids to hypothesize that a phytochemical, catechin in Acacia catechu and Piper nigrum might act as β-glucuronidase and aromatase inhibitor to balance estrogen levels of patients with menorrhagia and dysmenorrhea. ConclusionAs traditional phytomedicines are suffering from a lack of standardization parameters compared to modern synthetic drugs, the implementation of advanced technologies and emerging scientific strategies can significantly impact the scientific validity, quality, and standardization of traditional herbal medicines. The present study suggests further biological research towards a more updated standardized phytomedicine formulation that can effectively treat menorrhagia and dysmenorrhea.

Synthesis, Biological Evaluation, and In Silico Studies of Novel Coumarin-Based 4H,5H-pyrano[3,2-c]chromenes as Potent β-Glucuronidase and Carbonic Anhydrase Inhibitors.

The search for novel heterocyclic compounds with a natural product skeleton as potent enzyme inhibitors against clinical hits is our prime concern in this study. Here, a simple and facile two-step strategy has been designed to synthesize a series of novel coumarin-based dihydropyranochromenes (12a–12m) in a basic moiety. The synthesized compounds were thus characterized through spectroscopic techniques and screened for inhibition potency against the cytosolic hCA II isoform and β-glucuronidase. Few of these compounds were potent inhibitors of hCA II and β-glucuronidase with varying IC50 values ranging from 4.55 ± 0.22 to 21.77 ± 3.32 μM and 440.1 ± 1.17 to 971.3 ± 0.05 μM, respectively. Among the stream of synthesized compounds, 12e and 12i were the most potent inhibitors of β-glucuronidase, while 12h, 12i, and 12j showed greater potency against hCA II. In silico docking studies illustrated the significance of substituted groups on the pyranochromene skeleton and binding pattern of these highly potent compounds inside enzyme pockets.

The in vitro metabolism and in vivo pharmacokinetics of the bacterial β-glucuronidase inhibitor UNC10201652

In vitro incubation of the bacterial β-glucuronidase inhibitor UNC10201652 (4-(8-(piperazin-1-yl)-1,2,3,4-tetrahydro-[1,2,3]triazino[4',5':4,5]thieno[2,3-c]isoquinolin-5-yl)morpholine) with mouse, rat, and human liver microsomes and hepatocytes generated metabolites at multiple sites via deethylations, oxidations and glucuronidation. Two UNC10201652 metabolites were detected in human, and four in mouse and rat liver microsomal incubations. Intrinsic clearances of UNC10201652 in human, mouse, and rat liver microsomes were 48.1, 115, and 194 µL/min/mg respectively. Intrinsic clearances for human, mouse, and rat hepatocytes were 20.9, 116, and 140 µL/min/106 cells respectively and 24 metabolites were characterised: 9 for human and 11 for both rodent species. Plasma clearance was 324.8 mL/min/kg with an elimination half-life of 0.66 h following IV administration of UNC10201652 to Swiss Albino mice (3 mg/kg). Pre-treatment with 1-aminobenzotriazole (ABT) decreased clearance to 127.43 mL/min/kg, increasing the t 1/2 to 3.66 h. Comparison of profiles after oral administration of UNC10201652 to control and pre-treated mice demonstrated a large increase in C max (from 15.2 ng/mL to 184.0 ng/mL), a delay in Tmax from 0.25 to 1 h and increased AUC from 20.1 to 253 h ng/ml. ABT pre-treatment increased oral bioavailability from 15% to >100% suggesting that CYP450's contributed significantly to UNC10201652 clearance in mice.

Species differences in liver microsomal hydrolysis of acyl glucuronide in humans and rats

Acyl glucuronides (AGs) are known as one of the causes of idiosyncratic drug toxicity (IDT). Although AGs can be enzymatically hydrolysed by β-glucuronidase and esterase, much information on their characteristics and species differences is lacking. This study was aimed to clarify species differences in AG hydrolysis between human and rat liver microsomes (HLM and RLM). To evaluate the AG hydrolysis profile, and the contribution of β-glucuronidase and esterase towards AG hydrolysis in HLM and RLM, nonsteroidal anti-inflammatory drugs (NSAIDs) were used. AGs were incubated with 0.1 M Tris-HCl buffer (pH 7.4) and 0.3 mg/mL HLM or RLM in the absence or presence of β-glucuronidase inhibitor, D-saccharic acid 1,4-lactone (D-SL) and esterase inhibitor, phenylmethylsulfonyl fluoride (PMSF). AGs of mefenamic acid (MEF-AG) and etodolac (ETO-AG) showed significantly higher AG hydrolysis rates in RLM than in HLM. Esterases were found to serve as AG hydrolases dominantly in HLM, whereas both esterases and β-glucuronidase equally contribute to AG hydrolysis in RLM. However, MEF-AG and ETO-AG were hydrolysed only by β-glucuronidase. We demonstrated for the first time that the activity of AG hydrolases towards NSAID-AGs differs between humans and rats.

Synthesis of new 1,2-disubstituted benzimidazole analogs as potent inhibitors of β-Glucuronidase and in silico study

New benzimidazole analogues (1–18) were synthesized and characterized through different spectroscopic techniques such as 1H NMR, 13C NMR and HREI-MS. All analogues were screened for β-glucuronidase inhibitory potential. All analogues showed varied degree of inhibitory potentials with IC50 values ranging between 1.10 ± 0.10 to 39.60 ± 0.70 μM when compared with standard D-saccharic acid-1,4- lactone having IC50 value 48.30 μM. Analogues 17, 11, 9, 6, 1 and 13 having IC50 values 1.10 ± 0.10, 1.70 ± 0.10, 2.30 ± 0.10, 5.30 ± 0.20, 6.20 ± 0.20 and 8.10 ± 0.20 μM respectively, showed excellent β-glucuronidase inhibitory potential many folds better than the standard. All other analogues also showed good inhibitory potential better as compared to standard. Structure activity relationships (SAR) has been established for all compounds. The results from molecular docking studies supports the established SAR and developed a strong correlation with the results from in to vitro assay. The molecular docking results clearly highlighted how substituents like nitro and chloro affect the binding position of the active compounds in the active site. The docking results were also used to properly establish the effect of bulky substituents of least active compounds on reduced β-glucuronidase inhibitory activity. Compounds 1–18 were found non-toxic.

Studying in vivo and in vitro effects of a novel polyvinyl benzyl chloride-D-glucaro-1, 4-lactonate polymer-coated bile duct stent for anti-biliary mud deposition.

We aim to coat a novel polyvinyl benzyl chloride-D-glucaro-1, 4-lactonate polymer-coated bile duct stent for anti-biliary mud deposition and investigate its in vivo and in vitro impacts. Biliary mud deposition is a leading cause of plastic biliary stent obstruction after its placement. Orally administrated D-glucaro-1, 4-lactonate is a specific competitive inhibitor of β-glucuronidase that causes biliary mud deposition. In this study, the stents coated with polyvinyl benzyl chloride-D-glucaro-1,4-lactonate polymer (PVBC-DGL) were placed in an ex vivo bile duct model perfused with porcine bile and observed every week until completely blocked. Post establishing the model of bile duct stenosis in piglets, stents are observed through endoscopy, hematology, patency time, and pathological changes within 6 months. The 70% PVBC-DGL stents achieved the highest percentage of the inhibitory effect when the drugs were completely released in vitro. Results were obtained from 14 pigs (5: no coating (original), 4: 0% coating, and 5: 70% coating). The overall patency time of the stents was prolonged in all groups; however, the group with 70% coated stents showed a significantly prolonged patency time as compared to no coating (original) and the 0% coating groups in pigs (23.4 ± 1.8 vs 11.2 ± 2.1 w (p = 0.05); 23.4 ± 1.8 vs 10.5 ± 2.5 w (p = 0.05), respectively). The stents with 70% PVBC-DGL better prevent and control the deposition of bile mud and prolong the patency time of stent placement in the subject animals and may be proposed for further clinical trials in patients.

Molecular modeling and docking analysis of bis-indolymethanes derivatives as human β-glucuronidase enzyme inhibitors

β-glucuronidase enzyme is present mostly in mammals’ tissues. β-glucuronidase is present in kidney, bile, serum, urine and spleen. In eukaryotic and prokaryotic organisms, it is important in the process of breaking down of β-glucuronide. It also helps in the neutralization of reactivity of some metabolites that are associated to many diseases. The most stable geometry of the dataset were obtained adopting DFT method at B3LYP/6-31G* level of theory. The model was developed using MLR analysis adopting GFA method. Molecular docking was also performed to portray the binding mode of these bis-indolymethanes derivatives in the binding pocket of their target receptor (human β-glucuronidase). The selected model was assessed and chosen based on its statistical fitness with R2trng=0.907233, R2adj=0.881465, Qcv2=0.833795, and R2test=0.609841. And also, the significance and impart of each physicochemical parameters to the selected model were determine by their ME values. Molecular docking analysis revealed that amino acid such as ALA49, SER52, ASP53, PHE51, VAL96, LEU92, TYR188, TYR199 and PHE200 might be responsible for the most promised binding affinity of the reported docked ligands. The molecular docking results showed that the reported compounds were better than the standard β-glucuronidase inhibitor. The results of this findings paved way for designing novel β-lucuronidase inhibitors.

Amentoflavone from Selaginella tamariscina as a potent inhibitor of gut bacterial β-glucuronidase: Inhibition kinetics and molecular dynamics stimulation

Gut bacterial β-glucuronidase (GUS) plays a pivotal role in the metabolism and reactivation of a vast of glucuronide conjugates of both endogenous and xenobiotic compounds in the gastrointestinal tract of human, which has been implicated in certain drug-induced gastrointestinal tract (GI) toxicity in clinic. Inhibitors of gut microbial GUS exhibited great potentials in relieving the drug-induced GI toxicity. In this study, Selaginella tamariscina and its major biflavonoid amentoflavone (AMF) were evaluated for their inhibitory activity against Escherichia coli GUS. Two selective probe substrates for GUS (a specific fluorescent probe substrate for GUS, DDAOG and a classical drug substrate for GUS, SN38G) were used in parallel for charactering the inhibition behaviors. Both the extract of S. tamariscina and its major biflavonoid AMF displayed evident inhibitory effects on GUS, and the IC50 values of AMF against GUS mediated DDAOG and SN-38G hydrolysis were 0.62 and 0.49 μM, respectively. Inhibition kinetics studies indicated that AMF showed mixed type inhibition for GUS-mediated DDAOG hydrolysis, while displayed competitive type inhibition against GUS-mediated SN-38G hydrolysis, with the Ki values of 0.24 and 1.25 μM, respectively. Molecular docking studies and molecular dynamics stimulation results clarified the role of amino acid residues Leu361, Ile363, and Glu413 in the inhibition of AMF on GUS. These results provided some foundations for the potential clinical utility of S. tamariscina and its major biflavonoid AMF for treating drug-induced enteropathy.

One-pot synthesis of thioxo-tetrahydropyrimidine derivatives as potent β-glucuronidase inhibitor, biological evaluation, molecular docking and molecular dynamics studies

A series of N,N-diethyl phenyl thioxo-tetrahydropyrimidine carboxamide have been synthesized and investigated for their β-glucuronidase inhibitory activities. All molecules exhibited excellent inhibition with IC50 values ranging from 0.35 to 42.05 µM and found to be even more potent than the standard d-saccharic acid. Structure-activity relationship analysis indicated that the meta-aryl-substituted derivatives significantly influenced β-glucuronidase inhibitory activities while the para-substitution counterpart outperforming moderate potency. The most potent compound in this series was 4g bearing thiophene motif with IC50 of 0.35 ± 0.09 µM. To verify the SAR, molecular docking and molecular dynamics studies were also performed.

Abstract A108: Specific and traceless payload release from HER2-ADC containing LBG-linker prodrug by the action of lysosomal β-glucuronidase

Abstract Many antibody-drug conjugates (ADCs) have actively been evaluated in clinical trials since Mylotarg was approved in 2001, but only 4 more have been approved by the FDA. The main reason for the poor results is yet to overcome worse risk-benefit than the expected from ADC approach. Controlled payload release specifically at cancer cells would significantly reduce systemic toxicity. Herein, we report that LBG000-linker can be selectively cleaved from ADC to release free toxins in a traceless manner by β-glucuronidase. Lysosomal β-glucuronidase is overexpressed in many cancer cell lines, and its enzymatic activity is much higher in acidic condition than neutral pH. As a model reaction, an LBG-linker-MMAE conjugate, LCB14-0302, was incubated with a β-glucuronidase in vitro resulting clean release of free MMAE while other lysosomal enzymes such as cathepsin B or heparanase did not generate the free toxin. We also confirmed that there was no free MMAE found when β-glucuronidase inhibitor, β-D-glucuronic acid, was treated. Also, lysosomal extract incubation with LCB14-0302 clearly demonstrated the traceless toxin release by β-glucuronidase regardless of other enzymatic disturbance. Therefore, these results strongly suggest that LBG-linker is highly stable and specific to β-glucuronidase among hundreds of enzymes in the lysosome. Citation Format: Dong Yeon Shin, Taeik Jang, Ho Young Song, Sung Min Kim, Yun-Hee Park, Chang Sik Park, Hwanhee Oh, Juyuel Baek, Jeiwook Chae, Chang-Sun Lee. Specific and traceless payload release from HER2-ADC containing LBG-linker prodrug by the action of lysosomal β-glucuronidase [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A108. doi:10.1158/1535-7163.TARG-19-A108

Sesamin Catechol Glucuronides Exert Anti-inflammatory Effects by Suppressing Interferon β and Inducible Nitric Oxide Synthase Expression through Deconjugation in Macrophage-like J774.1 Cells.

Sesamin, a representative sesame lignan, has health-promoting activities. Sesamin is converted into catechol derivatives and further into their glucuronides or sulfates in vivo, whereas the biological activities of sesamin metabolites remain unclear. We examined the inhibitory effects of sesamin metabolites on the lipopolysaccharide (LPS)-induced nitric oxide (NO) production in mouse macrophage-like J774.1 cells and found that a monocatechol derivative SC1, (7α,7'α,8α,8'α)-3,4-dihydroxy-3',4'-methylenedioxy-7,9':7',9-diepoxylignane, has a much higher activity than sesamin and other metabolites. The inhibitory effects of SC1 glucuronides were time-dependently enhanced, associated with the intracellular accumulation of SC1 and the methylated form. SC1 glucuronides and SC1 attenuated the expression of inducible NO synthase (iNOS) and upstream interferon-β (IFN-β) in the LPS-stimulated macrophages. The inhibitory effects of SC1 glucuronides against NO production were canceled by the β-glucuronidase inhibitor and enhanced by the catechol-O-methyltransferase inhibitor. Our results suggest that SC1 glucuronides exert the anti-inflammatory effects by inhibiting the IFN-β/iNOS signaling through macrophage-mediated deconjugation.