ABSTRACT
The ß-glucuronidase, a lysosomal enzyme, catalyzes the cleavage of glucuronosyl-O-bonds. Its inhibitors play a significant role in different medicinal therapies as they cause a decrease in carcinogen-induced colonic tumors by reducing the level of toxic substances present in the intestine. Among those inhibitors, bisindole derivatives had displayed promising ß-glucuronidase inhibition activity. In the current study, hydrazone derivatives of bisindolymethane (1-30) were synthesized and evaluated for in vitro ß-glucuronidase inhibitory activity. Twenty-eight analogs demonstrated better activity (IC50 = 0.50-46.5 µM) than standard D-saccharic acid 1,4-lactone (IC50 = 48.4 ± 1.25 µM). Compounds with hydroxyl group like 6 (0.60 ± 0.01 µM), 20 (1.50 ± 0.10 µM) and 25 (0.50 ± 0.01 µM) exhibited the most potent inhibitory activity, followed by analogs with fluorine 21 (3.50 ± 0.10 µM) and chlorine 23 (8.20 ± 0.20 µM) substituents. The presence of hydroxyl group at the aromatic side chain was observed as the main contributing factor in the inhibitory potential. From the docking studies, it was predicted that the active compounds can fit properly in the binding groove of the ß-glucuronidase and displayed significant binding interactions with essential residues.
Subject(s)
Glycoproteins , Hydrazones , Indoles , Glucuronidase/antagonists & inhibitors , Glucuronidase/chemistry , Glycoproteins/chemical synthesis , Glycoproteins/chemistry , Hydrazones/chemical synthesis , Hydrazones/chemistry , Indoles/chemical synthesis , Indoles/chemistry , Molecular Docking SimulationABSTRACT
The inhibition of the ß-glucuronidase released from gut bacteria is associated with specific health-related benefits. Though a number of ß-glucuronidase inhibition assays are currently in use, none of them can directly measure the relevant activity of each single constituent in a complex mixture, without prior separation and tedious isolation of the pure compounds. Thus, the hyphenation of the high performance thin layer chromatography (HPTLC) technique with a ß-glucuronidase inhibition assay was investigated and successfully demonstrated for the first time. A colorimetric as well as fluorometric detection of the inhibitors was achieved using 5-bromo-4-chloro-3-indolyl-ß-D-glucuronide as a substrate. Hence, ß-glucuronidase inhibitors were detected as bright zones against an indigo blue or fluorescent background. The established method was optimized and validated employing the well-known inhibitor d-saccharic acid 1,4-lactone monohydrate. As proof of concept, the suitability of the new workflow was verified through analysis of two botanical extracts, Primula boveana and silymarin flavonolignans from Silybum marianum fruits. The found inhibitors were identified by spectroscopic methods; one of them, 3'-O-(ß-galactopyranosyl)-flavone, is here described as a newly isolated natural compound. The new hyphenation HPTLC-UV/Vis/FLD-ß-glucuronidase inhibition assay-HRMS covers four orthogonal dimensions, i.e. separation, spectral detection, biochemical activity and structural characterization, in a highly targeted time- and material-saving workflow for analysis of complex or costly mixtures.
ABSTRACT
Twenty-five thiadiazole derivatives 1-25 were synthesized from methyl 4-methoxybenzoate via hydrazide and thio-hydrazide intermediates, and evaluated for their potential against ß-glucuronidase enzyme. Most of the compounds including 1 (IC50â¯=â¯26.05⯱â¯0.60⯵M), 2 (IC50â¯=â¯42.53⯱â¯0.80⯵M), 4 (IC50â¯=â¯38.74⯱â¯0.70⯵M), 5 (IC50â¯=â¯9.30⯱â¯0.29⯵M), 6 (IC50â¯=â¯6.74⯱â¯0.26⯵M), 7 (IC50â¯=â¯18.40⯱â¯0.66⯵M), and 15 (IC50â¯=â¯18.10⯱â¯0.53⯵M) exhibited superior activity potential than the standard d-saccharic acid-1,4-lactone (IC50â¯=â¯48.4⯱â¯1.25⯵M). Molecular docking studies were conducted to correlate the in vitro results and to identify possible mode of interaction with enzyme active site.
Subject(s)
Enzyme Inhibitors/chemistry , Glucuronidase/antagonists & inhibitors , Thiadiazoles/chemistry , Catalytic Domain , Enzyme Inhibitors/isolation & purification , Enzyme Inhibitors/metabolism , Glucuronidase/chemistry , Glucuronidase/metabolism , Humans , Molecular Docking Simulation , Molecular Structure , Protein Binding , Structure-Activity Relationship , Thiadiazoles/isolation & purification , Thiadiazoles/metabolismABSTRACT
A new series of oxadiazole with thiadiazole moiety (6-27) were synthesized, characterized by different spectroscopic techniques and evaluated for ß-glucuronidase inhibitory potential. Sixteen analogs such as 6, 7, 8, 9, 10, 12, 13, 14, 17, 18, 20, 23, 24, 25, 26 and 27 showed IC50 values in the range of 0.96⯱â¯0.01 to 46.46⯱â¯1.10⯵M, and hence were found to have excellent inhibitory potential in comparison to standard d-saccharic acid 1,4-lactone (IC50â¯=â¯48.4⯱â¯1.25⯵M). Two analogs such as 16 and 19 showed moderate inhibitory potential while analogs 11, 15, 21 and 22 were found inactive. Our study identifies new series of potent ß-glucuronidase inhibitors for further investigation. Structure activity relationships were established for all compounds which showed that the activity is varied due to different substituents on benzene ring. The interaction of the compounds with enzyme active site were confirmed with the help of docking studies, which reveals that the electron withdrawing group and hydroxy group make the molecules more favorable for enzyme inhibition.
Subject(s)
Glycoproteins/therapeutic use , Molecular Docking Simulation/methods , Oxadiazoles/chemical synthesis , Glycoproteins/pharmacology , Oxadiazoles/chemistryABSTRACT
Chromen-4-one substituted oxadiazole analogs 1-19 have been synthesized, characterized and evaluated for ß-glucuronidase inhibition. All analogs exhibited a variable degree of ß-glucuronidase inhibitory activity with IC50 values ranging in between 0.8 ± 0.1-42.3 ± 0.8 µM when compared with the standard d-saccharic acid 1,4 lactone (IC50 = 48.1 ± 1.2 µM). Structure activity relationship has been established for all compounds. Molecular docking studies were performed to predict the binding interaction of the compounds with the active site of enzyme.
Subject(s)
Benzopyrans/chemical synthesis , Benzopyrans/pharmacology , Glycoproteins/chemical synthesis , Glycoproteins/pharmacology , Oxadiazoles/chemical synthesis , Oxadiazoles/pharmacology , Benzopyrans/chemistry , Glucuronidase/chemistry , Glucuronidase/metabolism , Glycoproteins/chemistry , Inhibitory Concentration 50 , Ligands , Molecular Docking Simulation , Oxadiazoles/chemistryABSTRACT
Amoxapine has been demonstrated to be a potent inhibitor of Escherichia coli ß-glucuronidase. This study aims to explore the factors causing unsatisfactory efficacy of amoxapine in alleviating CPT-11-induced gastrointestinal toxicity in mice and to predict the outcomes in humans. Amoxapine (100 µM) exhibited poor and varied inhibition on ß-glucuronidase activity in gut microbiota from 10 healthy individuals and their pool (pool, 11.9%; individuals, 3.6%-54.4%) with IC50 >100 µM and potent inhibition toward E. coli ß-glucuronidase (IC50 = 0.34 µM). p-Nitrophenol formation from p-nitrophenyl-ß-D-glucuronide by pooled and individual gut microbiota fitted classical Michaelis-Menten kinetics, showing similar affinity (Km = 113-189 µM) but varied catalytic capability (Vmax = 53-556 nmol/h/mg). Interestingly, amoxapine showed distinct inhibitory effects (8.7%-100%) toward ß-glucuronidases of 13 bacterial isolates (including four Enterococcus, three Streptococcus, two Escherichia, and two Staphylococcus strains; gus genes belonging to OTU1, 2 or 21) regardless of their genetic similarity or bacterial origin. In addition, amoxapine inhibited the growth of pooled and individual gut microbiota at a high concentration (6.3%-30.8%, 200 µM). Taken together, these findings partly explain the unsatisfactory efficacy of amoxapine in alleviating CPT-11-induced toxicity and predict a poor outcome of ß-glucuronidase inhibition in humans, highlighting the necessity of using a human gut microbiota community for drug screening.
Subject(s)
Amoxapine/pharmacology , Gastrointestinal Microbiome , Glucuronidase/antagonists & inhibitors , Glycoproteins/pharmacology , Amoxapine/chemistry , Bacteria/classification , Bacteria/enzymology , Bacteria/genetics , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Dose-Response Relationship, Drug , Glucuronidase/genetics , Glucuronidase/metabolism , Glycoproteins/chemistry , Humans , Hydrolysis , Kinetics , Molecular Structure , PhylogenyABSTRACT
Natural products are the main source of motivation to design and synthesize new molecules for drug development. Designing new molecules against ß-glucuronidase inhibitory is utmost essential. In this study indole analogs (1-35) were synthesized, characterized using various spectroscopic techniques including 1H NMR and EI-MS and evaluated for their ß-glucuronidase inhibitory activity. Most compounds were identified as potent inhibitors for the enzyme with IC50 values ranging between 0.50 and 53.40µM, with reference to standard d-saccharic acid 1,4-lactone (IC50=48.4±1.25µM). Structure-activity relationship had been also established. The results obtained from docking studies for the most active compound 10 showed that hydrogen bond donor features as well as hydrogen bonding with (Oε1) of nucleophilic residue Glu540 is believed to be the most importance interaction in the inhibition activity. It was also observed that hydroxyl at fourth position of benzylidene ring acts as a hydrogen bond donor and interacts with hydroxyl (OH) on the side chain of catalysis residue Tyr508. The enzyme-ligand complexed were being stabilized through electrostatic π-anion interaction with acid-base catalyst Glu451 (3.96Å) and thus preventing Glu451 from functioning as proton donor residue.
Subject(s)
Glucuronidase/antagonists & inhibitors , Glycoproteins/pharmacology , Indoles/pharmacology , Dose-Response Relationship, Drug , Glucuronidase/metabolism , Glycoproteins/chemical synthesis , Glycoproteins/chemistry , Humans , Indoles/chemical synthesis , Indoles/chemistry , Molecular Structure , Structure-Activity RelationshipABSTRACT
Biotransformation of a steroidal contraceptive drug, etonogestrel (1), (13-ethyl-17ß-hydroxy-11-methylene-18,19-dinor-17α-pregn-4-en-20-yn-3-one) was investigated with Cunninghamella blakesleeana and C. echinulata. Five metabolites 2-6 were obtained on incubation of 1 with Cunninghamella blakesleeana, and three metabolites, 2, 4, and 6 were isolated from the transformation of 1 with C. echinulata. Among them, metabolites 2-4 were identified as new compounds. Their structures were deduced as 6ß-hydroxy-11,22-epoxy-etonogestrel (2), 11,22-epoxy-etonogestrel (3), 10ß-hydroxy-etonogestrel (4), 6ß-hydroxy-etonogestrel (5), and 14α-hydroxy-etonogestrel (6). Compounds 1-6 were evaluated for various biological activities. Interestingly, compound 5 was found to be active against ß-glucuronidase enzyme with IC50 value of 13.97±0.12µM, in comparison to standard compound, d-saccharic acid 1,4-lactone (IC50=45.75±2.16µM). Intestinal bacteria produce ß-glucuronidase. Increased activity of ß-glucuronidase is responsible for the hydrolyses of glucuronic acid conjugates of estrogen and other toxic substances in the colon, which plays a key role in the etiology of colon cancer. Inhibition of ß-glucoronidase enzyme therefore has a therapeutic significance. Compounds 1-6 were also found to be non cytotoxic against 3T3 mouse fibroblast cell lines.
Subject(s)
Contraceptive Agents/metabolism , Cunninghamella/metabolism , Desogestrel/metabolism , Glucuronidase/metabolism , 3T3 Cells , Animals , Contraceptive Agents/chemistry , Desogestrel/chemistry , Magnetic Resonance Spectroscopy , Mice , Molecular StructureABSTRACT
A series of compounds consisting of 25 novel oxadiazole-benzohydrazone hybrids (6-30) were synthesized through a five-step reaction sequence and evaluated for their ß-glucuronidase inhibitory potential. The IC50 values of compounds 6-30 were found to be in the range of 7.14-44.16µM. Compounds 6, 7, 8, 9, 11, 13, 18, and 25 were found to be more potent than d-saccharic acid 1,4-lactone (48.4±1.25µM). These compounds were further subjected for molecular docking studies to confirm the binding mode towards human ß-d-glucuronidase active site. Docking study for compound 13 (IC50=7.14±0.30µM) revealed that it adopts a binding mode that fits within the entire pocket of the binding site of ß-d-glucuronidase. Compound 13 has the maximum number of hydrogens bonded to the residues of the active site as compared to the other compounds, that is, the ortho-hydroxyl group forms hydrogen bond with carboxyl side chain of Asp207 (2.1Å) and with hydroxyl group of Tyr508 (2.6Å). The other hydroxyl group forms hydrogen bond with His385 side chain (2.8Å), side chain carboxyl oxygen of Glu540 (2.2Å) and Asn450 side-chain's carboxamide NH (2.1Å).
Subject(s)
Glucuronidase/antagonists & inhibitors , Hydrazones/pharmacology , Oxadiazoles/pharmacology , Catalytic Domain , Humans , Hydrazones/chemical synthesis , Hydrazones/chemistry , Hydrogen Bonding , Molecular Docking Simulation , Oxadiazoles/chemical synthesis , Oxadiazoles/chemistryABSTRACT
A series of 15 novel compounds incorporating the thieno[2,3-b]thiophene moiety were synthesized. The chemical structures of these compounds were deduced from elemental analyses, (1)H NMR, (13)C NMR, and ESI-mass spectral data. The enzyme inhibition potential of these compounds was evaluated, in vitro, against ß-glucuronidase, xanthine oxidase, and α-chymotrypsin enzymes. The cytotoxicity was evaluated by a cell viability assay utilizing the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye. Among the compounds tested, compound 3 was the most potent ß-glucuronidase inhibitor with an IC50 value of 0.9 ± 0.0138 µM; it was much more active than the standard, d-saccharic acid 1,4-lactone (IC50=45.75 ± 2.16 µM). Compound 12, on the other hand, was the most potent as a xanthine oxidase inhibitor with an IC50 of 14.4 ± 1.2 µM. With the characterization of their mechanism of action and with further testing, these compounds could be useful candidates as anticancer drugs. In addition, the newly synthesized compounds were subjected to POM analyses to get insights about their degree of their toxicity.
Subject(s)
Glucuronidase/antagonists & inhibitors , Glycoproteins/chemical synthesis , Glycoproteins/pharmacology , Pyridines/chemistry , Pyridines/pharmacology , Thiophenes/chemistry , Thiophenes/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Crystallography, X-Ray , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Glucuronidase/metabolism , Glycoproteins/chemistry , Humans , Models, Molecular , Molecular Structure , Structure-Activity RelationshipABSTRACT
2-Arylquinazolin-4(3H)-ones 1-25 were synthesized by reacting anthranilamide with various benzaldehydes using CuCl2·2H2O as a catalyst in ethanol under reflux. Synthetic 2-arylquinazolin-4(3H)-ones 1-25 were evaluated for their ß-glucuronidase inhibitory potential. A trend of inhibition IC50 against the enzyme in the range of 0.6-198.2µM, was observed and compared with the standard d-saccharic acid 1,4-lactone (IC50=45.75±2.16µM). Compounds 13, 19, 4, 12, 14, 22, 23, 25, 15, 8, 17, 11, 21, 1, 3, 18, 9, 2, and 24 with the IC50 values within the range of 0.6-44.0µM, indicated that the compounds have superior activity than the standard. The compounds showed no cytotoxic effects against PC-3 cells. A structure-activity relationship is established.
Subject(s)
Enzyme Inhibitors/pharmacology , Glucuronidase/metabolism , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Escherichia coli/enzymology , Molecular Structure , Structure-Activity RelationshipABSTRACT
Biotransformation of the anabolic steroid dianabol (1) by suspended-cell cultures of the filamentous fungi Cunninghamella elegans and Macrophomina phaseolina was studied. Incubation of 1 with C. elegans yielded five hydroxylated metabolites 2-6, while M. phaseolina transformed compound 1 into polar metabolites 7-11. These metabolites were identified as 6ß,17ß-dihydroxy-17α-methylandrost-1,4-dien-3-one (2), 15α,17ß-dihydroxy-17α-methylandrost-1,4-dien-3-one (3), 11α,17ß-dihydroxy-17α-methylandrost-1,4-dien-3-one (4), 6ß,12ß,17ß-trihydroxy-17α-methylandrost-1,4-dien-3-one (5), 6ß,15α,17ß-trihydroxy-17α-methylandrost-1,4-dien-3-one (6), 17ß-hydroxy-17α-methylandrost-1,4-dien-3,6-dione (7), 7ß,17ß,-dihydroxy-17α-methylandrost-1,4-dien-3-one (8), 15ß,17ß-dihydroxy-17α-methylandrost-1,4-dien-3-one (9), 17ß-hydroxy-17α-methylandrost-1,4-dien-3,11-dione (10), and 11ß,17ß-dihydroxy-17α-methylandrost-1,4-dien-3-one (11). Metabolite 3 was also transformed chemically into diketone 12 and oximes 13, and 14. Compounds 6 and 12-14 were identified as new derivatives of dianabol (1). The structures of all transformed products were deduced on the basis of spectral analyses. Compounds 1-14 were evaluated for ß-glucuronidase enzyme inhibitory activity. Compounds 7, 13, and 14 showed a strong inhibition of ß-glucuronidase enzyme, with IC50 values between 49.0 and 84.9 µM.
Subject(s)
Biotransformation , Fungi/metabolism , Methandrostenolone/chemistry , Animals , Caenorhabditis elegans/drug effects , Fungi/enzymology , Fungi/growth & development , Glucuronidase/antagonists & inhibitors , Glucuronidase/metabolism , HydroxylationABSTRACT
Bisindole analogs 1-17 were synthesized and evaluated for their in vitro ß-glucuronidase inhibitory potential. Out of seventeen compounds, the analog 1 (IC50=1.62±0.04 µM), 6 (IC50=1.86±0.05 µM), 10 (IC50=2.80±0.29 µM), 9 (IC50=3.10±0.28 µM), 14 (IC50=4.30±0.08 µM), 2 (IC50=18.40±0.09 µM), 19 (IC50=19.90±1.05 µM), 4 (IC50=20.90±0.62 µM), 7 (IC50=21.50±0.77 µM), and 3 (IC50=22.30±0.02 µM) showed superior ß-glucuronidase inhibitory activity than the standard (d-saccharic acid 1,4-lactone, IC50=48.40±1.25 µM). In addition, molecular docking studies were performed to investigate the binding interactions of bisindole derivatives with the enzyme. This study has identified a new class of potent ß-glucouronidase inhibitors.