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We synthesised eight derivatives of 4-aminoquinolines differing in the substituents attached to the C(4)-amino group and C(7) carbon of 4-aminoquinoline, and tested their potency to inhibit human AChE and BChE. All of the compounds reversibly inhibited both enzymes with dissociation inhibition (Ki) constants from 0.50 to 50 µM exhibiting selectivity. In other words, for all compounds, AChE exhibited higher affinity than BChE. The most potent inhibitors of AChE were compounds with an octyl chain or adamantane, regardless of the group in position C(7). The shortening of the chain length caused the AChE inhibition decrease by 5-20 times. Docking studies made it clear that the high AChE affinity resulted from simultaneous interactions of the quinoline group with aromatic residues of both the catalytic active site and the peripheral site. In conclusion, the inhibition potency and selectivity classify several novel compounds as leads for further modification and optimization towards the development of new inhibitors of AChE and potential drugs for treatment of neurodegenerative diseases. 13th International Meeting on Cholinesterases and and the 7th International Conference on Paraoxonases, 9th to 14th of September 2018 Hradec Králové, Czech Republic

We investigated the influence of bronchodilating β2-agonists on the activity of human acetylcholinesterase (AChE) and usual, atypical and fluoride-resistant butyrylcholinesterase (BChE). We determined the inhibition potency of racemate and enantiomers of fenoterol as a resorcinol derivative, isoetharine and epinephrine as catechol derivatives and salbutamol and salmeterol as saligenin derivatives. All of the tested compounds reversibly inhibited cholinesterases with Ki constants ranging from 9.4 μM to 6.4 mM and had the highest inhibition potency towards usual BChE, but generally none of the cholinesterases displayed any stereoselectivity. Kinetic and docking results revealed that the inhibition potency of the studied compounds could be related to the size of the hydroxyaminoethyl chain on the benzene ring. The additional π–π interaction of salmeterol’s benzene ring and Trp286 and hydrogen bond with His447 probably enhanced inhibition by salmeterol which was singled out as the most potent inhibitor of all the cholinesterases.

We investigated the influence of bronchodilating β2-agonists on the activity of human acetylcholinesterase (AChE) and usual, atypical and fluoride-resistant butyrylcholinesterase (BChE). We determined the inhibition potency of racemate and enantiomers of fenoterol as a resorcinol derivative, isoetharine and epinephrine as catechol derivatives and salbutamol and salmeterol as saligenin derivatives. All of the tested compounds reversibly inhibited cholinesterases with Ki constants ranging from 9.4 μM to 6.4 mM and had the highest inhibition potency towards usual BChE, but generally none of the cholinesterases displayed any stereoselectivity. Kinetic and docking results revealed that the inhibition potency of the studied compounds could be related to the size of the hydroxyaminoethyl chain on the benzene ring. The additional π–π interaction of salmeterol’s benzene ring and Trp286 and hydrogen bond with His447 probably enhanced inhibition by salmeterol which was singled out as the most potent inhibitor of all the cholinesterases.

Catalytic activity of acetylcholinesterase (EC 3.1.1.7 ; AChE) was inhibited with oximes HI-6, K114, K127 and K203, and inhibition constants were determined. Dissociation constants for reversible enzyme-inhibitor complex (KI) were determined in the presence of acetylthiocholine. Based on the mixed inhibition model dissociation constants were 0.020 mM for HI-6, 0.0021 mM for K114, 0.175 mM for K127, and 0.036 mM for K203. Therefore, the most potent inhibitor of AChE was K114, while the weakest was K127. Molecular modelling of AChE-oxime complexes was used to determine aminoacid residues of the active site involved in the interactions. Bis-oxime K114 achieved the best stabilisation in the active site due to π -π interaction between its three aromatic rings and Tyr124, Tyr341 and Trp86, and hydrogen bonds formed by its oxime groups. Mono-oximes HI-6 and K203 that inhibited enzyme with similar potency, showed similar position of their pyridinium rings in the active site. K127 also formed several hydrogen bonds with residues of the active site, but due to its long linker more likely was stabilised at the peripheral site (Tyr124).

Liver fibrosis is a frequent event which follows chronic damage to the liver, characterized by excess deposition of connective tissue in extracellular matrix and a distortion of the liver tissue architecture by forming a fibrous scar. The aim of this study was to investigate the hepatoprotective effects of anthocyanidin delphinidin in carbon tetrachloride (CCl4)-induced liver fibrosis in mice. Male Balb/C mice were treated with CCl4 (20% CCl4 in olive oil, 2 mL/kg) intraperitoneally (i.p.), twice a week for 7 weeks. Delphinidin was administered i.p. once daily for next 2 weeks, in doses of 10 and 25 mg/kg of body weight. The CCl4 control group has been observed for spontaneous reversion of fibrosis. CCl4-administration increased serum transaminase and alkaline phosphatase levels, and oxidative stress in the liver. Delphinidin has attenuated oxidative stress, increased matrix metalloproteinase-9 and metallothionein I/II expression, and restored hepatic architecture in a dose-dependent manner. Hepatic overexpression of tumor necrosis factor-α and transforming growth factor-β1 has been completely withdrawn by delphinidin. Concomitantly, the expression of α-smooth muscle actin indicated returning of hepatic stellate cells (HSC) into inactive state. Our results suggest the therapeutic effects of delphinidin in CCl4-induced liver fibrosis by promoting extracellular matrix degradation, HSC inactivation and down-regulation of fibrogenic stimuli, with strong enhancement of hepatic regenerative capability.

Selected flavonoids: galangin, kaempferol, quercetin, myricetin, fisetin, apigenin, luteolin and rutin, reversibly inhibited human butyrylcholinesterase (BChE, EC 3.1.1.8). Inhibition potency of the flavonoids we attributed to their chemical structure, i.e., the number of OH groups and their side on the phenyl ring. The most potent BChE inhibitor among the tested flavonoids was galangin, which showed 12 times higher preference for binding to BChE (Ki = 7 μ mol/L) than to the related enzyme human acetylcholinesterase (AChE, EC 3.1.1.7). Docking study showed that flavonoids bind to the BChE active site by forming multiple hydrogen bonds and π -π interactions. The UV-VIS (200-500 nm) absorption spectra of the flavonoid phosphate buffer solution (pH 7.4), with the exception of rutin, revealed time dependant changes indicating precipitation of flavonoids or in the case of myricetin, a change in the chemical structure resulting in a BChE non-inhibiting specie. Selected flavonoids showed no cytotoxic effect on HepG2 and A549 cell lines at concentrations up to 200 μ mol/L. Cytotoxicity was observed only for fisetin, apigenin and luteolin in the THP-1 cell line with IC50 of 30, 60 and 70 μ mol/L, respectively. Galangin is therefore singled out as a promising hit in the search for new BChE inhibitors.

Introduction: The process of digestion takes place in the intestinal lumen, mediated by a large number of hydrolases secreted in the gastrointestinal tract or produced by the enterocytes in the brush border. Loss of intestinal brush border membrane (BBM) enzymes in aged animals could result in impaired digestion and absorption of all nutrients, which could consequently lead to malnutrition in older age. Objective: The objective of this study was to determine and describe the age-related changes in intestinal BBM enzyme activities which occur in C57Bl/6 mice. Methods: Wild type C57Bl/6 male mice aged 2-weeks, 1-, 2-, 3-, 6- and 12-months were used in this study. Three intestinal segments were analyzed: jejunum, duodenum and ileum. The relationship between ageing and BBM enzymes was characterized using a multiple-sampling technique that quantifies intestinal enzyme activity. Specific dipeptidyl peptidase IV/CD26, disaccharidases (lactase, sucrase and maltase) and alkaline phosphatase activities were determined spectrophotometrically. Results: An emphasized correlation between analyzed BBM enzyme activities and animal age was found. Our study revealed that total intestinal dipeptidyl peptidase IV/CD26, lactase, sucrase, maltase and alkaline phosphatase activities decline significantly with age (P

Non-ribosomal peptide synthetases (NRPS) are molecular machines that synthesize peptide-based natural products, many of which are important pharmaceuticals with antibiotic, immunosuppressor and antitumor activities. A typical NRPS assembly line consists of a number of catalytic modules, each responsible for the incorporation of a single amino acid into the growing peptide product. Modules themselves comprise distinct catalytic domains, such as the adenylation domain (A) that, firstly, selects a specific amino acid monomer to be activated by ATP and, secondly, transfers it to the phosphopantetheinyl arm of the adjacent thiolation domain. Recent biochemical and structural evidence support the idea that the A domain uses a rotational movement of its two subdomains to adapt a single active site for the two partial reactions in the catalytic cycle. It has long been recognized that A domains share a set of 10 conserved motifs (A1-A10), most of which where assigned a particular role in substrate binding and/or catalysis. To our knowledge, this is the first study aimed at elucidating the role of the A9 motif. By analyzing homology models of the A domain from tyrocidine synthetase 1 from Bacillus brevis, it could be observed that during the rotational movement of the subdomains, the A9 motif drastically changes its position relative to the active site. The role of the conserved amino acid residues in this region and its importance for the two partial reactions of the catalytic cycle was examined by site-directed mutagenesis and biochemical characterization of mutant enzymes.