Antiepileptic drugs: Impacts on human serum paraoxonase‐1

Serum paraoxonase (PON1) is a key enzyme related to high‐density lipoprotein (HDL)‐cholesterol particle. It can prevent the oxidation of low‐density lipoprotein (LDL) and HDL. The present article focuses on the in vitro inhibition role of some antiepileptic drugs (AEDs) such as valproic acid, gabapentin, primidone, phenytoin, and levetiracetam on human paraoxonase (hPON1). Therefore, PON1 was purified from human serum with a specific activity of 3976.36 EU/mg and 13.96% yield by using simple chromatographic methods. The AEDs were tested at various concentrations, which showed reduced in vitro hPON1 activity. IC₅₀ values for gabapentin, valproic acid, primidone, phenytoin, and levetiracetam were found to be 0.35, 0.67, 0.87, 6.3, and 53.3 mM, respectively. K_i constants were 0.261 ± 0.027, 0.338 ± 0.313, 0.410 ± 0.184, 10.3 ± 0.001, and 43.01 ± 0.003 mM, respectively. Gabapentin exhibited effective inhibitory activity as compared with the other drugs. The inhibition mechanisms of all compounds were noncompetitive.     

Assessment of the inhibitory effects and molecular docking of some sulfonamides on human serum paraoxonase 1

Paraoxonase‐1 (PON1) is an organophosphate hydrolyzer and antiatherogenic enzyme. Due to the PON1's crucial functions, inhibitors and activators of PON1 must be known for pharmacological applications. In this study, we investigated the in vitro effects of some sulfonamides compounds on human serum PON1 (hPON1). For this aim, we purified the hPON1 from human serum with high specific activity by using simple chromatographic methods, and after the purification processes, we investigated in vitro interactions between the enzyme and some sulfonamides (2‐amino‐5‐methyl‐1,3‐benzenedisulfonamide, 2‐chloro‐4‐sülfamoilaniline, 4‐amino‐3‐methylbenzenesulfanilamide, sulfisoxazole, sulfisomidine, and 5‐amino‐2‐methylbenzenesulfonamide). IC₅₀, K_i values, and inhibition types were calculated for each sulfonamide. 2‐amino‐5‐methyl‐1,3‐benzenedisulfonamide and 2‐chloro‐4‐sülfamoilaniline exhibited noncompetitive inhibition effect, whereas 4‐amino‐3‐methylbenzenesulfanilamide, sulfisoxazole, and sulfisomidine exhibited mixed type inhibition. On the other hand, 5‐amino‐2‐methylbenzenesulfonamide showed competitive inhibition and so molecular docking studies were performed for this compound in order to assess the probable binding mechanism into the active site of hPON1.     

Amphenicol and macrolide derived antibiotics inhibit paraoxonase enzyme activity in human serum and human hepatoma cells (HepG2) in vitro

Human serum paraoxonase (hPON1) was separately purified by ammonium sulfate precipitation and hydrophobic interaction chromatography. The in vitro effects of commonly used antibiotics, namely clarithromycin and chloramphenicol, on purified human serum paraoxonase enzyme activity (serum hPON1) and human hepatoma (HepG2) cell paraoxonase enzyme activity (liver hPON1) were determined. Serum hPON1 and liver hPON1 were determined using paraoxon as a substrate and IC(50) values of these drugs exhibiting inhibition effects were found from graphs of hydratase activity (%) by plotting concentration of the drugs. We determined that chloramphenicol and clarithromycin were effective inhibitors of serum hPON1.     

An alternative purification method for human serum paraoxonase 1 and its interactions with anabolic compounds

In this study, an alternative purification method for human paraoxonase 1 (hPON1) enzyme was developed using two-step procedures, namely, ammonium sulfate precipitation and Sepharose-4B-l-tyrosine-3-aminophenantrene hydrophobic interaction chromatography. SDS-polyacrylamide gel electrophoresis of the enzyme indicates a single band with an apparent M_W of 43?kDa. The enzyme was purified 219-fold with a final specific activity of 4?408?400?U/mg and a yield of 10%. Furthermore, we examined the in vitro effects of some anabolic compounds, such as zeranol, 17 β-estradiol, diethylstilbestrol, oxytocin, and trenbolone on the enzyme activity to understand the better inhibitory properties of these molecules. The five anabolic compounds dose dependently decreased the activity of hPON1 with inhibition constants in the millimolar–micromolar range. The results show that these compounds exhibit inhibitory effects on hPON1 at low concentrations with IC₅₀ values ranging from 0.064 to 16.900?µM.     

Human serum paraoxonase-1 (hPON1): in vitro inhibition effects of moxifloxacin hydrochloride,levofloxacin hemihidrate,cefepime hydrochloride,cefotaxime sodium and ceftizoxime sodium

Abstract

In this study, we investigated the effects of antibacterial drugs (moxifloxacin hydrochloride, levofloxacin hemihidrate, cefepime hydrochloride, cefotaxime sodium and ceftizoxime sodium) on human serum paraoxonase-1 (hPON1) enzyme activity from human serum in vitro conditions. For this purpose, hPON1 enzyme was purified from human serum using simple chromatographic methods. The antibacterial drugs exhibited inhibitory effects on hPON1 at low concentrations. K_i constants were calculated to be 2.641?±?0.040?mM, 5.525?±?0.817?mM, 35.092?±?1.093?mM, 252.762?±?5.749?mM and 499.244?±?10.149?mM, respectively. The inhibition mechanism of moxifloxacin hydrochloride was competitive, whereas levofloxacin hemihidrate, cefepime hydrochloride, cefotaxime sodium and ceftizoxime sodium were noncompetitive inhibitors.     

Effect of some analgesics on Paraoxonase-1 purified from human serum

The in vitro effects of the analgesic drugs, lornoxicam, indomethacin, tenoxicam, diclofenac sodium, ketoprofen and lincomycine, on the activity of purified human serum paraoxonase (hPON1) (EC 3.1.8.1.) were evaluated. hPON1 was purified from human serum with a final specific activity of 3840 U mg^?1 and a purity of 25.3 % using simple chromatographic methods, including DEAE-Sephadex anion exchange and Sepharose 4B-L-tyrozine-1-napthylamine hydrophobic interaction chromatography. SDS-polyacrylamide gel electrophoresis indicated a single protein band corresponding to hPON1. The six analgesics dose-dependently decreased in vitro hPON1 activity, with IC₅₀ values for lornoxicam, indomethacin, tenoxicam, diclofenac sodium, ketoprofen and lincomycine of 0.136, 0.195, 0.340, 1.639, 6.23 and 9.638 mM, respectively. K_i constants were 0.009, 0.097, 0.306, 0.805, 13.010 and 11.116 mM, respectively. Analgesics showed different inhibition mechanisms: lornoxicam, diclofenac sodium and lincomycine were uncompetitive, indomethacin and tenoxicam were competitive, ketoprofen was noncompetitive. According to the results, inhibition potency was lornoxicam>indomethacin>tenoxicam> diclofenac sodium>ketoprofen> lincomycine.     

Differential effects of some antibiotics on paraoxonase enzyme activity on human hepatoma cells (HepG2) in vitro

Serum paraoxonase (aryldialkylphosphatase, EC 3.1.8.1., PON1) is an esterase protein synthesised by the liver and released into the serum, where it is associated with HDL lipoproteins. In this study, we have determined the in vitro effects of the following antibiotics: sodium ampicillin, ciprofloxacin, Rifamycin SV and clindamiycin phosphate, on human hepatoma (HepG2) cells (liver hPON1). All the antibiotics caused a dose-dependent and time-dependent decrease in the paraoxonase activity while Rifamycin SV was the most effective antibiotic due to its low 50% inhibition concentration (IC₅₀) value. Liver hPON1 activity was determined using paraoxon as a substrate. The IC₅₀ values of the drugs were calculated from graphs of hydratase activity (%) by plotting concentration of the drugs that showed an inhibition effect.     

Anticancer potency of small linear and cyclic tetrapeptides and pharmacokinetic investigations of peptide binding to human serum albumin

We have in the present study explored the anticancer activity against human Burkitt's lymphoma cells (Ramos) of a series of small linear and cyclic tetrapeptides containing a β^2,2‐amino acid with either two 2‐naphthyl‐methylene or two para‐CF₃‐benzyl side chains, along with their interaction with the main plasma protein human serum albumin (HSA). The cyclic and more amphipathic tetrapeptides revealed a notably higher anticancer potency against Ramos cells [50% inhibitory concentration (IC₅₀) 11–70 μM] compared to the linear tetrapeptide counterparts (IC₅₀ 18.7 to >413 μM). The most potent cyclic tetrapeptide c3 had a 16.5‐fold preference for Ramos cells compared to human red blood cells, whereas the cyclic tetrapeptide c1 both showed low hemolytic activity and displayed the overall highest (2.9‐fold) preference for Ramos cells (IC₅₀ 23 μM) compared to healthy human lung fibroblast cells (MRC‐5). Investigating the interaction of selected tetrapeptides and recently reported hexapeptides with HSA revealed that the peptides bind to drug site II of HSA in the 22–28 μM range, disregarding size and overall structure. NMR and in silico molecular docking experiments identified the lipophilic residues as responsible for the interaction, but in vitro studies showed that the anticancer potency of the peptides varied in the presence of HSA and that c3 remained the most potent peptide. Based on our findings, we call for implementing serum albumin binding in development of anticancer peptides, as it may have implications for future administration and systemic distribution of peptide‐based cancer drugs. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Purification of PON1 from Human Serum and Assessment of Enzyme Kinetics Against Metal Toxicity

Paraoxonase-1 (PON1) is an organophosphate hydrolyser enzyme which has also antioxidant properties in metabolism. Due to its crucial functions, inhibition of the enzyme is undesirable and very dangerous. PON1 enzyme activity should not be altered in any case. Inhibitory investigations of this enzyme are therefore important and useful. Metal toxicology of enzymes has become popular in the recent years. Here, we report the in vitro inhibitory effects of some metal ions, including Pb⁺², Cr⁺², Fe⁺², and Zn⁺², on the activity of human serum PON1 (hPON1; EC 3.1.8.1.). For this purpose, we purified the enzyme from human serum and analyzed the alterations in the enzyme activity in the presence of metal ions. The results show that metal ions exhibit inhibitory effects on hPON1 at low concentrations with IC ₅₀ values ranging from 0.838 to 7.410 mM. Metal ions showed different inhibition mechanisms: lead and iron were competitive, chrome was noncompetitive, and zinc was uncompetitive. Lead was determined to be the most effective inhibitor.     

Comparison of Aromatase Activity in Human Prostatic,Testicular and Placental Tissues

Abstract

The aromatase enzyme was quantified by the release of tritiated water from [1β-³H] androstenedione. Tritiated water was released by the crude homogenates in 4 of 18 samples of benign prostatic hyperplasia tissue and one of 5 samples of prostate carcinoma tissue. However, this apparent aromatase activity was not inhibited by 4-hydroxyandrostenedione (0.5 and 5.0μM), and none of the particulate fractions (100,000 g pellet) prepared from each of the prostatic tissues exhibited aromatase activity. Particulate fractions from rat ovary (n = 3) and human testes (n = 6) displayed significant aromatase activity (mean values of 9.9 and 0.033 nmol estrone formed/g protein/h, respectively). The testicular aromatase was inhibited by aminoglutethimide, 4-hydroxyandrostenedione and CGS 16949A with IC₅₀ values of 6.4, 0.17 and 0.0017 μM. respectively. These are of a similar order to values obtained with the aromatase enzyme from human placental microsomes (14, 0.43 and 0.0075μM, respectively).     

In vitro and in vivo effects of some benzodiazepine drugs on human and rabbit erythrocyte carbonic anhydrase enzymes

Carbonic anhydrase inhibitors (CAIs) are a class of pharmaceuticals used as antiglaucoma agents, diuretics and antiepileptics. Thus, discovery of novel CAIs has become of great importance in the recent years. In the current study, in vitro and in vivo inhibition effects of benzodiazepine drugs, diazepam and midazolam, on human erythrocytes carbonic anhydrase I and II isozymes were investigated. After purification of the isoenzymes, in vitro inhibition assays were performed and K_i values were determined to be of 141.5 μM and 40.7 μM for hCA I and of 5.11 μM and 0.58 μM against hCA II by the esterase activity assay, respectively. The drugs showed strong inhibitory effects on hCA II, in the same range as the clinically used sulphonamide acetazolamide. For in vivo studies, five adult male New Zealand White rabbits (3–4.2?kg) were selected for intravenous administrations of the drugs (2?mg/kg and 0.2?mg/kg body weight, respectively). The enzyme was significantly inhibited by 2?mg/kg diazepam (p?<?0.05), and 0.2?mg/kg midazolam (p?<?0.05) for up to 30?min following intravenous administration.     

Novel amides of 1,1‐bis‐(carboxymethylthio)‐1‐arylethanes: Synthesis,characterization, acetylcholinesterase,butyrylcholinesterase, and carbonic anhydrase inhibitory properties

The thiolation reaction was carried out in a benzene solution at 80°C and p‐substituted ketones and mercaptoacetic acid in a molar ratio (1:4) of in the presence of a catalytic amount of toluene sulfonic acids. The enzyme inhibition activities of the novel amides of 1,1‐bis‐(carboxymethylthio)‐1‐arylethanes derivatives were investigated. These novel amides of 1,1‐bis‐(carboxymethylthio)‐1‐arylethanes derivatives showed good inhibitory action against acetylcholinesterase (AChE) butyrylcholinesterase (BChE), and human carbonic anhydrase I and II isoforms (hCA I and II). AChE inhibitors, interacting with the enzyme as their primary target, are applied as relevant drugs and toxins. Many clinically established drugs are carbonic anhydrase inhibitors, and it is highly anticipated that many more will eventually find their way into the market. The novel synthesized compounds inhibited AChE and BChE with K_i values in the range of 0.64–1.47 nM and 9.11–48.12 nM, respectively. On the other hand, hCA I and II were effectively inhibited by these compounds, with K_i values between 63.27–132.34 and of 29.63–127.31 nM, respectively.     

Inhibition Mode of Soybean Lipoxygenase‐1 by Quercetin

Quercetin noncompetitively inhibited the peroxidation of linoleic acid catalyzed by soybean lipoxygenase‐1 (EC 1.13.11.12, Type 1) with an IC₅₀ value of 4.8 μM (1.45 μg/ml). This inhibition is considered to proceed in sequential order, by initially reducing the ferric form of the enzyme to an inactive ferrous form and then, by chelating the iron of the active site of the enzyme. In the pseudoperoxidase assay, quercetin was slowly oxidized by hydroperoxides to a rather stable intermediate, 2‐(3,4‐dihydroxybenzoyl)‐2,4,6‐trihydroxybenzofuran‐3(2H)‐one, and this oxidized intermediate still inhibited the enzymatic oxidation, probably as a chelator. Rutin and kaempferol also exhibited lipoxygenase‐1 inhibitory activity, but to a much lesser extent than quercetin.     

Inactivation of acetylcholinesterase by various fluorophores

The inhibition of recombinant mouse acetylcholinesterase (rMAChE) and electric eel acetylcholinesterase (EEAChE) by seven, structurally different chromophore-based (dansyl, pyrene, dabsyl, diethylamino- and methoxycoumarin, Lissamine rhodamine B, and Texas Red) propargyl carboxamides or sulfonamides was studied. Diethylaminocoumarin, Lissamine, and Texas Red amides inhibited rMAChE with IC₅₀ values of 1.00 μM, 0.05 μM, and 0.70 μM, respectively. Lissamine and Texas Red amides inhibited EEAChE with IC₅₀ values of 3.57 and 10.4 μM, respectively. The other chromophore amides did not inhibit either AChE. The surprising inhibitory potency of Lissamine was examined in further detail against EEAChE and revealed a mixed-type inhibition with K_i?=?11.7 μM (competitive) and K_i′?=?24.9 μM (noncompetitive), suggesting that Lissamine binds to free enzyme and enzyme–substrate complex.     

Purification and partial characterization of rat kidney histamine-N-methyltransferase

Histamine-N-methyltransferase (EC 2.1.1.8) was purified 1700-fold with a yield of 9% from rat kidney. Purification included ammonium sulfate precipitation, linear gradient DEAE-cellulose chromotography and S-adenosylhomocysteine affinity chromotography. The purified enzyme preparation showed a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of 35 000. The isoelectric point of the enzyme was at pH 5.2. The purified enzyme preparation did not contain detectable amounts of histamine. The purified enzyme was totally inhibited in 100 μM parahydroxymercuric benzoate and in 10 μM iodoacetamide, and it was found to be stabilized with dithiothreitol (1 mM), suggesting that the enzyme has an SH-group in the active center. The K_m values for histamine and S-adenosylmethionine were 6.0 and 7.1 μM, respectively. 50% inhibition of histamine-N-methyltransferase was obtained at 28 μM S-adenosylhomocysteine and 100 μM methylhistamine. The purified enzyme was slightly inhibited in 1 mM methylthioadenosine. Histamine in concentrations higher than 25 μM caused substrate inhibition.     

Purification and Characterisation of the Enantiospecific Dioxygenases from Delftia acidovorans MC1 Initiating the Degradation of Phenoxypropionate and Phenoxyacetate Herbicides

After cultivation on (R,S)‐2‐(2,4‐dichlorophenoxy)propionate, two α‐ketoglutarate‐dependent dioxygenases were isolated and purified from Delftia acidovorans MC1, catalysing the cleavage of the ether bond of various phenoxyalkanoate herbicides. One of these enzymes showed high specificity for the cleavage of the R‐enantiomer of substituted phenoxypropionate derivatives: the K_m values were 55 μM and 30 μM, the k_cat values 55 min^–1 and 34 min^–1 with (R)‐2‐(2,4‐dichlorophenoxy)propionate [(R)‐2,4‐DP] and (R)‐2‐(4‐chloro‐2‐methylphenoxy)propionate, respectively. The other enzyme predominantly utilised the S‐enantiomers with K_m values of 49 μM and 22 μM, and k_cat values of 50 min^–1 and 46 min^–1 with (S)‐2‐(2,4‐dichlorophenoxy)propionate [(S)‐2,4‐DP] and (S)‐2‐(4‐chloro‐2‐methylphenoxy)propionate, respectively. In addition, it cleaved phenoxyacetate herbicides (i.e. 2,4‐dichlorophenoxyacetate: K_m = 123 μM, k_cat = 36 min^–1) with significant activity. As the second substrate, only α‐ketoglutarate served as an oxygen acceptor for both enzymes. The enzymes were characterised by excess substrate inhibition kinetics with apparent K_i values of 3 mM with (R)‐2,4‐DP and 1.5 mM with (S)‐2,4‐DP. The reaction was strictly dependent on the presence of Fe²⁺ and ascorbate; other divalent cations showed inhibitory effects to different extents. Activity was completely extinguished within 2 min in the presence of 100 μM diethylpyrocarbonate (DEPC).     

Inhibitory effects of some drugs on carbonic anhydrase enzyme purified from Kangal Akkaraman sheep in Sivas,Turkey

In this study, carbonic anhydrase (CA) enzyme was purified and characterized from blood samples of Kangal Akkaraman sheep and inhibitory properties on certain antibiotics were examined. CA purification was composed of preparation of the hemolysate and conducting the Sepharose‐4B‐tyrosine‐sulfanilamide affinity gel chromatography in having specific activity of 11626 EU mg^?1, yield of 14.40%, and 242.76‐fold purification. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis was performed to assess the enzyme purity and a single band was observed. Some antibiotics were exhibited in vitro inhibition on the CA activity. IC₅₀ values of these inhibitors were calculated by plotting activity percentage. IC₅₀ values of certain drugs (dexamethasone; caffeine; metamizole sodium; tetramisol; ceftiofur HCl; ivermectin; tavilin 50; penokain G; neosym; and sulfamezathine) were found as 0.38, 8.24, 285.53, 114.77, 5.33, 2.76, 27.58, 213.50, 208.28, and 36.60 μM, respectively. K_i values of different drugs on Kangal Akkaraman sheep blood CA activity were found in the range of 0.21 ± 0.038–266.64 ± 37.11 μM.     

A new ferulic acid ester, a new ellagic acid derivative, and other constituents from pachycentria formosana: effects on neutrophil pro-inflammatory responses

A new ferulic acid ester derivative, tetracosane‐1,24‐diyl di[(Z)‐ferulate] ( 1 ), and a new ellagic acid derivative, 3,4 : 3′,4′‐bis(O,O‐methylene)ellagic acid ( 2 ), have been isolated from leaves and twigs of Pachycentria formosana, together with eight known compounds. Their structures were determined by in‐depth spectroscopic and mass‐spectrometric analyses. Among the isolated compounds, oleanolic acid ( 6 ), ursolic acid acetate ( 7 ), and 3‐epibetulinic acid ( 9 ) exhibited potent inhibition (IC₅₀ values ≤21.8 μM ) of O₂^⋅− generation by human neutrophils in response to N‐formyl‐L ‐methionyl‐L ‐leucyl‐L ‐phenylalanine/cytochalasin B (fMLP/CB). In addition, oleanolic acid ( 6 ), 3‐O‐[(E)‐feruloyl]ursolic acid ( 8 ), 3‐epibetulinic acid ( 9 ), and lawsonic acid ( 10 ) also inhibited fMLP/CB‐induced elastase release with IC₅₀ values ≤18.6 μM .     

Purification of glutathione S‐transferase enzyme from quail liver tissue and inhibition effects of (3aR,4S,7R,7aS)‐2‐(4‐((E)‐3‐(aryl)acryloyl)phenyl)‐3a,4,7,7a‐tetrahydro‐1H‐4,7‐methanoisoindole‐1,3(2H)‐dione derivatives on the enzyme activity

The use of quail meat and eggs has made this animal important in recent years, with its low cost and high yields. Glutathione S‐transferases (GST, E.C.2.5.1.18) are an important enzyme family, which play a critical role in detoxification system. In our study, GST was purified from quail liver tissue with 47.88‐fold purification and 12.33% recovery by glutathione agarose affinity chromatography. The purity of enzyme was checked by SDS‐PAGE method and showed a single band. In addition, inhibition effects of (3aR,4S,7R,7aS)‐2‐(4‐((E)‐3‐(aryl)acryloyl)phenyl)‐3a,4,7,7a‐tetrahydro‐1H‐4,7methanoisoindole‐1,3(2H)‐dion derivatives ( 1a–g ) were investigated on the enzyme activity. The inhibition parameters (IC₅₀ and K_i values) were calculated for these compounds. IC₅₀ values of these derivatives ( 1a–e ) were found as 23.00, 15.75, 115.50, 10.00, and 28.75 μM, respectively. K_i values of these derivatives ( 1a–e ) were calculated in the range of 3.04 ± 0.50 to 131.50 ± 32.50 μM. However, for f and g compounds, the inhibition effects on the enzyme were not found.     

Grape seed extract enhances eNOS expression and NO production through regulating calcium‐mediated AKT phosphorylation in H2O2‐treated endothelium

GSE (grape seed extract) has been shown to exhibit protective effects against cardiovascular events and atherosclerosis, although the underlying molecular mechanisms of action are unknown. Herein, we assessed the ability of GSE to enhance eNOS (endothelial nitric oxide synthase) expression and NO (nitric oxide) production in H₂O₂ (hydrogen peroxide)‐treated HUVECs (human umbilical vein endothelial cells). GSE enhanced eNOS expression and NO release in H₂O₂‐treated cells in a dose‐dependent manner. GSE inhibited intracellular ROS (reactive oxygen species) and reduced intracellular calcium in a dose‐dependent manner in H₂O₂‐treated cells, as shown by confocal microscopy. ROS was inhibited in cells pretreated with 5.0 μM GSE, 2.0 μM TG (thapsigargin) and 20.0 μM 2‐APB (2‐aminoethoxydiphenyl borate) instead of 0.25 μM extracellular calcium. In addition, GSE enhanced eNOS expression and reduced ROS production via increasing p‐AKT (AKT phosphorylation) with high extracellular calcium (13 mM). In conclusion, GSE protected against endothelial injury by up‐regulation of eNOS and NO expression via inhibiting InsP₃Rs (inositol 1,4,5‐trisphosphate receptors)‐mediated intracellular excessive calcium release and by activating p‐AKT in endothelial cells.