Inhibition Effects of Some Non-Proteinogenic Amino Acid Derivatives on Carbonic Anhydrase Isoenzymes and Acetylcholinesterase: An In Vitro Inhibition and Molecular Modeling Studies.

Amino acid derivatives are molecules of interest for medicinal chemistry and drug design studies due to their important chemical properties. In this study, the inhibition effects of some non-proteinogenic amino acid derivatives (hippuric acid (A), N-(9-Fluorenylmethoxycarbonyl)-D-valine (B), N-Z-(1-Benzotriazolylcarbonyl) methylamine (C), (S)-N-Z-1-Benzotriazolylcarbonyl-2-phenylethylamine (D)) on carbonic anhydrase I (hCA-I), II (hCA-II) isoenzymes and acetylcholinesterase (AChE) activity, whose inhibitors are of vital pharmacological importance, were examined. While carbonic anhydrase (CA) inhibitors are effective molecule candidates for the treatment of many diseases from glaucoma to cancer, acetylcholinesterase inhibitors are target molecules for the treatment of Alzheimer's disease. According to the results of this study, compound D had a strong inhibitory effect on hCA-I (IC50: 0.840 µM) and hCA-II (IC50: 0.661 µM), while compound B (IC50: 100 µM) showed a strong inhibitory effect on AChE activity. In addition, inhibition results were supported by molecular modeling studies. We hope that the obtained results will contribute to the synthesis of new and effective amino acid derivative inhibitors for CA and AChE.

Depletion of Tip60/Kat5 affects the hepatic antioxidant system in mice.

Tat-interactive protein 60 kDa (TIP60, also known as lysine acetyltransferase 5 [KAT5]) is a member of the MYST protein family with histone acetyltransferase activity. Recent studies have reported that TIP60 has multiple functions in many signal transduction mechanisms, especially p53-mediated apoptosis. Although the activation of apoptosis signaling pathways requires the presence of cellular reactive oxygen species (ROS) at a certain level, an imbalance between the production and consumption of ROS in cells results in oxidative stress (OS). In this study, we investigated for the first time how the absence of the Tip60 gene in the liver affects gene expression, enzyme activity, and protein expression of the hepatic antioxidant members localized in the cytoplasm, including superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx), and glutathione S-transferase (GST). First, we successfully generated liver-specific Tip60 knockout mice (mutants) using Cre/LoxP recombination. The reduced glutathione level and nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) expression, a marker of OS, increased significantly in the Tip60 mutant liver. Gene expression, activity, and protein expression of the enzymatic antioxidant system, including SOD, CAT, GR, GPx, and GST were investigated in mutants and control groups. Despite a significant correlation between the gene, enzyme activity, and protein content for CAT and GR, this was not true for SOD and GPx. The overall results suggest that TIP60 acts on the hepatic antioxidant system both at the gene and protein levels, but the actual effect of the deletion of Tip60 is observed at the protein level, especially for SOD and GPx.

Synthesis, characterizations of aryl-substituted dithiodibenzothioate derivatives, and investigating their anti-Alzheimer's properties.

The main objective of the present study was to synthesize potential inhibitor/activators of AChE and hCA I-II enzymes, which are thought to be directly related to Alzheimer's disease. Dithiodibenzothioate compounds were synthesized by thioesterification. Six different thiolate compounds produced were characterized by 1H-, 13C-NMR, FT-IR, LC-MS/MS methods. HOMO-LUMO calculations and electronic properties of all synthesized compounds were comprehensively illuminated with a semi-empirical molecular orbital (SEMO) package for organic and inorganic systems using Austin Model 1 (AM1)-Hamiltonian as implemented in the VAMP module of Materials Studio. In addition, the inhibition effects of these compounds for AChE and hCA I-II in vitro conditions were investigated. It was revealed that TE-1, TE-2, TE-3, TE-4, TE-5, and TE-6 compounds inhibited the AChE under in vitro conditions. TE-1 compound activated the enzyme hCA I while TE-2, TE-3 TE-4 compounds inhibited it. TE-5 and TE-6, on the other hand, did not exhibit a regular inhibition profile. Similarly, TE-1 activated the hCA II enzyme whereas TE-2, TE-3, TE-4, and TE-5 compounds inhibited it. TE-6 compound did not have a consistent inhibition profile for hCA II. Docking studies were performed with the compounds against AChE and hCA I-II receptors using induced-fit docking method. Molecular Dynamics (MD) simulations for best effective three protein-ligand couple were conducted to explore the binding affinity of the considered compounds in semi-real in-silico conditions. Along with the MD results, TE-1-based protein complexes were found more stable than TE-5. Based on these studies, TE-1 compound could be considered as a potential drug candidate for AD.Communicated by Ramaswamy H. Sarma.

The role of Bax/Bcl-2 and Nrf2-Keap-1 signaling pathways in mediating the protective effect of boric acid on acrylamide-induced acute liver injury in rats.

INTRODUCTION: This study aims to investigate whether boric acid (BA) can protect rats from acrylamide (AA)-induced acute liver injury. MATERIALS AND METHODS: AA was used to induce acute liver injury. Thirty rats were divided into five group including Group 1 (saline), Group 2 (AA), Group 3 (20 mg/kg BA), Group 4 (10 mg/kg BA+AA) and Group 5 (20 mg/kg BA+AA). Their blood and liver were harvested to be kept for analysis. Liver function enzyme activities were performed by spectrophotometric method. Catalase (CAT), superoxide dismutase (SOD) activity, and malondialdehyde levels were determined by colorimetric method. The in-silico studies were performed using the "blind docking" method. RESULTS: Administration AA to rats, biochemical parameters, liver histology, and expression levels of apoptotic markers were negatively affected. However, after the administration of BA, the altered biochemical parameters, liver histology, and expression levels of apoptotic markers were reversed. Moreover, the mechanisms of AA-induced deterioration in the levels of SOD, CAT, and Nrf2-Keap-1 and the mechanisms of the protective effect of BA against these deteriorations were explained by in silico studies. CONCLUSION: Thus, the present study could explain the interactions between AA and thiol-containing amino acid residues of Keap-1, the effect of BA on these interactions, and the biochemical toxicity caused by the AA. In this sense, this work is the first of its kind in the literature. Based on the biochemical, histopathological, and in silico results, it can be suggested that BA has the potential to be used as a protective agent against AA-induced liver injury.

Investigation of in vitro and in silico effects of some novel carbazole Schiff bases on human carbonic anhydrase isoforms I and II.

Carbonic anhydrases (CAs, EC4.2.1.1) are metalloenzymes that catalyse reversible hydration reaction of carbon dioxide to bicarbonate and protons. In recent years, there has been a great interest in inhibitors/activators of carbonic anhydrase isoenzymes. Therefore, we investigated the effects of four different carbazole Schiff base derivatives, which are believed to have a potential to be used as a drug, on human carbonic anhydrase (hCA) isoenzymes I and II under in vitro conditions. The IC50 values of carbazole Schiff base derivatives were found to be in the range of 32.09-151.2 µM for hCA isoenzyme I and 21.82-40.54 µM for hCA isoenzyme II. Among all compounds, (E)-3-(((9-Octyl-9H-carbazole-3-yl)imino)methyl)benzene-1,2-diol (C3) had the strongest inhibitory effect on hCA isoenzyme II. It was determined that 2,3,4-trimethoxy and 4-hydroxy phenyl containing carbazole compounds have selective inhibition against hCA II isoenzyme. Docking studies were performed against hCA I and II receptors using induced-fit docking method. The compounds had affinity scores varying from -7.74 ± 0.27 to -6.27 ± 0.07 kcal/mol for hCA I and from -8.04 ± 0.17 to -7.27 ± 0.18 kcal/mol for hCA II.Communicated by Ramaswamy H. Sarma.

Inhibitory properties of some heavy metals on carbonic anhydrase I and II isozymes activities purified from Van Lake fish (Chalcalburnus Tarichi) gill.

In this study, CA I and II isoenzymes were purified from Van Lake fish gills by using Sepharose-4B-L-tyrosine-sulfanilamide affinity chromatography and to determine the effects of some metals on the enzyme activities. For purified CA I isoenzyme, yield, specific activity, and purification fold were obtained as 42.07%, 4948.12 EU/mg protein, and 116.61 and for CA II isoenzyme, 7%, 1798.56 EU/mg protein, and 42.38 respectively. Activity of CA was determined by measuring "CO2-hydratase activity". Purity control was checked by SDS-PAGE. In vitro inhibitory effect of Cu2+, Ag+, Cd2+, Ni2+ metal ions, and arsenic (V) oxide were also examined for both isozymes activities. Whereas Cu2+, Ag+, Cd2+, and Ni2+ ions showed inhibitory effects on both isozymes, arsenic (V) oxide showed activation effect. IC50 values were calculated by drawing activity %-[I] graphs for metal ions exhibiting inhibitory effects. IC50 values were determined as 3.39, 6.38, 13.52, and 206 µM for CA I isozyme and 6.16, 20.29, 46, and 223 µM for CA II isozyme respectively.

The determination of the carbonic anhydrases activators in vitro effect of mixed donor crown ethers.

Carbonic anhydrases (CAs) play an important function in various physiological and pathological processes. Therefore, many researchers work in this field in order to design and synthesize new drugs. Both inhibitors and activators of CAs, which are associated with the diagnosis and treatment of many diseases, are very important. The emergence of the use of CA activators in the treatment of Alzheimer has led many scholars to work on this issue. In this study, CA activators and inhibitors are determined. The crown ethers compounds (1, 2, 3, 6, 7, 8, and 9) were found to cause activation on enzyme activities of hCA I and II. The AC50 values on hCA I and II of the compounds are in the range of 4.6565-374.979 µM. The 4 (IC50 ; 1.301 and 3.215 µM for hCA I and II) and 5 (IC50 ; 73.96 and 378.5 µM for hCA I and II) compounds were found to cause inhibition on enzyme activities of hCA I and II.

Investigation of the Effect of Some Optically Active Imine Compounds on the Enzyme Activities of hCA-I and hCA-II under In Vitro Conditions: An Experimental and Theoretical Study.

Inhibitors of carbonic anhydrase (hCA; EC 4.2.1.1) are used as medicines for many diseases. Therefore, they are very important. In this study, a known series of Schiff bases were synthesized and their effects on the activities of hCA-I and hCA-II, which are cytosolic isoenzymes of carbonic anhydrase, were investigated under in vitro conditions. The synthesized compounds (H1, H2, H3, and H4) were found to cause inhibition on enzyme activities of hCA-1 and hCA-II. IC50 values of H1, H2, H3, and H4 compounds were 140, 88, 201, and 271 µM for hCA-I enzyme activity and 134, 251, 79, and 604 µM for hCA-II enzyme activity, respectively. The synthesized Schiff bases were characterized by several methods, including (1) H NMR, FT-IR, elemental analysis, and polarimetric measurements. Correlation coefficient square values (R(2) ) of comparison of the theoretical and experimental (1) H NMR shifts for H1, H2, H3, and H4 compounds were found as 0.9781, 0.9814, 0.9758, and 0.8635, respectively.

Purification and characterization of glucose 6-phosphate dehydrogenase enzyme from rainbow trout (Oncorhynchus mykiss) liver and investigation of the effects of some metal ions on enzyme activity.

Glucose 6-phosphate dehydrogenase (d-glucose 6-phosphate: NADP(+) oxidoreductase, EC 1.1.1.49; G6PD) is a key enzyme that is localized in all mammal tissues, especially in cytoplasmic sections and that catalyzes the first step of pentose phosphate metabolic pathway. In this study, G6PD enzyme was purified 1444-fold with a yield of 77% from rainbow trout liver using 2',5'-ADP-sepharose-4B affinity chromatography. Moreover, a purity check of the enzyme was performed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Some characteristic features like optimal pH, stable pH, optimal temperature and optimal ionic strength were determined for the purified enzyme. In addition to this, in vitro effects of ions like silver nitrate (Ag(+)), thallium sulphate (TI(+)), cobalt (II) nitrate (Co(2+)) and arsenic (V) oxide (As(5+)) on enzyme activity were researched. Half-maximal inhibitory concentration (IC50) values of Ag(+), Co(2+) and As(5+) metal ions, which showed an inhibitory effect, were found to be 0.0044, 0.084 and 4.058 mM, respectively; and their inhibition constants (K i) were found to be 0.0052 ± 0.00042, 0.087 ± 0.015700 and 4.833 ± 1.753207 mM, respectively. Tl(+) not exhibited inhibitory effect on the enzyme activity.

Purification and characterization of glutathione S-transferase from turkey liver and inhibition effects of some metal ions on enzyme activity.

The glutathione S-transferases (EC 2.5.1.18) were purified and characterized from turkey liver for the first time. The enzyme was purified 252.7-fold with a yield of 45%, with a specific activity of 164.31 U/mg from turkey liver. The purity of the enzyme was determined by SDS-PAGE and showed two bands nearly 26 kDa and 24 kDa on the gel. The native molecular mass of the enzyme was found to be approximately 53 kDa by Sephadex G-100 gel filtration chromatography. Optimal pH, stable pH, optimal temperature, optimum ionic strength, K(m) and V(max) values for GSH and CDNB were also determined for the enzyme as 7.3, 8.5, 50 °C, 600 mM, 0.154 mM, 0.380 mM, 1.803 EU/ml, and 2.125 EU/ml, respectively. Additionally, inhibitory effects of metal ions (Cu(2+), Hg(2+), Fe(2+), Zn(2+), Ag(+), Mg(2+), Ni(2+), and Mn(2+)) were examined the enzyme's activity in vitro by performing Lineweaver-Burk graphs and plotting activity% vs., respectively.

In vitro effects of some drugs on human erythrocyte glutathione reductase.

The effects of ketotifen, meloxicam, phenyramidol-HCl and gadopentetic acid on the enzyme activity of GR were studied using human erythrocyte glutathione reductase (GR) enzymes in vitro. The enzyme was purified 209-fold from human erythrocytes in a yield of 19% with 0.31 U/mg. The purification procedure involved the preparation of haemolysate, ammonium sulphate precipitation, 2'',5'-ADP Sepharose 4B affinity chromatography and Sephadex G-200 gel filtration chromatography. Purified enzyme was used in the in vitro studies. In the in vitro studies, IC(50) values and K(i) constants were 0.012 mM and 0.0008 ± 0.00021 mM for ketotifen; 0.029 mM and 0.0061 ± 0.00127 mM for meloxicam; 0.99 mM and 0.4340 ± 0.0890 mM for phenyramidol-HCl; 138 mM and 28.84 ± 4.69 mM for gadopentetic acid, respectively, showing the inhibition effects on the purified enzyme. Phenyramidol-HCl showed competitive inhibition, whereas the others showed non-competitive inhibition.

The effects of chemical and radioactive properties of Tl-201 on human erythrocyte glutathione reductase activity.

AIM: The aim of the study was to evaluate the inhibitory effects of thallium-201 ((201)Tl) solution on human erythrocyte glutathione reductase (GR) activity. METHODS: Erythrocyte GR was initially purified by 2',5'-adenosine diphosphate Sepharose-4B affinity and Sephadex G-200 gel filtration chromatography. The purification was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which showed a single band for the final enzyme preparation. The in vitro and in vivo effects of the (201)Tl solution including Tl(+), Fe(+3) and Cu(+2) metals and the in vitro effects of the radiation effect of the (201)Tl solution and nonradioactive Tl(+), Fe(+3) and Cu(+2) metals on human erythrocyte GR enzyme were studied. Enzyme activity was determined with the Beutler method at 340 nm using a spectrophotometer. All purification procedures were carried out at (+)4 °C. RESULTS: Glutathione reductase was purified 2033-fold at a yield of 28.17%. (201)Tl solution and radiation exposure had inhibitory effects on the enzyme activity. Besides, effects of nonradioactive Tl(+), Fe(+3) and Cu(+2) were studied on enzyme activity in vitro. Furthermore, seven human patients were also used for in vivo studies of (201)Tl solution. CONCLUSION: It was detected in in vitro and in vivo studies that the human erythrocyte GR enzyme is inhibited due to the radiation effect of (201)Tl solution.

Effects of some drugs on human erythrocyte glucose 6-phosphate dehydrogenase: an in vitro study.

Inhibitory effects of some drugs on glucose 6-phosphate dehydrogenase from the erythrocytes of human have been investigated. For this purpose, at the beginning, erythrocyte glucose 6-phosphate dehydrogenase was purified 2256 times in a yield of 44.22% by using ammonium sulphate precipitation and 2', 5'-ADP Sepharose 4B affinity gel. Temperature of +4°C was maintained during the purification process. Enzyme activity was determined with the Beutler method by using a spectrophotometer at 340 nm. This method was utilized for all kinetic studies. Ketotifen, dacarbazine, thiocolchicoside, meloxicam, methotrexate, furosemide, olanzapine, methylprednizolone acetate, paricalcitol, ritodrine hydrochloride, and gadobenate-dimeglumine were used as drugs. All the drugs indicated the inhibitory effects on the enzyme. Ki constants for glucose 6-phosphate dehydrogenase were found by means of Lineweaver-Burk graphs. While methylprednizolone acetate showed competitive inhibition, the others displayed non-competitive inhibition. In addition, IC(50) values of the drugs were determined by plotting Activity% vs [I].

Effects of some drugs on human erythrocyte 6-phosphogluconate dehydrogenase: an in vitro study.

The inhibitory effects of some drugs on 6-phosphogluconate dehydrogenase from human erythrocytes have been investigated. For this purpose, initially, erythrocyte 6-phosphogluconate dehydrogenase was purified 3364 times in a yield of 58% by using ammonium sulfate precipitation and 2',5'-ADP Sepharose 4B affinity gel. A temperature of +4 degrees C was maintained during the purification process. Enzyme activity was determined with the Beutler method by using a spectrophotometer at 340 nm. This method was utilized for all kinetic studies. Many commonly used drugs were investigated in this study. Some drugs (ketotifen (K(i): 8.3 +/- 1.7 microM), dacarbazine (K(i): 10.1 +/- 0.7 microM), meloxicam (K(i): 50.9 +/- 13.2 microM), furosemide (K(i): 127 +/- 37.8 microM), methotrexate (K(i): 136.7 +/- 25.3 microM), metochloropramide hydrochloride (K(i): 2.1113 +/- 0.6979 mM), ritodrine hydrochloride (K(i): 6.0353 +/- 1.2783 mM), and gadopentetic acid (K(i): 73.4 +/- 21.9 mM)) inhibited enzyme activity in vitro. K(i) constants for the enzyme were found by means of Lineweaver-Burk graphs. All drugs showed non-competitive inhibition. In addition, IC(50) values of the drugs were determined by plotting activity percent vs [I].