Structure-Activity Relationship of Methyl 4-Aminobenzoate Derivatives as Being Drug Candidate Targeting Glutathione Related Enzymes: in Vitro and in Silico Approaches.

A thiol compound, glutathione, is essential for healthy cell defence against xenobiotics and oxidative stress. Glutathione reductase (GR) and glutathione S-transferase (GST) are two glutathione-related enzymes that function in the antioxidant and the detoxification systems. In this study, potential inhibitory effects of methyl 4-aminobenzoate derivatives on GR and GST were examined in vitro. GR and GST were isolated from human erythrocytes with 7.63 EU/mg protein and 5.66 EU/mg protein specific activity, respectively. It was found that compound 1 (methyl 4-amino-3-bromo-5-fluorobenzoate with Ki value of 0.325±0.012 µM) and compound 5 (methyl 4-amino-2-nitrobenzoate with Ki value of 92.41±22.26 µM) inhibited GR and GST stronger than other derivatives. Furthermore, a computer-aided method was used to predict the binding affinities of derivatives, ADME characteristics, and toxicities. Derivatives 4 (methyl 4-amino-2-bromobenzoate) and 6 (methyl 4-amino-2-chlorobenzoate) were estimated to have the lowest binding energies into GR and GST receptors, respectively according to results of in silico studies.

Design and Synthesis of Novel Dual Cholinesterase Inhibitors: In Vitro Inhibition Studies Supported with Molecular Docking.

The major cholinesterase enzymes, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), are important in the therapy of Alzheimer's disease (AD) based on the cholinergic hypothesis. As a result, in recent years, the investigation of dual cholinesterase inhibition methods has become important among scientists. In this study, novel N-(4-chlorobenzyl)-3,4-dimethoxy-N-(m-substituted)benzamide derivatives were synthesized. Then, inhibitory properties of these derivatives were examined in human AChE and BuChE in vitro and possible interactions were determined by molecular docking studies. All benzamide derivatives were exhibited dual inhibitory character and high BBB permeability. The most effective inhibitor was found as N7 for both AChE and BuChE with IC50 values of 1.57 and 2.85 µM, respectively. Besides the most potent inhibitor was predicted as N7 in terms of binding energies with -12.18 kcal/mol and -9.92 kcal/mol, respectively. The reason for these results is that bromine (N7) is the bulkiest molecule among the other substituted groups. These derivatives could be exploited to develop new medications for the treatment of central nervous system-related diseases as AD by acting as dual inhibitors of AChE and BChE.

In vitro and in silico interactions of antiulcer, glucocorticoids and urological drugs on human carbonic anhydrase I and II isozymes.

Carbonic anhydrases (CAs, Enzyme Commission 4.2.1.1) convert carbon dioxide to bicarbonate in metabolism and use Zn2+ ions as a cofactor for their catalytic activity. The activators or inhibitors of CA-I and CA-II, which are the most abundant CA isozymes in erythrocytes, have pharmacological applications in medicine. So, investigation of drug-protein interaction of these isozymes is significant. On this basis, the objective of this study was to clarify the primer effects of widely used drugs on the activity of human CA-I and CA-II enzymes and elucidate the inhibition mechanism through molecular docking studies. For this aim isozymes were purified from human erythrocytes by affinity chromatography technique. Then inhibition profiles of antiulcer, glucocorticoids, and urological drugs were investigated. As a result, while budesonide had the highest inhibitory potency on hydratase activity of hCA-I with the IC50 of 0.08 mM, levofloxacin showed the highest inhibition effect on hCA-II with the IC50 of 0.886 mM. The most effective inhibitor on the esterase activity of isozymes was found as fluticasone propionate with the Ki values of 0.0365 ± 0.016 mM and 0.054 ± 0.018 mM respectively. However, by molecular docking study, it was estimated that budesonide showed maximum inhibition potency for both isozymes with the free binding energy of -7.58 and -6.97 kcal/mol, respectively. Consequently, it was observed that some of the drugs studied did not show any inhibitory effect. Drug-enzyme interactions were also estimated by molecular docking. This study could contribute to the discovery of new drug candidates and as well as target proteins.

Antibacterial properties and carbonic anhydrase inhibition profiles of azido sulfonyl carbamate derivatives.

Aim: The aim of this study was to identify inhibition of carbonic anhydrase I and II (CA I and II) isozymes by azido sulfonyl carbamates through both in vitro and in silico approaches and also to determine the drug-likeness properties and antibacterial activities of azido sulfonyl carbamates. Methods & Results:In vitro inhibition and molecular docking studies of azido sulfonyl carbamate derivatives (1-4) on isozymes were performed. Except for derivative 4, all derivatives inhibited human CA I and II. Almost all compounds had antibacterial effects. The docking results showed that compound 3 had the best results, with binding energy of -8.20 kcal/mol for human CA I and -8.24 kcal/mol for human CA II. Conclusion: Molecule 4 inhibited only CA I. Its usage as a potential chemotherapeutic agent specific to the CA I isozyme may be considered.

A study on the effects of inhibition mechanism of curcumin, quercetin, and resveratrol on human glutathione reductase through in vitro and in silico approaches.

Glutathione reductase (GR) is a major antioxidant enzyme essential to maintain GSH/GSSG ratio by catalyzing recovery of reduced glutathione (GSH) from oxidized glutathione (GSSG). Because of this vital task, the inhibition of GR is an important target in the treatment of many diseases, so we aimed to identify natural and new GR inhibitors to be guide for drug design.For this purpose, two different approaches were used. The first one is in vitro inhibition, the first phase of which was the purification of the enzyme from human erythrocyte by 2', 5'-ADP Sepharose 4B affinity chromatography, and then the in vitro inhibition effects of curcumin, quercetin, and resveratrol were examined. The second one is in silico study, which was performed to elucidate the drug-likeness, active site identification and inhibition mechanisms of these compounds.hGR was isolated from human erythrocytes with 7.036 EU/mg protein specific activity and 48.97% yield. Then, IC50 values were as 17.25 ± 3.8 µM, 57.8 ± 14.2 µM, and 520 ± 96.7 µM for curcumin, quercetin, and resveratrol respectively. Docking studies of compounds were performed against hGR receptors with induced-fit docking method. The compound showed Glide score as 10.519 kcal/mol, -9.789, and -8.133 respectively.In conclusion, it was seen that curcumin is the much better inhibitor than quercetin and resveratrol for hGR according to both in vitro and in silico studies. Curcumin, a potential inhibitor of hGR, can be used in drug design to target the glutathione system in cellular injury.Communicated by Ramaswamy H. Sarma.

Radical Scavenging and Antiacetylcholinesterase Activities of Ethanolic Extracts of Carob, Clove, and Linden.

CONTEXT: Acetylcholine (ACh) breaks down in a very short time in diseases related to memory loss. It's a neurotransmitter involved in cholinergic transmission in the brain. Acetylcholinesterase (AChE) hydrolyzes ACh. When AChE is inhibited, the ACh levels increase in the cholinergic synapses. The investigation of natural AChE inhibitors with minimal side effects has become important. CONTEXT: Objective • This study intended to determine the total phenolic content, total flavonoid contents, radical scavenging activities, and antiacetylcholinesterase activities of ethanolic extracts of carob pods (ceratonia siliqua), clove buds (eugenia aromatica), and linden flowers (tilia cordata). CONTEXT: Design • The research team designed an in-vitro study. CONTEXT: Setting • The study took place at a biochemistry research laboratory where purification of enzymes and studies on their kinetic properties and inhibitions are carried out. CONTEXT: Outcome measures • The antioxidant properties of the extracts including the total phenolic content (TPC), total flavonoid content (TFC), and free radical scavenging activities, were determined. The AChE enzyme was partially purified by DE-52 anion exchange chromatography from human erythrocytes. Besides, The AChE inhibitory properties of the ethanolic extracts were investigated. CONTEXT: Results • The TPCs of the carob pods, clove buds, and linden flowers were 46.78 ± 0.020, 103.57 ± 0.020, and 28.81 ± 0.031, mg GAE/L, respectively. The TFCs were 27.35 ± 0.021, 30.85 ± 0.017, and 32.12 ± 0.022 mg QE/L, respectively. While the extracts of carob pods and linden flowers inhibited AChE, with IC50s of 0.838 mg/ml and 0.156 mg/ml, respectively, clove buds didn't show inhibitory effect. CONTEXT: Conclusion • Although the clove buds had the maximum TPC; 1,1-diphenyl-2-picryl hydrazyl (DPPH); and 2,2'-azino-bis [3-ethylbenzothiazoline-6-sulphonic acid] (ABTS+) radical scavenging activity, it didn't show anticholinesterase activity.

Effects of different LED light spectra on rainbow trout (Oncorhynchus mykiss): in vivo evaluation of the antioxidant status.

Rainbow trout (Oncorhynchus mykiss) farming is one of the major aquacultures in Turkey. Some conditions in fish farming can induce oxidative stress leading to the deterioration in properties such as appearance/color, texture, and flavor in fish meat. This situation may cause the consumer not to prefer edible fish. Although there are some studies on the impacts of light intensity on fish welfare, the changes in the antioxidant enzyme activities have not been elucidated. In the current study, it was intended to examine in rainbow trout how cultivating under different wavelengths affects the antioxidant enzymes and acetylcholine esterase (AChE) activity, because its activity is associated with oxidative stress, and also the determination of which light is suitable for fish welfare was aimed. Rainbow trout larvae were grown under four lights with different wavelengths: natural sunlight and incandescent long-wave (red light), medium-wave (green light), and short-wave (blue light) LED light. The experiment lasted for 64 days. Biochemical assays were carried on in the brain, gill, and liver of rainbow trout. Antioxidant enzymes and AChE activity, which play an important role in the central nervous system, were assayed. In gill tissues, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glucose 6 phosphate dehydrogenase (G6PD), glutathione reductase (GR), glutathione S-transferase (GST), and AChE activities increased under all three light wavelengths. In the liver, while activities of antioxidant enzymes and AChE decreased in red light, all of them increased in blue and green light. In the brain, GPx, GST, G6PD, and SOD activities were reduced but AChE activity did not alter under all three light sources. In conclusion, light sources with different spectral structures caused important changes in the activities of antioxidant enzymes in rainbow trout. On this basis, it may be thought that this may be a response to the changing redox status of a cell. Based on our results, blue light sources may be suggested for fish welfare in rainbow trout culture, and providing fish welfare by changing light sources can be easy and cheap in fish farming.

A Comparison of the Inhibitory Effects of Anti-Cancer Drugs on Thioredoxin Reductase and Glutathione S-Transferase in Rat Liver.

BACKGROUND: While Thioredoxin Reductase (TrxR) plays an important role in regulation of the intracellular redox balance and various signalling pathways, Glutathione S-Transferase (GSTs) enzymes belong to the detoxification family that catalyse the conjugation of glutathione with various endogenous and xenobiotic electrophiles. Since TrxR and GSTs are overexpressed in many cancer cells, they have been identified as potential targets to develop chemotherapeutic strategies. METHOD: The mitochondrial TrxR (TrxR2) enzyme and the cytosolic GST enzyme was purified from rat liver via affinity chromatography. After the purification, the in vitro inhibition effects of some anticancer drugs (cisplatin, calcium folinate, carboplatin, epirubicin hydrochloride, doxorubicin hydrochloride, paclitaxel, etoposide, fluorouracil, and methotrexate) were investigated on both enzymes. Since only methotrexate inhibits both enzymes among all the anticancer drugs, a molecular docking study was performed to determine the binding site and the binding affinity of methotrexate to the enzymes. RESULTS: Firstly, TrxR2 and GST were found to have a specific activity of 0.436, 1765 EU/mg proteins with a yield of 39.20%, 31.28% and 207.6, 3516.6 of purification fold, respectively. While TrxR2 was strongly inhibited by all of the anticancer drugs, GST was not inhibited by any of the anticancer drugs except methotrexate. CONCLUSION: Both enzymes were inhibited by only methotrexate in rat liver, and methotrexate was well placed in the active sites of both proteins. Therefore, it may be argued that methotrexate may be a more effective anticancer drug than all other drugs used in this study against the multi drug resistance that will occur during chemotherapy.