Human Glutathione Reductase Research Articles

Flavan-Benzofurans from Artocarpus lacucha: Their Intracellular Antioxidant Activity and Molecular Docking to Glutathione Reductase.

Phytochemical investigation of Artocarpus lacucha Buch.-Ham (Moraceae) leaves led to the identification of three of the rarely found flavan-benzofuranes named artocarpinol C (1), 3-epi-artocarpinol C (2), and artocarpinol D (6) along with six known flavan derivatives. Thus, a total of six artocarpinols are now described. All their chemical structures and absolute configurations were established by one dimensional (1D)- and two-dimensional (2D) NMR, infrared (IR), electronic circular dichroism (ECD), high-resolution electrospray ionisation mass spectrometry (HR-ESI-MS), and optical rotation (OR). Density functional theory (DFT) calculations based on the B3LYP theory level were conducted to determine the stereochemistry at positions 2 and 3 in the C-ring. All compounds exhibited in vitro radical scavenging activities, and compounds 3 and 5 demonstrated pronounced intracellular antioxidative effects in colon carcinoma cells (SW480), as determined by the DCFH-DA assay. Compounds 3 and 5 exhibited further high affinities for binding to the active site of human glutathione reductase. These molecular properties are discussed with regard to possible applications.

The role of the active site lysine residue on FAD reduction by NADPH in glutathione reductase

Glutathione reductase (GR) is a two dinucleotide binding domain flavoprotein (tDBDF) that catalyzes the reduction of glutathione disulfide to glutathione coupled to the oxidation of NADPH to NADP+. An interesting feature of GR and other tDBDFs is the presence of a lysine residue (Lys-66 in human GR) at the active site, which interacts with the flavin group, but has an unknown function. To better understand the role of this residue, the dynamics of GR was studied using molecular dynamics simulations, and the reaction mechanism of FAD reduction by NADPH was studied using QM/MM molecular modeling. The two possible protonation states of Lys-66 were considered: neutral and protonated. Molecular dynamics results suggest that the active site is more structured for neutral Lys-66 than for protonated Lys-66. QM/MM modeling results suggest that Lys-66 should be in its neutral state for a thermodynamically favorable reduction of FAD by NADPH. Since the reaction is unfavorable with protonated Lys-66, the reverse reaction (the reduction of NADP+ by FADH−) is expected to take place. A phylogenetic analysis of various tDBDFs was performed, finding that an active site lysine is present in different the tDBDFs enzymes, suggesting that it has a conserved biological role. Overall, these results suggest that the protonation state of the active site lysine determines the energetics of the reaction, controlling its reversibility.

An in-silico approach to identify bioactive phytochemicals from Houttuynia cordata Thunb. As potential inhibitors of human glutathione reductase

Cellular infections are central to the etiology of various diseases, notably cancer and malaria. Counteracting cellular oxidative stress via the inhibition of glutathione reductase (GR) has emerged as a promising therapeutic strategy. Houttuynia cordata, a medicinal plant known for its potent antioxidant properties, has been the focus of our investigation. In this study, we conducted comprehensive in silico analyses involving the phytochemical constituents of H. cordata to identify potential natural GR inhibitors. Our methodological approach encompassed multiple in silico techniques, including molecular docking, molecular dynamics simulations, MMPBSA analysis, and dynamic cross-correlation analysis. Out of 13 docked phytochemicals, Quercetin, Quercitrin, and Sesamin emerged as particularly noteworthy due to their exceptional binding affinities for GR. Notably, our investigation demonstrated that Quercetin and Sesamin exhibited promising outcomes compared to the well-established pharmaceutical agent N-acetylcysteine (NAC). Molecular dynamics analyses provided insights into the ability of these phytochemicals to induce structural compaction and stabilization of the GR protein, as evidenced by changes in radius of gyration and solvent-accessible surface area. Moreover, MMPBSA analysis highlighted the crucial roles of specific residues, namely Gly27, Gly28, Ser51, His52, and Val61, in mediating essential interactions with these phytochemicals. Furthermore, an assessment of Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADME-Tox) profiles underscored the favourable drug-like attributes of these phytochemicals. Thus, the current findings underscore the immense potential of Houttuynia cordata phytochemicals as potent antioxidants with the capacity to combat a spectrum of maladies, including malaria and cancer. This study not only unveils novel therapeutic avenues but also underscores the distinctive outcomes and paramount significance of harnessing H. cordata phytochemicals for their efficacious antioxidant properties. Communicated by Ramaswamy H. Sarma

Ameliorative Effect of Ethanolic Extract of Moringa oleifera Leaves in Combination with Curcumin against PTZ-Induced Kindled Epilepsy in Rats: In Vivo and In Silico.

The ameliorative effect of ethanolic extract of M. oleifera (MOEE) leaves in combination with curcumin against seizures, cognitive impairment, and oxidative stress in the molecular docking of PTZ-induced kindled rats was performed to predict the potential phytochemical effects of MOEE and curcumin against epilepsy. The effect of pretreatment with leaves of M. oleifera ethanolic extracts (MOEE) (250 mg/kg and 500 mg/kg, orally), curcumin (200 mg/kg and 300 mg/kg, orally), valproic acid used as a standard (100 mg/kg), and the combined effect of MOEE (250 mg/kg) and curcumin (200 mg/kg) at a low dose on Pentylenetetrazole was used for (PTZ)-induced kindling For the development of kindling, individual Wistar rats (male) were injected with pentyletetrazole (40 mg/kg, i.p.) on every alternate day. Molecular docking was performed by the Auto Dock 4.2 tool to merge the ligand orientations in the binding cavity. From the RCSB website, the crystal structure of human glutathione reductase (PDB ID: 3DK9) was obtained. Curcumin and M. oleifera ethanolic extracts (MOEE) showed dose-dependent effects. The combined effects of MOEE and curcumin leaves significantly improved the seizure score and decreased the number of myoclonic jerks compared with a standard dose of valproic acid. PTZ kindling induced significant oxidative stress and cognitive impairment, which was reversed by pretreatment with MOEE and curcumin. Glutathione reductase (GR) is an enzyme that plays a key role in the cellular control of reactive oxygen species (ROS). Therefore, activating GR can uplift antioxidant properties, which leads to the inhibition of ROS-induced cell death in the brain. The combination of the ethanolic extract of M. oleifera (MOEE) leaves and curcumin has shown better results than any other combination for antiepileptic effects by virtue of antioxidant effects. As per the docking study, chlorogenic acid and quercetin treated with acombination of curcumin have much more potential.

Phenothiazine-based virtual screening, molecular docking, and molecular dynamics of new trypanothione reductase inhibitors of Trypanosoma cruzi.

Phenothiazine derivatives can unselectively inhibit the trypanothione-dependent antioxidant system enzyme trypanothione reductase (TR). A virtual screening of 2163 phenothiazine derivatives from the ZINC15 and PubChem databases docked on the active site of T. cruzi TR showed that 285 compounds have higher affinity than the natural ligand trypanothione disulfide. 244 compounds showed higher affinity toward the parasite's enzyme than to its human homolog glutathione reductase. Protein-ligand interaction profiling predicted that the main interactions for the top scored compounds were with residues important for trypanothione disulfide binding: Phe396, Pro398, Leu399, His461, Glu466, and Glu467, particularly His461, which participates in catalysis. Two compounds with the desired profiles, ZINC1033681 (Zn_C687) and ZINC10213096 (Zn_C216), decreased parasite growth by 20 % and 50 %, respectively. They behaved as mixed-type inhibitors of recombinant TR, with Ki values of 59 and 47 μM, respectively. This study provides a further understanding of the potential of phenothiazine derivatives as TR inhibitors.

Inhibition of Leishmania infantum Trypanothione Reductase by New Aminopropanone Derivatives Interacting with the NADPH Binding Site

As a result of the paucity of treatment, Leishmaniasis continues to provoke about 60,000 deaths every year worldwide. New molecules are needed, and drug discovery research is oriented toward targeting proteins crucial for parasite survival. Among them, trypanothione reductase (TR) is of remarkable interest owing to its vital role in Leishmania species protozoan parasite life. Our previously identified compound 1 is a novel chemotype endowed with a unique mode of TR inhibition thanks to its binding to a formerly unknown but druggable site at the entrance of the NADPH binding cavity, absent in human glutathione reductase (hGR). We designed and synthesized new 3-amino-1-arylpropan-1-one derivatives structurally related to compound 1 and evaluated their potential inhibition activity on TR from Leishmania infantum (LiTR). Cluster docking was performed to assess the binding poses of the compounds. The newly synthesized compounds were screened at a concentration of 100 μM in in vitro assays and all of them proved to be active with residual activity percentages lower than 75%. Compounds 2a and 2b were the most potent inhibitors found, suggesting that an additional aromatic ring might be promising for enzymatic inhibition. Further structure-activity relationships are needed to optimize our compounds activity.

The X-ray crystal structures, molecular docking and biological activities of two novel Cu(II) and Zn(II) complexes with a ligand having a potentially N4O2 donor set and two nitro phenyl rings as pendant arms

A new ligand (L) containing two nitro phenyl rings as side chains was synthesized. The reaction of this ligand with copper(II) and zinc(II) metal ions gave complexes with different coordination environments. The free ligand and the metal complexes were characterized using a number of spectroscopic methods. The crystal structure of [ZnLBr]ClO4 showed that the Zn(II) ion was in a distorted square pyramidal environment. The crystal structure of [CuL](ClO4)2 showed that the Cu(II) ion is in a tetragonally distorted octahedral environment. Molecular docking studies with DNA indicated that the binding of L, [ZnLBr]ClO4 and [CuL](ClO4)2 involved the major groove of DNA, H-bonds and Vander Waals interactions. In contrast, the molecular docking of L, [ZnLBr]ClO4 and [CuL](ClO4)2 with human glutathione reductase (GR) showed that the dominant interactions of these compounds with GR were H-bonding, vander Waals and hydrophobic interactions. The antioxidant activity of the synthesized complexes was analyzed by the free radical scavenging activity using 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay with [CuL](ClO4)2 showing maximum activity. In addition, in vitro anticancer activity of the complexes against human breast MCF-7 cancer cell line was confirmed through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay. All compounds showed a dose-dependent inhibitory effect on the growth of breast cancer cells with the inhibition activity of [CuL](ClO4)2 being more active than the other synthesized compounds. Furthermore, results from the antibacterial activity screening of the compounds against two Gram-positive and Gram-negative pathogenic bacteria by the micro-broth dilution and disk diffusion methods indicated that [CuL](ClO4)2 complex had the strongest antibacterial potential.

New Schiff bases with a 2,6-bis(2-aminophenylthio)pyridine moiety acting as glutathione reductase activator and inhibitors: Synthesis and molecular docking studies

2,6-bis(2-aminophenylthio)pyridine was synthesized and identified by x-ray diffraction method. Its new Schiff bases (H2L1, H2L2, L3 and L4) were synthesized and characterized by elemental analysis, FT-IR, LC-MS, 1H NMR and 13C NMR techniques. in vitro glutathione reductase activities of the compounds were tested on yeast and human glutathione reductase. L4 enhanced both glutathione reductase activities, resulting in AC50 values of 15.06 µM and 15.89 µM, respectively. H2L1, H2L2 and L3 were found to be the inhibitors in the range of 50.09 – 55.23 µM for yeast glutathione reductase, and in the range of 56.12–66.87 µM for human glutathione reductase. According to molecular docking analysis at the xanthine binding site in human glutathione reductase (PDB: 1XAN), 2,6-bis(2-aminophenylthio)pyridine is predicted to have antimalarial property due to having a higher XP docking score than the malaria drug Chloroquine. Also, five binding pockets at the human glutathione reductase (PDB:1GRA) were identified using Sitemap analysis for Schiff bases which are non-competitive inhibitors. IFD-Docking scores were found to correlate with experimental IC50 value of H2L1, H2L2 and L3. Based on the fact that Schiff bases have higher IFD docking scores at binding pocket-1 than at the active site, it can be predicted that Schiff bases prefer to bind to binding pocket-1 in the presence of natural substrate. The difference in glutathione reductase activities of Schiff bases was attributed to the fact that the conformation of the activator L4-human GR complex was different from that of other inhibitor Schiff bases-human glutathione reductase complexes. ADMET calculations predicted that synthesized ligands obey the Lipinski Rule (rule of five) and Jorgensen Rule (rule of three).

Profiling of phytochemical and antioxidant activity of wild mushrooms: Evidence from the in vitro study and phytoconstituent's binding affinity to the human erythrocyte catalase and human glutathione reductase.

This study was undertaken to evaluate the appearance of phytochemicals and antioxidant activity of seven wild mushrooms of the University of Chittagong campus. Phytochemical screening was performed using standard methods, whereas DPPH radical scavenging assay was used to elucidate the antioxidant effect. Besides, in silico studies were implemented using the targets of human erythrocyte catalase 3‐amino‐1,2,4‐triazole, human glutathione reductase, and selected compounds. Again, the absorption, distribution, metabolism, elimination and toxicity (ADME/T) analysis has been determined by using online tools. Both Ganoderma lucidum (Curtis) Karst. and Ganoderma applanatum (Pers.) Pat. showed a significant (p < .001) increase in the percentage of scavenging activity at 400 μg/ml concentration when compared with ascorbic acid. The methanol extract of G. lucidum, G. applanatum, and Rhodofomes cajanderi (P. Karst.) B. K. Cui, M. L. Han & Y. C. Dai showed strong antioxidant activity with an IC50 value. In addition, molecular docking studies of the previously isolated compounds from three selective mushrooms revealed that the targeted compounds along with positive controls were able to interact strongly (range: −3.498 to −8.655) with the enzymes. The study concludes that the G . lucidum, G. applanatum, and R. cajanderi mushrooms can be a strong source in the management of oxidative stress‐induced diseases.

Computational modelling of nanotube delivery of anti-cancer drug into glutathione reductase enzyme

Density functional theory method combined with docking and molecular dynamics simulations are used to understand the interaction of carmustine with human glutathione reductase enzyme. The active site of the enzyme is evaluated by docking simulation is used for molecular dynamics simulation to deliver the carmustine molecule by (5,5) single walled carbon nanotube (SWCNT). Our model of carmustine in the active site of GR gives a negative binding energy that is further refined by QM/MM study in gas phase and solvent phase to confirm the stability of the drug molecule inside the active site. Once released from SWCNT, carmustine forms multiple polar and non-polar hydrogen bonding interactions with Tyr180, Phe209, Lys318, Ala319, Leu320, Leu321, Ile350, Thr352 and Val354 in the range of 2–4 Å. The SWCNT vehicle itself is held fix at its place due to multiple pi-pi stacking, pi-amide, pi-sigma interactions with the neighboring residues. These interactions in the range of 3–5 Å are crucial in holding the nanotube outside the drug binding region, hence, making an effective delivery. This study can be extended to envisage the potential applications of computational studies in the modification of known drugs to find newer targets and designing new and improved controlled drug delivery systems.

Identification of novel thiadiazin derivatives as potentially selective inhibitors towards trypanothione reductase from Trypanosoma cruzi by molecular docking using the numerical index poses ratio Pr and the binding mode analysis

Chagas disease is a serious health problem in Central and South America for which effective treatment is not currently available. This illness is caused by the protozoa Trypanosoma cruzi, a species that relies on a thiol-based metabolism to regulate oxidative stress. Trypanothione reductase enzyme plays a central role in the metabolic pathway of the parasite. In this work, a virtual screening of a library of novel thiadiazine derivatives against trypanothione reductase using molecular docking was performed. Four different series of hybrid ligands having in the structure one or two peptoid moieties (series I and II) or the tetrazole ring (series III and IV) were considered. An ad hoc numerical index called poses ratio was introduced to interpret the results of the docking analysis and to establish relevant structure-interaction relationships. In addition, six binding modes were found for the ligands with the highest populated conformational clusters after applying contact-based analysis. The most regular and relevant were binding modes I and II, found mainly for ligands from series I. A subsequent molecular docking on human glutathione reductase enzyme allowed to assess the possible cytotoxicity of the ligands towards human cells. A selective binding profile was found for ligands with interactions in the Hydrophobic cleft, the spermidine and the Z subsites inside the active site of trypanothione reductase. At the end of the study, new thiadiazine-based compounds were identified as plausible candidates to selectively inhibit the parasitic enzyme.Graphic abstract

Carbazole derivatives: Synthesis, spectroscopic characterization, antioxidant activity, molecular docking study, and the quantum chemical calculations

A various carbazole derivatives were synthesized for the synthesis 2,3-Dihydro-1H-carbazol-4(9H)-one O-acetyl oxime (3), ethyl 2-(2,3,4,9-tetrahydrospiro[carbazole-1,2′-[1,3]dithiolane]-2-yl)acetate (5), 2- hydroxyethyl {2,3,4,9-tetrahydrospiro[1H-carbazole-1,2′[1,3]dithiolane-4-one]-2-yl}-acetamide (7). These products (3, 5, and 7) were characterized by the spectroscopic techniques (IR, 1H NMR, 13C NMR and elemental analysis). Then, the observed FT-IR and NMR peaks of the studied compounds were compared with the calculated values. The FMO analyses disclosed that the (5) would prefer intermolecular interactions more than intramolecular interactions because of its higher energy gap. NBO analyses displayed that the n → П* and П → П* interactions were responsible for the lowering of the stabilization energy. The molecular docking studies showed that (5) exhibited highest binding affinity with the human glutathione reductase at binding site (−7.21 kcal/mol). Also, the antioxidant activities were investigated using the CUPRAC method, and TEAC coefficient implied that compound (3) have an antioxidant property. In addition, docking calculations of the compound (3), (5), and (7) were performed on bacterial tyrosinase enzyme and human glutathione reductase protein.

Molecular Insight of the Possible Inhibition Mechanism of Therapeutic Cephalosporin Derivatives against Human Glutathione Reductase Enzyme

Glutathione reductase is a key enzyme for glutathione metabolism. Inhibition of the enzyme activity related to various health problems. Therefore, determination of inhibitors of the enzyme and its possible inhibition mechanism are quite important. Some cephalosporins have exhibited potent inhibitory effect against human glutathione reductase (hGR). In order to understand the inhibition mechanism of the cephalosporins, we carried out molecular docking studies with Glide docking and Induced-fit Docking methods. Binding sites of hGR were predicted and the best suitable binding site of the drugs was identified with the Glide docking method. The binding affinity of the drugs was calculated with the induced-fit docking method. The best binding site of the drugs was detected as a part of the catalytic active site for Cefoperazone, Cefodizime, and Ceftazidime, dimerization site for Cefotaxime, Ceftriaxone, and Cefuroxime, and aromate binding site for Ceftizoxime. The Binding affinity of the Cefoperazone was calculated as -10.643 kcal/mol. The results have indicated that hGR enzyme would be inhibited with different mechanisms because of its several druggable sites. These findings would be helpful for designing new inhibitors for hGR enzyme and understanding of potential inhibition mechanism of its other known inhibitors.

The In Vitro and In Silico Inhibition Mechanism of Glutathione Reductase by Resorcinol Derivatives: A Molecular Docking Study

Glutathione reductase (GR), which is a major antioxidant enzyme, is targeted in the treatment of many diseases due to the dual role of its product, reduced glutathione (GSH), in infected cells. This study's goal was to introduce new GR inhibitors to the literature. The inhibition profiles of the inhibitor were studied via two different approaches: in vitro and in silico. To this end, firstly, GR was isolated from human erythrocytes, following which the in vitro inhibition impacts of resorcinol derivatives and in silico inhibition mechanisms of them were elucidated. GR was obtained with a specific activity of 12.49 EU/mg protein and a 27.67% yield. Afterward, IC50 values was found to range from 0.014 to 1.500 mM. The docking studies of the compounds were performed against hGR receptors, and 4-hexylresorcinol had the lowest docking score of -4.276 kcal/mol. In conclusion, it was determined that 4-hexylresorcinol is the most effective inhibitor among resorcinol derivatives for human erythrocyte GR in accordance with both in vitro and in silico studies. 4-Hexylresorcinol may be utilized in drug design to target the recovery of GSH.

Virtual Compound Screening and Molecular Dynamics to Identify New Inhibitors for Human Glutathione Reductase

Background: Oxidative stress is a defense mechanism against malarial intracellular parasite infection. On the other hand, the Human glutathione reductase enzyme reduces oxidative stress in the cells, making the inhibitors of this enzyme a promising candidate for malaria treatment. Objective: Rational drug design was used in this work to plan new human glutathione reductase inhibitors. Methods: Virtual screening was performed using the ZINC database and molecular docking was used to detect appropriate human glutathione reductase inhibitors. Based on the docking scores obtained, the top three highest-ranked ligands were selected for the molecular dynamics simulation study. The MD simulation was performed for each complex in a length of 100 ns. Results: RMSD, RMSF and hydrogen bond analyzes were performed on the derived trajectories. Molecular mechanics generalized born surface area (MM-GBSA) and pairwise per-residue free energy decomposition analyzes were performed for the determination of binding free energy and the determination of dominant residues involved in the binding process, respectively. The binding free energy analysis showed that the molecule of 3-((7-(furan-2-ylmethyl)-5,6-diphenyl-7H-pyrrolo[2,3- d] pyrimidin-4-yl) amino) propan-1-ol is the most potent inhibitor among the molecules considered against human glutathione reductase enzyme. Conclusion: This molecule can be considered a novel candidate for antimalarial treatments.

Spiro-containing derivatives show antiparasitic activity against Trypanosoma brucei through inhibition of the trypanothione reductase enzyme.

Trypanothione reductase (TR) is a key enzyme that catalyzes the reduction of trypanothione, an antioxidant dithiol that protects Trypanosomatid parasites from oxidative stress induced by mammalian host defense systems. TR is considered an attractive target for the development of novel anti-parasitic agents as it is essential for parasite survival but has no close homologue in humans. We report here the identification of spiro-containing derivatives as inhibitors of TR from Trypanosoma brucei (TbTR), the parasite responsible for Human African Trypanosomiasis. The hit series, identified by high throughput screening, was shown to bind TbTR reversibly and to compete with the trypanothione (TS2) substrate. The prototype compound 1 from this series was also found to impede the growth of Trypanosoma brucei parasites in vitro. The X-ray crystal structure of TbTR in complex with compound 1 solved at 1.98 Å allowed the identification of the hydrophobic pocket where the inhibitor binds, placed close to the catalytic histidine (His 461') and lined by Trp21, Val53, Ile106, Tyr110 and Met113. This new inhibitor is specific for TbTR and no activity was detected against the structurally similar human glutathione reductase (hGR). The central spiro scaffold is known to be suitable for brain active compounds in humans thus representing an attractive starting point for the future treatment of the central nervous system stage of T. brucei infections.

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

Characterisation of naturally occurring isothiocyanates as glutathione reductase inhibitors

Glutathione reductase (GR), an essential antioxidant enzyme against oxidative stress, has become an attractive drug target for the development of anticancer and antimalarial drugs. In this regard, we evaluated the naturally occurring isothiocyanates as promising GR inhibitors and elucidated the mechanism of action. It was found that benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC) inhibited yeast GR (yGR) and human GR (hGR) in a time- and concentration-dependent manner. The Ki and kinact of BITC against yGR were determined to be 259.87 µM and 0.0266 min−1, respectively. The GR inhibition occurred only in the presence of NADPH and persisted after extensive dialysis. The tandem mass spectrometric analysis revealed that Cys61 rather than Cys66 at the active site of yGR was mono-benzyl thiocarbamoylated by BITC. Inhibition of intracellular GR by BITC and PEITC in cultured cancer cells was also observed. BITC and PEITC were evaluated as competitive and irreversible inhibitors of GR.

Targeting a Large Active Site: Structure-Based Design of Nanomolar Inhibitors of Trypanosoma brucei Trypanothione Reductase.

Trypanothione reductase (TR) plays a key role in the unique redox metabolism of trypanosomatids, the causative agents of human African trypanosomiasis (HAT), Chagas' disease, and leishmaniases. Introduction of a new, lean propargylic vector to a known class of TR inhibitors resulted in the strongest reported competitive inhibitor of Trypanosoma (T.) brucei TR, with an inhibition constant Ki of 73 nm, which is fully selective against human glutathione reductase (hGR). The best ligands exhibited in vitro IC50 values (half-maximal inhibitory concentration) against the HAT pathogen, T. brucei rhodesiense, in the mid-nanomolar range, reaching down to 50 nm. X-Ray co-crystal structures confirmed the binding mode of the ligands and revealed the presence of a HEPES buffer molecule in the large active site. Extension of the propargylic vector, guided by structure-based design, to replace the HEPES buffer molecule should give inhibitors with low nanomolar Ki and IC50 values for in vivo studies.

Inhibitory Effects of Usnic and Carnosic Acid on Some Metabolic Enzymes: An In vitro Study.

Natural products are produced via primary and secondary metabolism in different organisms. The compounds obtained via secondary metabolism are not essential for the survival of the organism, but they can have a different value for humans. The objective of this study was to examine inhibitory effects of Usnic Acid (UA), a well-known lichen secondary metabolite, and Carnosic Acid (CA), the primary antioxidant compound of Rosmarinus officinalis L., on purified Human Paraoxonase, (PON1), Glutathione Reductase (GR) and Glutathione S-Transferase (GST). These enzymes have antioxidant properties and a protective effect on the oxidation of free radicals. Hence, deficiencies of such enzymes inside cells can result in a buildup of toxic substances and cause some metabolic disorders. UA and CA were tested in various concentrations against human GST, PON1, and GR activity in vitro and they reduced human GST, PON1, and GR activity. UA Ki constants were calculated as 0.012±0.0019, 0.107±0.06 and 0.21±0.1 mM for GST, PON1, and GR enzymes. CA Ki constants were determined as 0.028±0.009, 0.094±0.03 and 0.79±0.33 mM, for GST, PON1, and GR enzymes. UA and CA showed competitive inhibition for GR and GST enzymes, while they exhibited non-competitive inhibition for PON1. These findings indicate that UA and CA could be useful in drug development studies.