Naegleria fowleri: a free-living highly pathogenic amoeba contains trypanothione/trypanothione reductase and glutathione/glutathione reductase systems

This paper presents definitive data showing that the thiol-bimane compound isolated and purified by HPLC from Naegleria fowleri trophozoites unequivocally corresponds by matrix assisted laser-desorption ionization-time-of-flight MS, to the characteristic monoprotonated ion of trypanothione-(bimane)(2) [M(+)H(+)] of m/z 1104.57 and to the trypanothione-(bimane) of m/z 914.46. The trypanothione disulfide T(S)(2) was also found to have a molecular ion of m/z 723.37. Additionally HPLC demonstrated that thiol-bimane compounds corresponding to cysteine and glutathione were present in Naegleria. The ion patterns of the thiol-bimane compounds prepared from commercial trypanothione standard, Entamoeba histolytica and Crithidia luciliae are identical to the Naegleria thiol-bimane compound. Partially purified extracts from N. fowleri showed the coexistence of glutathione and trypanothione reductases activities. There is not doubt that the thiol compound trypanothione, which was previously thought to occur only in Kinetoplastida, is also present in the human pathogens E. histolytica and N. fowleri, as well as in the non-pathogenic euglenozoan E. gracilis. The presence of the trypanothione/trypanothione reductase system in N. fowleri creates the possibility of using this enzyme as a new "drug target" for rationally designed drugs to eliminate the parasite, without affecting the human host.     

Redox enzyme engineering: conversion of human glutathione reductase into a trypanothione reductase

The substrate specificity of the human enzyme glutathione reductase was changed from its natural substrate glutathione to trypanothione [N1,N8-bis(glutathionyl)spermidine] by site-directed mutagenesis of two residues. The glutathione analogue, trypanothione, is the natural substrate for trypanothione reductase, an enzyme found in trypanosomatids and leishmanias, the causative agents of diseases such as African sleeping sickness, Chagas disease, and Oriental sore. The rational bases for our mutational experiments were the availability of a high-resolution X-ray structure for human glutathione reductase with bound substrates, the active site sequence comparisons of human glutathione reductase and the trypanothione reductases from Trypanosoma congolense and Trypanosoma cruzi, a complementary set of mutants in T. congolense trypanothione reductase, and the properties of substrate analogues of trypanothione. Mutation of two residues, A34----E34 and R37----W37, in the glutathione-binding site of human glutathione reductase switches human glutathione reductase into a trypanothione reductase with a preference for trypanothione over glutathione by a factor of 700 using kcat/Km as a criterion.     

Docking and molecular dynamics studies on triclosan derivatives binding to FabI

FabI, enoyl-ACP reductase (ENR), is the rate-limiting enzyme in the last step for fatty acids biosynthesis in many bacteria. Triclosan (TCL) is a commercial bactericide, and as a FabI inhibitor, it can depress the substrate (trans-2-enoyl-ACP) binding with FabI to hinder the fatty acid synthesis. The structure-activity relationship between TCL derivatives and FabI protein has already been acknowledged, however, their combination at the molecular level has never been investigated. This paper uses the computer-aided approaches, such as molecular docking, molecular dynamics simulation, and binding free energy calculation based on the molecular mechanics/Poisson-Bolzmann surface area (MM/PBSA) method to illustrate the interaction rules of TCL derivatives with FabI and guide the development of new derivatives. The consistent data of the experiment and corresponding activity demonstrates that electron-withdrawing groups on side chain are better than electron-donating groups. 2-Hydroxyl group on A ring, promoting the formation of hydrogen bond, is vital for bactericidal effect; and the substituents at 4-position of A ring, 2′-position and 4′-position of B ring benefit antibacterial activity due to forming a hydrogen bond or stabilizing the conformation of active pocket residues of receptor. While the substituents at 3′-position and 5′-position of B ring destroy the π-π stacking interaction of A ring and NAD⁺ which depresses the antibacterial activity. This study provides a new sight for designing novel TCL derivatives with superior antibacterial activity.     

Investigation of trypanothione reductase inhibitory activity by 1,3,4-thiadiazolium-2-aminide derivatives and molecular docking studies

The biological activities of a series of mesoionic 1,3,4-thiadiazolium-2-aminide derivatives have been studied. The most active compounds (MI-HH; MI-3-OCH(3); MI-4-OCH(3) and MI-4-NO(2)) were evaluated to determine their effect on trypanothione reductase (TryR) activity in Leishmania sp. and Trypanosoma cruzi. Among the assayed compounds, only MI-4-NO(2) showed enzyme inhibition effect on extracts from different cultures of parasites, which was confirmed using the recombinant enzyme from T. cruzi (TcTryR) and Leishmania infantum (LiTryR). The enzyme kinetics determined with LiTryR demonstrated a non-competitive inhibition profile of MI-4-NO(2). A molecular docking study showed that the mesoionic compounds could effectively dock into the substrate binding site together with the substrate molecule. The mesoionic compounds were also effective ligands of the NADPH and FAD binding sites and the NADPH binding site was predicted as the best of all three binding sites. Based on the theoretical results, an explanation at the molecular level is proposed for the MI-4-NO(2) enzyme inhibition effect. Given TryR as a molecular target, it is important to continue the study of mesoionic compounds as part of a drug discovery campaign against Leishmaniasis or Chagas' disease.     

Docking small-molecule ligands into active sites

Docking involves the development of computer algorithms that evaluate the binding modes of putative ligands in receptor sites. The principal advances of the past year have been the development of new algorithmic approaches, several of which incorporate conformational flexibility, and the increased use of docking to identify leads in drug-discovery programmes.     

Synthesis of asymmetric disulfides as potential alternative substrates for trypanothione reductase and glutathione reductase: Part 2

Summary The synthesis of asymmetrical disulfides, based on Zervas' inter-mediate, monocarbobenzoxy-L-cystine, has been developed. A series of substrate analogues of trypanothione disulfide (TSST) and glutathione disulfide (GSSG) are described, where the spermidine ring of (TSST) has been replaced by 3-dimethylaminopropylamine (DMAPA). The free amino group in Zervas' product was condensed with phenylalanyl, tryptophanyl or glutamyl residues, while the carbobenzoxy group was unaffected under the reaction conditions employed. The same synthetic approach was applied in the design of analogues of glutathione disulfide (GSSG).     

Mutational analysis of parasite trypanothione reductase: acquisition of glutathione reductase activity in a triple mutant

African trypanosomes contain a cyclic derivative of oxidized glutathione, N1,N8-bis(glutathionyl)spermidine, termed trypanothione. This is the substrate for the parasite enzyme trypanothione reductase, a key enzyme in disulfide/dithiol redox balance and a target enzyme for trypanocidal therapy. Trypanothione reductase from these and related trypanosomatid parasites is structurally homologous to host glutathione reductase but the two enzymes show mutually exclusive substrate specificities. To assess the basis of host vs parasite enzyme recognition for their disulfide substrates, the interaction of bound glutathione with active-site residues in human red cell glutathione reductase as defined by prior X-ray analysis was used as the starting point for mutagenesis of three residues in trypanothione reductase from Trypanosoma congolense, a cattle parasite. Mutation of three residues radically alters enzyme specificity and permits acquisition of glutathione reductase activity at levels 10(4) higher than in wild-type trypanothione reductase.     

Synthesis of symmetric disulfides as potential alternative substrates for trypanothione reductase and glutathione reductase: Part 1

Summary The synthesis of a series of symmetrical disulfides as potential substrates of trypanothione reductase and glutathione reductase was described. The key intermediate in the synthetic approach was the choice of S-^tbutylmercapto-L-cysteine (1). The spermidine ring in the native substrate, trypanothione disulfide (TSST), was replaced with 3-dimethyl-aminopropylamine (DMAPA), while the-Glu moiety was replaced by phenylalanyl or tryptophanyl residues. The same modifications in the-Glu moiety of glutathione disulfide (GSSG) were applied.     

Febrifugine analogues as Leishmania donovani trypanothione reductase inhibitors: binding energy analysis assisted by molecular docking,ADMET and molecular dynamics simulation

Visceral leishmaniasis affects people from 70 countries worldwide, mostly from Indian, African and south American continent. The increasing resistance to antimonial, miltefosine and frequent toxicity of amphotericin B drives an urgent need to develop an antileishmanial drug with excellent efficacy and safety profile. In this study we have docked series of febrifugine analogues (n = 8813) against trypanothione reductase in three sequential docking modes. Extra precision docking resulted into 108 ligands showing better docking score as compared to two reference ligand. Furthermore, 108 febrifugine analogues and reference inhibitor clomipramine were subjected to ADMET, QikProp and molecular mechanics, the generalized born model and solvent accessibility study to ensure the toxicity caused by compounds and binding-free energy, respectively. Two best ligands (FFG7 and FFG2) qualifying above screening parameters were further subjected to molecular dynamics simulation. Conducting these studies, here we confirmed that 6-chloro-3-[3-(3-hydroxy-2-piperidyl)-2-oxo-propyl]-7-(4-pyridyl) quinazolin-4-one can be potential drug candidate to fight against Leishmania donovani parasites.     

Two interacting binding sites for quinacrine derivatives in the active site of trypanothione reductase: a template for drug design

Trypanothione reductase is a key enzyme in the trypanothione-based redox metabolism of pathogenic trypanosomes. Because this system is absent in humans, being replaced with glutathione and glutathione reductase, it offers a target for selective inhibition. The rational design of potent inhibitors requires accurate structures of enzyme-inhibitor complexes, but this is lacking for trypanothione reductase. We therefore used quinacrine mustard, an alkylating derivative of the competitive inhibitor quinacrine, to probe the active site of this dimeric flavoprotein. Quinacrine mustard irreversibly inactivates Trypanosoma cruzi trypanothione reductase, but not human glutathione reductase, in a time-dependent manner with a stoichiometry of two inhibitors bound per monomer. The rate of inactivation is dependent upon the oxidation state of trypanothione reductase, with the NADPH-reduced form being inactivated significantly faster than the oxidized form. Inactivation is slowed by clomipramine and a melarsen oxide-trypanothione adduct (both are competitive inhibitors) but accelerated by quinacrine. The structure of the trypanothione reductase-quinacrine mustard adduct was determined to 2.7 A, revealing two molecules of inhibitor bound in the trypanothione-binding site. The acridine moieties interact with each other through pi-stacking effects, and one acridine interacts in a similar fashion with a tryptophan residue. These interactions provide a molecular explanation for the differing effects of clomipramine and quinacrine on inactivation by quinacrine mustard. Synergism with quinacrine occurs as a result of these planar acridines being able to stack together in the active site cleft, thereby gaining an increased number of binding interactions, whereas antagonism occurs with nonplanar molecules, such as clomipramine, where stacking is not possible.     

Molecular studies on trypanothione reductase, a target for antiparasitic drugs

Trypanosoma and Leishmania are parasitic protozoa that cause a variety of diseases, which include African sleeping sickness and oriental sore. Attempts to determine pharmaceutically exploitable differences between host and parasite biochemistry have identified the unique trypanothione pathway as a possible target. This pathway includes the enzyme trypanothione reductase, the parasite analogue of glutathione reductase.     

Titration studies on the active sites of pig heart lipoamide dehydrogenase and yeast glutathione reductase as monitored by the charge transfer absorbance

Macroscopic pKa values associated with the influence of pH on the visible spectrum of 2-electron reduced pig heart lipoamide dehydrogenase and yeast glutathione reductase have been determined by monitoring changes in the principal flavin band near 460 nm and the charge transfer band at 540 nm. The ionization of at least three active site amino acid side chains can influence the spectra over the range of pH studied: the two nascent thiols (interchange thiol and electron transfer thiol) and the histidine residue which acts as the base catalyst in lipoamide dehydrogenase and the acid catalyst in glutathione reductase thiol-disulfide interchange reactions. These systems are analogous to, but more complex than, those in glyceraldehyde-3-phosphate dehydrogenase and papain where a single thiol and a histidine residue in a relatively apolar milieu form a thiolate-imidazolium ion pair which is favored over the thiol-imidazole prototropic tautomer. In an effort to more nearly mimic the papain titrations, the macroscopic pKa values were determined on reduced glutathione reductase which had been monoalkylated with iodoacetamide under conditions known to favor the reaction of the interchange thiol by at least 10 to 1 (Arscott, L. D., Thorpe, C., and Williams, C. H., Jr. (1981) Biochemistry 20, 1513-1520). Like papain and glyceraldehyde-3-phosphate dehydrogenase, alkylated glutathione reductase showed two macroscopic pKa values, at pH 3.7 and pH 9.1, and by analogy, these were associated primarily with the thiol and the imidazole, respectively. Results with the native enzymes depended on the wavelength monitored. Glutathione reductase had pKa values at 4.8, 7.1, and 9.2 when monitored at 540 nm and 5.1 and 8.2 when monitored at 462 nm. Lipoamide dehydrogenase had pKa values at 4.4 and 8.7 when monitored at 529 nm and 3.9, 7.0, and 9.3 when monitored at 455 nm.     

Steady-state kinetic studies of glutathione reductase

The steady-state kinetic studies of yeast glutathione reductase, performed when [GSSG] = 10[NADPH] in the assay mixture, show that at concentrations of GSSG under 450 microM the enzymatic mechanism pathway is ping-pong. Furthermore, in the case of higher values, the enzymatic kinetics follows a sequential pathway. However when the glutathione reductase reaction passes to the ping-pong mechanism, the inhibition effect by excess of NADPH is stronger than when the reaction takes place over the sequential mechanism.     

Docking and molecular dynamics simulation studies on glycation-induced conformational changes of human paraoxonase 1

Human paraoxonase 1 (huPON1) is a calcium-dependent esterase responsible for hydrolysis of a wide variety of substrates including organophosphates, esters, lactones, and paraoxon. Although its natural substrate is unknown, the action of PON as an antioxidant is well documented. Because recent reports have suggested glycation may induce reduced PON activity in diabetes, we investigated the structural features of huPON1 and its glycated mutant by template-based modeling, docking, and molecular dynamics (MD) simulations. Our results corroborated the importance of the His115–His134 dyad in both the lactonase and paraoxonase activity of huPON1. Structural alterations in the glycated model reflected weak interactions between the docked substrate and the active site cleft. We also used MD simulation to gain insight into glycation-induced conformational changes of huPON1 and the implication of this on depleted enzymatic activity. The catalytic calcium found on the surface interacts with the side chain oxygen of residues, including Asn224, Asn270, Asn168, Asp269, and Glu53, and this interaction with the respective residues undergoes minor displacement on glycation. The root-mean-square fluctuation had high motional flexibility in the non-glycated model whereas the conformation of the glycated structure was comparatively stable. Our findings emphasize the consequence of glycation-induced alterations and their effect on overall enzymatic activity.     

Cell-free production of active E. coli thioredoxin reductase and glutathione reductase

Escherichia coli thioredoxin reductase (TR) and glutathione reductase (GR) are dimeric proteins that require a flavin adenine dinucleotide (FAD) cofactor for activity. A cell-free protein synthesis (CFPS) reaction supplemented with FAD was used to produce TR at 760 microg/ml with 89% of the protein being soluble. GR accumulated to 521 microg/ml in a cell-free reaction with 71% solubility. The TR produced was fully active with a specific activity of 1390 min(-1). The GR had a specific activity of 139 U/mg, which is significantly more active than reported for GR purified from cells. The specific activity for both TR and GR decreased without FAD supplementation. This research demonstrates that CFPS can be used to produce enzymes that are multimeric and require a cofactor.     

Correlation of binding constants and molecular modelling of inhibitors in the active sites of aldose reductase and aldehyde reductase

Aldose reductase (ALR2) belongs to the aldo–keto reductase (AKR) superfamily of enzymes, is the first enzyme involved in the polyol pathway of glucose metabolism and has been linked to the pathologies associated with diabetes. Molecular modelling studies together with binding constant measurements for the four inhibitors Tolrestat, Minalrestat, quercetin and 3,5-dichlorosalicylic acid (DCL) were used to determine the type of inhibition, and correlate inhibitor potency and binding energies of the complexes with ALR2 and the homologous aldehyde reductase (ALR1), another member of the AKR superfamily. Our results show that the four inhibitors follow either uncompetitive or non-competitive inhibition pattern of substrate reduction for ALR1 and ALR2. Overall, there is correlation between the IC₅₀ (concentration giving 50% inhibition) values of the inhibitors for the two enzymes and the binding energies (ΔH) of the enzyme–inhibitor complexes. Additionally, the results agree with the detailed structural information obtained by X-ray crystallography suggesting that the difference in inhibitor binding for the two enzymes is predominantly mediated by non-conserved residues. In particular, Arg312 in ALR1 (missing in ALR2) contributes favourably to the binding of DCL through an electrostatic interaction with the inhibitor’s electronegative halide atom and undergoes a conformational change upon Tolrestat binding. In ALR2, Thr113 (Tyr116 in ALR1) forms electrostatic interactions with the fluorobenzyl moiety of Minalrestat and the 3- and 4-hydroxy groups on the phenyl ring of quercetin. Our modelling studies suggest that Minalrestat’s binding to ALR1 is accompanied by a conformational change including the side chain of Tyr116 to achieve the selectivity for ALR1 over ALR2.     

Density functional theory studies of model complexes for molybdenum-dependent nitrate reductase active sites

Molybdenum and tungsten complexes as models for the active sites of assimilatory or dissimilatory nitrate reductases (NR) were computed at the CPCM-B98/SDDp//B3LYP/Lanl2DZp* plus zero point energy level of density functional theory. The ligands were chosen on the basis of available experimental protein or small chemical model structures. A water molecule is found to bind to assimilatory NR models [(Me₂C₂S₂)MO(YMe)]⁻ (−11.5 kcal mol⁻¹ for M is Mo, Y is S) and may be replaced by nitrate (−4.5 kcal mol⁻¹) (but a hydroxy group may not). Nature’s choice of M is Mo and Y is S for NR has the largest activation energy for protein-free models (13.3 kcal mol⁻¹) and the least exothermic reaction energy for the nitrate reduction (−14.9 kcal mol⁻¹) compared with M is W and Y is O or Se alternatives. Water binding to dissimilatory NR model complexes [(Me₂C₂S₂)₂M(YR)]⁻ is considerably endothermic (10.3 kcal mol⁻¹); nitrate binding is only slightly so (1.5 kcal mol⁻¹ for RY⁻ is MeS⁻). The exchange of an oxo ligand (assimilatory NR) for a dithiolato ligand (dissimilatory NR model) reduces the exothermicity (−8.6 kcal mol⁻¹ relative to the fivefold-coordinate reduced complex) and raises the barrier for oxygen atom transfer (OAT) in the nitrate complex (19.2 kcal mol⁻¹). Not for the mono but only for the bisdithiolato complexes hydrogen bonding involving the coordinated substrate may significantly lower the OAT barrier as shown by explicitly adding water molecules. Substitution of tungsten for molybdenum generally lowers OAT activation energies and makes nitrate reduction reaction energies more negative. Bidentate carboxylato binding identified in Escherichia coli NarGHI is the preferred binding mode also for an acetato model. However, one dithiolato ligand folds when the Mo^VI center is bare of a good π-donor ligand, e.g., an oxo group. Computations on [(mnt)₂Mo^IV(YR)(PPh₃)]⁻ [mnt is (CN)₂C₂S₂ ²⁻] gave a smaller nitrate reduction activation energy for RY⁻ is Cl⁻, compared with RY⁻ is PhS⁻, although experimentally only the phenyl thiolato complex and not the chloro complex was found to be a functional NR model. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Comparative studies of glutathione reductase and lipoamide dehydrogenase

1. Glutathione reductase and lipoamide dehydrogenase are structurally and mechanistically related flavoenzymes catalyzing various one and two electron transfer reactions between NAD(P)H and substrates with different structures. 2. The two enzymes differ in their coenzyme and functional specificities. Lipoamide dehydrogenase shows higher coenzyme preference while glutathione reductase displays greater functional specificity. 3. Binding preference of the two flavoenzymes for nicotinamide coenzymes is demonstrated by 31P-NMR spectroscopy. 4. The presence of arginines in glutathione reductase which is inactivated by phenyl glyoxal, is likely to be responsible for the NADPH-activity of glutathione reductase. 5. The substrate binding sites of the two enzymes are similar, though their functional details differ. 6. The active-site histidine of glutathione reductase functions primarily as the proton donor during catalysis. While the active-site histidine of lipoamide dehydrogenase stabilizes the thiolate anion intermediate and relays a proton in the catalytic process.     

Docking and molecular dynamics studies of new potential inhibitors of the human epidermal receptor 2

Compounds similar to lapatinib and gefitinib have been investigated as potential inhibitors of the intracellular receptor tyrosine kinase (RTK) domain of the human epidermal receptor 2 (HER2), which is a promising molecular target to the drug design of new chemotherapies for breast, lung, ovarian and colorectal cancers. In this study, we have searched potential HER2 inhibitors used for treatment of other illnesses such as hepatitis, bacterial infections and sexual impotence screened in the DrugBank. The compounds selected were subjected to virtual screening docking in order to evaluate the main interactions between them and the RTK domain of HER2. The selected compounds were investigated by flexible docking, molecular dynamics studies and ΔG _bind calculations. The results suggest that antrafenine, saprisartan, reserpine, irinotecan and udenafil are potential candidates to inhibit the RTK domain of HER2.     

Developing imidazole analogues as potential inhibitor for Leishmania donovani trypanothione reductase: virtual screening,molecular docking,dynamics and ADMET approach

Visceral leishmaniasis (VL) affects Indian subcontinent, African and South American continent, and it covers 70 countries worldwide. Visceral form of leishmaniasis is caused by Leishmania donovani in Indian subcontinent which is lethal if left untreated. Extensive resistance to antileishmanial drugs such as sodium stibogluconate, pentamidine and miltefosine and their decreased efficacy has been reported in the endemic region. Amphotericin B drug has shown good antileishmanial activity with significant toxicity, but its cost of treatment has limited the outreach of this treatment to affected people living in endemic zone. So, there is an urgent need to identify new antileishmanial drugs with excellent activity and minimal toxicity issues. Trypanothione reductase, a component of antioxidant system, is necessary for parasite growth and survival to raise infection. To develop potential inhibitor, we docked nine hundred and eighty-four 5-nitroimidazole analogues along with clomipramine which is a well-known inhibitor for TR. Total one hundred and forty-seven 5-nitroimidazole analogues with better docking score than clomipramine were chosen for ADMET and QikProp studies. Among these imidazole analogues, total twenty-four imidazole analogues and clomipramine were chosen on the basis of their ADMET, QikProp, and prime MM-GBSA study. Later on, two analogues with best MM-GBSA dG bind were undergone molecular dynamic simulation to ensure protein–ligand interactions. Using above approach, we confirm that ethyl 2-acetyl-5-[4-butyl-2-(3-hydroxypentyl)-5-nitro-1H-imidazol-1-yl]pent-2-enoate can be a drug candidate against L. donovani for the treatment of VL in the Indian subcontinent.