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Alan H Fairlamb - One of the best experts on this subject based on the ideXlab platform.
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comparative structural kinetic and inhibitor studies of trypanosoma brucei Trypanothione reductase with t cruzi
Molecular and Biochemical Parasitology, 2010Co-Authors: Deuan C Jones, Antonio Ariza, Winghuen A Chow, Alan H FairlambAbstract:As part of a drug discovery programme to discover new treatments for human African trypanosomiasis, recombinant Trypanothione reductase from Trypanosoma brucei has been expressed, purified and characterized. The crystal structure was solved by molecular replacement to a resolution of 2.3 A and found to be nearly identical to the T. cruzi enzyme (root mean square deviation 0.6 A over 482 Cα atoms). Kinetically, the Km for Trypanothione disulphide for the T. brucei enzyme was 4.4-fold lower than for T. cruzi measured by either direct (NADPH oxidation) or DTNB-coupled assay. The Km for NADPH for the T. brucei enzyme was found to be 0.77 μM using an NADPH-regenerating system coupled to reduction of DTNB. Both enzymes were assayed for inhibition at their respective S = Km values for Trypanothione disulphide using a range of chemotypes, including CNS-active drugs such as clomipramine, trifluoperazine, thioridazine and citalopram. The relative IC50 values for the two enzymes were found to vary by no more than 3-fold. Thus Trypanothione reductases from these species are highly similar in all aspects, indicating that they may be used interchangeably for structure-based inhibitor design and high-throughput screening.
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leishmania Trypanothione synthetase amidase structure reveals a basis for regulation of conflicting synthetic and hydrolytic activities
Journal of Biological Chemistry, 2008Co-Authors: Paul K Fyfe, Alan H Fairlamb, Sandra L. Oza, William N HunterAbstract:The bifunctional Trypanothione synthetase-amidase catalyzes biosynthesis and hydrolysis of the glutathione-spermidine adduct Trypanothione, the principal intracellular thiol-redox metabolite in parasitic trypanosomatids. These parasites are unique with regard to their reliance on Trypanothione to determine intracellular thiol-redox balance in defense against oxidative and chemical stress and to regulate polyamine levels. Enzymes involved in Trypanothione biosynthesis provide essential biological activities, and those absent from humans or for which orthologues are sufficiently distinct are attractive targets to underpin anti-parasitic drug discovery. The structure of Leishmania major Trypanothione synthetase-amidase, determined in three crystal forms, reveals two catalytic domains. The N-terminal domain, a cysteine, histidine-dependent amidohydrolase/peptidase amidase, is a papain-like cysteine protease, and the C-terminal synthetase domain displays an ATP-grasp family fold common to C:N ligases. Modeling of substrates into each active site provides insight into the specificity and reactivity of this unusual enzyme, which is able to catalyze four reactions. The domain orientation is distinct from that observed in a related bacterial glutathionylspermidine synthetase. In Trypanothione synthetase-amidase, the interactions formed by the C terminus, binding in and restricting access to the amidase active site, suggest that the balance of ligation and hydrolytic activity is directly influenced by the alignment of the domains with respect to each other and implicate conformational changes with amidase activity. The potential inhibitory role of the C terminus provides a mechanism to control relative levels of the critical metabolites, Trypanothione, glutathionylspermidine, and spermidine in Leishmania.
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Phenotypic analysis of Trypanothione synthetase knockdown in the African trypanosome
Biochemical Journal, 2005Co-Authors: Mark R. Ariyanayagam, Sandra L. Oza, Maria Lucia S. Güther, Alan H FairlambAbstract:Trypanothione plays a pivotal role in defence against chemical and oxidant stress, thiol redox homoeostasis, ribonucleotide metabolism and drug resistance in parasitic kinetoplastids. In Trypanosoma brucei, Trypanothione is synthesized from glutathione and spermidine by a single enzyme, TryS (Trypanothione synthetase), with glutathionylspermidine as an intermediate. To examine the physiological roles of Trypanothione, tetracycline-inducible RNA interference was used to reduce expression of TRYS. Following induction, TryS protein was reduced >10-fold and growth rate was reduced 2-fold, with concurrent 5–10-fold decreases in glutathionylspermidine and Trypanothione and an up to 14-fold increase in free glutathione content. Polyamine levels were not significantly different from non-induced controls, and neither was the intracellular thiol redox potential, indicating that these factors are not responsible for the growth defect. Compensatory changes in other pathway enzymes were associated with prolonged suppression of TryS: an increase in Trypanothione reductase and γ-glutamylcysteine synthetase, and a transient decrease in ornithine decarboxylase. Depleted Trypanothione levels were associated with increases in sensitivity to arsenical, antimonial and nitro drugs, implicating Trypanothione metabolism in their mode of action. Escape mutants arose after 2 weeks of induction, with all parameters, including growth, returning to normal. Selective inhibitors of TryS are required to fully validate this novel drug target.
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Trypanothione biosynthesis in Leishmania major.
Molecular and biochemical parasitology, 2005Co-Authors: Sandra L. Oza, Susan Wyllie, Matthew P. Shaw, Alan H FairlambAbstract:Abstract Trypanothione plays a crucial role in regulation of intracellular thiol redox balance and in defence against chemical and oxidant stress. Crithidia fasciculata requires two enzymes for the formation of Trypanothione, namely glutathionylspermidine synthetase (GspS; EC 6.3.1.8) and a glutathionylspermidine-dependent Trypanothione synthetase (TryS; EC 6.3.1.9), whereas Trypanosoma cruzi and Trypanosoma brucei use a broad-specificity Trypanothione synthetase to make Trypanothione from glutathione (GSH) and spermidine. Here, we report the identification of two genes in Leishmania major with similarity to previously identified GSPS and TRYS . GSPS is an apparent pseudogene containing two frame shift mutations and two stop codons, whereas TRYS is in a single open-reading frame. The enzyme encoded by TRYS was expressed and found to catalyse formation of Trypanothione with GSH and either spermidine or glutathionylspermidine. When GSH is varied as substrate the enzyme displays substrate inhibition (apparent K m = 89 μM, K i s = 1 mM, k cat = 2 s −1 ). At a fixed GSH concentration, the enzyme obeys simple hyperbolic kinetics with the other substrates with apparent K m values for spermidine, glutathionylspermidine and MgATP of 940, 40 and 63 μM, respectively. Immunofluorescence and sub-cellular fractionation studies indicate that TryS localises to the cytosol of L. major promastigotes. Phylogenetic analysis of the GspS and TryS amino acid sequences suggest that in the trypanosomatids, TryS has evolved to replace the GspS/TryS complex in C. fasciculata . It also appears that the L. major still harbours a redundant GSPS pseudogene that may be currently in the process of being lost from its genome.
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dual action of antimonial drugs on thiol redox metabolism in the human pathogen leishmania donovani
Journal of Biological Chemistry, 2004Co-Authors: Susan Wyllie, Mark Cunningham, Alan H FairlambAbstract:Abstract Despite extensive use of antimonial compounds in the treatment of leishmaniasis, their mode of action remains uncertain. Here we show that trivalent antimony (SbIII) interferes with Trypanothione metabolism in drug-sensitive Leishmania parasites by two inherently distinct mechanisms. First, SbIII decreases thiol buffering capacity by inducing rapid efflux of intracellular Trypanothione and glutathione in approximately equimolar amounts. Second, SbIII inhibits Trypanothione reductase in intact cells resulting in accumulation of the disulfide forms of Trypanothione and glutathione. These two mechanisms combine to profoundly compromise the thiol redox potential in both amastigote and promastigote stages of the life cycle. Furthermore, we demonstrate that sodium stibogluconate, a pentavalent antimonial used clinically for the treatment for leishmaniasis, induces similar effects on thiol redox metabolism in axenically cultured amastigotes. These observations suggest ways in which current antimony therapies could be improved, overcoming the growing problem of antimony resistance.
Mark Bradley - One of the best experts on this subject based on the ideXlab platform.
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Substrate Specificity of Trypanothione Reductase
European journal of biochemistry, 1997Co-Authors: Ian R. Marsh, Mark BradleyAbstract:Trypanothione redutase, one of the family of flavin-dependent disulfide oxidoreductases, catalyses the reduction of Trypanothione disulfide [N1,N8-bis(glutathionyl)spermidine] and related glutathionyl-polyamine disulfides. A series of subtly different, designed substrate analogues based on Trypanothione were prepared by means of a solid-phase approach and used to study the catalytic efficiency of the parasitic enzyme. Kinetic analysis showed that the size of the polyamine bridge was relatively unimportant, for catalysis, while replacement of the ammonium bridge was much more dramatic. The highly charged glutathionylspermidine disulfide had the largest Km of all the substrates tested. N1-acetylcysteinylglycinyl-N8-glutathionyl spermidine and N1-glutathionyl-N8-acetylcysteinylglycinylspermidine both showed a tenfold reduction in catalytic efficiency compared with Trypanothione.
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Kinetic isotope effect analysis of the reaction catalyzed by Trypanosoma congolense Trypanothione reductase
Biochemistry, 1992Co-Authors: Betty N. Leichus, Christopher T Walsh, Kari C. Nadeau, Mark Bradley, John S BlanchardAbstract:African trypanosomes are devoid of glutathione reductase activity, and instead contain a unique flavoprotein variant, Trypanothione reductase, which acts on a cyclic derivative of glutathione, Trypanothione. The high degree of sequence similarity between Trypanothione reductase and glutathione reductase, as well as the obvious similarity in the reactions catalyzed, led us to investigate the pH dependence of the kinetic parameters, and the isotopic behavior of Trypanothione reductase
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Kinetic isotope effect analysis of the reaction catalyzed by Trypanosoma congolense Trypanothione reductase.
Biochemistry, 1992Co-Authors: Betty N. Leichus, Christopher T Walsh, Kari C. Nadeau, Mark Bradley, John S BlanchardAbstract:African trypanosomes are devoid of glutathione reductase activity, and instead contain a unique flavoprotein variant, Trypanothione reductase, which acts on a cyclic derivative of glutathione, Trypanothione. The high degree of sequence similarity between Trypanothione reductase and glutathione reductase, as well as the obvious similarity in the reactions catalyzed, led us to investigate the pH dependence of the kinetic parameters, and the isotopic behavior of Trypanothione reductase. The pH dependence of the kinetic parameters V, V/K for NADH, and V/K for oxidized Trypanothione has been determined for Trypanothione reductase from Trypanosoma congolense. Both V/K for NADH and the maximum velocity decrease as single groups exhibiting pK values of 8.87 +/- 0.09 and 9.45 +/- 0.07, respectively, are deprotonated. V/K for oxidized Trypanothione, T(S)2, decreases as two groups exhibiting experimentally indistinguishable pK values of 8.74 +/- 0.03 are deprotonated. Variable magnitudes of the primary deuterium kinetic isotope effects on pyridine nucleotide oxidation are observed on V and V/K when different pyridine nucleotide substrates are used, and the magnitude of DV and D(V/K) is independent of the oxidized Trypanothione concentration at pH 7.25. Solvent kinetic isotope effects, obtained with 2',3'-cNADPH as the variable substrate, were observed on V only, and plots of V versus mole fraction of D2O (i.e., proton inventory) were linear, and yielded values of 1.3-1.6 for D2OV. Solvent kinetic isotope effects obtained with alternate pyridine nucleotides as substrates were also observed on V, and the magnitude of D2OV decreases for each pyridine nucleotide as its maximal velocity relative to that of NADPH oxidation decreases.(ABSTRACT TRUNCATED AT 250 WORDS)
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Molecular Studies on Trypanothione Reductase: An Antiparasitic Target Enzyme
Current Topics in Cellular Regulation, 1992Co-Authors: Christopher T Walsh, Mark Bradley, Kari C. NadeauAbstract:Publisher Summary This chapter discusses molecular studies on Trypanothione reductase. Investigations into the biochemistry of these parasites have quite surprisingly shown that they are devoid of the almost ubiquitous enzyme glutathione reductase. Instead they contain an enzyme that carries out an analogous role to that of glutathione reductase, the NADPH-dependent reduction of Trypanothione, a novel glutathione analog unique to the trypanosomatid parasites. By a combination of site-directed mutagenesis and the synthesis of substrate analogs, intimate details of the catalytic function of Trypanothione reductase can be determined. Additional evidence for the role of the two residues E18 and W21R in Trypanothione reductase has also recently been determined by an inverse site-directed mutagenesis approach with human glutathione reductase and its conversion to a Trypanothione reductase by modification of the residues A34 and R37 into glutamic and tryptophan residues, respectively. Undoubtedly, a crystal structure of this mutant human GR with bound Trypanothione or an X-ray structure of one of the available Trypanothione reductases will broaden the understanding and the potential for inhibition of this antiparasitic target enzyme.
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Redox enzyme engineering: conversion of human glutathione reductase into a Trypanothione reductase.
Biochemistry, 1991Co-Authors: Mark Bradley, Bücheler Us, Walsh CtAbstract: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.
Kenneth T. Douglas - One of the best experts on this subject based on the ideXlab platform.
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Synthesis of substrate analogues for Trypanothione reductase
International Journal of Peptide and Protein Research, 2009Co-Authors: Abdussalam F. El-waer, T. J. Benson, Kenneth T. DouglasAbstract:The synthesis and chemical characterisation of a range of substrate analogues for Trypanothione reductase are described, with the spermidine portion of Trypanothione replaced by the 3-dimethylaminopropylamide moiety. Using 1-hydroxybenzotriazole/N-hydroxysuccinimide coupling, products were obtained which had a range of replacements of the γ-glutamyl groups of the enzyme substrate. The materials were characterised by FPLC, 1 H /13 C NMR spectroscopy and FAB mass spectroscopy
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Vanadate as a Futile, Superoxide Ion-producing Substrate of Trypanothione Reductase from Trypanosoma cruzi
Chemistry Letters, 2005Co-Authors: M. O.f. Khan, Seheli Parveen, Gavin Malcolm Seddon, Kenneth T. DouglasAbstract:Recombinant Trypanosoma cruzi Trypanothione reductase can oxidize NADPH in a futile cycle in the presence of vanadate ion as a result of the generation of reactive oxygen and other radical species from the active site dithiol and the NADPH. This process is inhibited by competitive inhibitors of Trypanothione reductase catalyzed reduction of Trypanothione disulfide.
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Peptoid inhibition of Trypanothione reductase as a potential antitrypanosomal and antileishmanial drug lead.
Amino Acids, 2002Co-Authors: Cecil Chan, Alan H Fairlamb, James H. Mckie, Kenneth T. DouglasAbstract:One route to the design of lead compounds for rational drug design approaches to developing drugs against trypanosomiasis, Chagas' disease and leishmaniasis is to develop novel inhibitors of the parasite-specific enzyme Trypanothione reductase. A lead inhibitor based on a peptoid structure was designed in the present study based on the known strong competitive inhibition of Trypanothione reductase by N-benzoyl-Leu-Arg-Arg-β-naphthylamide and N-benzyloxycarbonyl-Ala-Arg-Arg-4-methoxy-β-naphthylamide. In the target peptoid the arginyl residues were replaced by alkylimidazolium units and the benzyloxycarbonyl group by the benzylaminocarbonyl function. The peptoid was synthesised using t-butoxycarbonyl protection chemistry and couplings were activated by 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate. The resulting peptoid was shown to be a competitive inhibitor of recombinant Trypanothione reductase from Trypanosoma cruzi with a Ki value of 179 μM and with only weak inhibition of human erythrocyte glutathione reductase (the inhibition of glutathione reductase was at least 291-fold weaker than of Trypanothione reductase).
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phenothiazine inhibitors of Trypanothione reductase as potential antitrypanosomal and antileishmanial drugs
Journal of Medicinal Chemistry, 1998Co-Authors: Cecil Chan, Jacqui Garforth, Peter Rock, James H. Mckie, Rabih Jaouhari, Kenneth T. Douglas, Vanessa Yardley, Simon L Croft, Peter Speers, Alan H FairlambAbstract:Given the role of Trypanothione in the redox defenses of pathogenic trypanosomal and leishmanial parasites, in contrast to glutathione for their mammalian hosts, selective inhibitors of Trypanothione reductase are potential drug leads against trypanosomiasis and leishmaniasis. In the present study, the rational drug design approach was used to discover tricyclic neuroleptic molecular frameworks as lead structures for the development of inhibitors, selective for Trypanothione reductase over host glutathione reductase. From a homology-modeled structure for Trypanothione reductase, replaced in the later stages of the study by the X-ray coordinates for the enzyme from Crithidia fasciculata, a series of inhibitors based on phenothiazine was designed. These were shown to be reversible inhibitors of Trypanothione reductase from Trypanosoma cruzi, linearly competitive with Trypanothione as substrate and noncompetitive with NADPH, consistent with ping-pong bi bi kinetics. Analogues, synthesized to define structure...
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Fluphenazine photoaffinity labelling of binding sites for phenothiazine inhibitors of Trypanothione reductase
Chemical Communications, 1996Co-Authors: Cecil Chan, Jacqui Garforth, Mark S. Bolgar, Simon J. Gaskell, Kenneth T. Douglas, Alan H FairlambAbstract:Photolysis of fluphenazine, a competitive inhibitor of Trypanothione reductase in the presence of Trypanothione reductase leads to irreversible, time-dependent inactivation, which is not dependent on the presence of molecular oxygen in the medium and can be pretected against by the presence of Trypanothione substrate; MALDI and electrospray mass spectrometric analyses shows that 2–5 equiv. of the phenothiazine are incorporated per enzyme subunit.
Christopher T Walsh - One of the best experts on this subject based on the ideXlab platform.
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cloning and characterization of the two enzymes responsible for Trypanothione biosynthesis in crithidia fasciculata
Journal of Biological Chemistry, 1998Co-Authors: Emmanuel Tetaud, Christopher T Walsh, Kari C. Nadeau, Faouzi Manai, Michael P Barrett, Alan H FairlambAbstract:Abstract Protozoa of the order Kinetoplastida differ from other organisms in their ability to conjugate glutathione (γ-Glu-Cys-Gly) and spermidine to form Trypanothione (N 1,N 8-bis(glutathionyl)spermidine), which is involved in maintaining intracellular thiol redox and in defense against oxidants. In this study, the genes from Crithidia fasciculata, Cf-GSS and Cf-TRS, which encode, respectively, glutathionylspermidine synthetase (EC 6.3.1.8) and Trypanothione synthetase (EC 6.3.1.9) have been cloned and expressed. The deduced amino acid sequence of both Cf-GSSand Cf-TRS share 50% sequence similarity with theEscherichia coli glutathionylspermidine synthetase/amidase. Both genes are present as single copies in the C. fasciculata genome. When expressed in E. coli andSaccharomyces cerevisiae, neither protein was present in an active soluble form. However, thiol analysis of S. cerevisiae demonstrated that cells transformed with theCf-GSS gene contained substantial amounts of glutathionylspermidine, whereas cells expressing both theCf-GSS and Cf-TRS genes contained glutathionylspermidine and Trypanothione, confirming that these genes encode the functional glutathionylspermidine and Trypanothione synthetases from C. fasciculata. The translation products of Cf-GSS and Cf-TRS show significant homology to the amidase domain present in E. coliglutathionylspermidine synthetase, which can catalyze both synthesis and degradation of glutathionylspermidine. Glutathionylspermidine synthetase isolated from C. fasciculata was found to possess a similar amidase activity.
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Kinetic isotope effect analysis of the reaction catalyzed by Trypanosoma congolense Trypanothione reductase
Biochemistry, 1992Co-Authors: Betty N. Leichus, Christopher T Walsh, Kari C. Nadeau, Mark Bradley, John S BlanchardAbstract:African trypanosomes are devoid of glutathione reductase activity, and instead contain a unique flavoprotein variant, Trypanothione reductase, which acts on a cyclic derivative of glutathione, Trypanothione. The high degree of sequence similarity between Trypanothione reductase and glutathione reductase, as well as the obvious similarity in the reactions catalyzed, led us to investigate the pH dependence of the kinetic parameters, and the isotopic behavior of Trypanothione reductase
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Kinetic isotope effect analysis of the reaction catalyzed by Trypanosoma congolense Trypanothione reductase.
Biochemistry, 1992Co-Authors: Betty N. Leichus, Christopher T Walsh, Kari C. Nadeau, Mark Bradley, John S BlanchardAbstract:African trypanosomes are devoid of glutathione reductase activity, and instead contain a unique flavoprotein variant, Trypanothione reductase, which acts on a cyclic derivative of glutathione, Trypanothione. The high degree of sequence similarity between Trypanothione reductase and glutathione reductase, as well as the obvious similarity in the reactions catalyzed, led us to investigate the pH dependence of the kinetic parameters, and the isotopic behavior of Trypanothione reductase. The pH dependence of the kinetic parameters V, V/K for NADH, and V/K for oxidized Trypanothione has been determined for Trypanothione reductase from Trypanosoma congolense. Both V/K for NADH and the maximum velocity decrease as single groups exhibiting pK values of 8.87 +/- 0.09 and 9.45 +/- 0.07, respectively, are deprotonated. V/K for oxidized Trypanothione, T(S)2, decreases as two groups exhibiting experimentally indistinguishable pK values of 8.74 +/- 0.03 are deprotonated. Variable magnitudes of the primary deuterium kinetic isotope effects on pyridine nucleotide oxidation are observed on V and V/K when different pyridine nucleotide substrates are used, and the magnitude of DV and D(V/K) is independent of the oxidized Trypanothione concentration at pH 7.25. Solvent kinetic isotope effects, obtained with 2',3'-cNADPH as the variable substrate, were observed on V only, and plots of V versus mole fraction of D2O (i.e., proton inventory) were linear, and yielded values of 1.3-1.6 for D2OV. Solvent kinetic isotope effects obtained with alternate pyridine nucleotides as substrates were also observed on V, and the magnitude of D2OV decreases for each pyridine nucleotide as its maximal velocity relative to that of NADPH oxidation decreases.(ABSTRACT TRUNCATED AT 250 WORDS)
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Molecular Studies on Trypanothione Reductase: An Antiparasitic Target Enzyme
Current Topics in Cellular Regulation, 1992Co-Authors: Christopher T Walsh, Mark Bradley, Kari C. NadeauAbstract:Publisher Summary This chapter discusses molecular studies on Trypanothione reductase. Investigations into the biochemistry of these parasites have quite surprisingly shown that they are devoid of the almost ubiquitous enzyme glutathione reductase. Instead they contain an enzyme that carries out an analogous role to that of glutathione reductase, the NADPH-dependent reduction of Trypanothione, a novel glutathione analog unique to the trypanosomatid parasites. By a combination of site-directed mutagenesis and the synthesis of substrate analogs, intimate details of the catalytic function of Trypanothione reductase can be determined. Additional evidence for the role of the two residues E18 and W21R in Trypanothione reductase has also recently been determined by an inverse site-directed mutagenesis approach with human glutathione reductase and its conversion to a Trypanothione reductase by modification of the residues A34 and R37 into glutamic and tryptophan residues, respectively. Undoubtedly, a crystal structure of this mutant human GR with bound Trypanothione or an X-ray structure of one of the available Trypanothione reductases will broaden the understanding and the potential for inhibition of this antiparasitic target enzyme.
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MUTATIONAL ANALYSIS OF PARASITE Trypanothione REDUCTASE : ACQUISITION OF GLUTATHIONE REDUCTASE ACTIVITY IN A TRIPLE MUTANT
Biochemistry, 1991Co-Authors: Francis X Sullivan, Susan B. Sobolov, Mark Bradley, Christopher T WalshAbstract:African trypanosomes contain a cyclic derivative of oxidized glutathione, N 1 , N 8 -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. 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 from Trypanosoma congolense, a cattle parasite
Marcelo A. Comini - One of the best experts on this subject based on the ideXlab platform.
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5-Vinylquinoline-substituted nitrofurans as inhibitors of Trypanothione reductase and antitrypanosomal agents
Chemija, 2020Co-Authors: Diego Benítez, Marcelo A. Comini, Jonas Šarlauskas, žilvinas Anusevicius, Valė Miliukienė, Eglė Miliuvienė, Narimantas ČėnasAbstract:Trypanothione reductase (TR) and Trypanothione synthase (TS) are critical for the maintenance of thiol-redox homeostasis and antioxidant protection in trypanosomal parasites, which cause African sleeping sickness and Chagas disease. Both enzymes are absent in mammals. Thus, the design of efficient and specific TR and TS inhibitors represents one of the pathways for a development of new antitrypanosomal drugs. 5-Vinylquinoline-substituted nitrofurans (n = 7), studied in this work, acted as un- or noncompetitive to Trypanothione inhibitors of Trypanosoma congolense TR. Their inhibition constants (Ki) varied from 2.3 µM to 150 µM. We for the first time observed a parallelism between their antitrypanosomal in vitro activity and their efficacy as TR inhibitors. The inhibition of TS appears not to be a significant factor of trypanocidal activity of examined compounds.
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5-Substituted 3-chlorokenpaullone derivatives are potent inhibitors of Trypanosoma brucei bloodstream forms
Bioorganic & Medicinal Chemistry, 2016Co-Authors: Oliver C.f. Orban, Ricarda S. Korn, Diego Benítez, Lutz Preu, Nadège Loaëc, Marcelo A. Comini, Andrea Medeiros, Oliver Koch, Laurent Meijer, Conrad KunickAbstract:Abstract Trypanothione synthetase is an essential enzyme for kinetoplastid parasites which cause highly disabling and fatal diseases in humans and animals. Inspired by the observation that N(5)-substituted paullones inhibit the Trypanothione synthetase from the related parasite Leishmania infantum, we designed and synthesized a series of new derivatives. Although none of the new compounds displayed strong inhibition of Trypanosoma brucei Trypanothione synthetase, several of them caused a remarkable growth inhibition of cultivated Trypanosoma brucei bloodstream forms. The most potent congener 3a showed antitrypanosomal activity in double digit nanomolar concentrations and a selectivity index of three orders of magnitude versus murine macrophage cells.
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genetic and chemical analyses reveal that Trypanothione synthetase but not glutathionylspermidine synthetase is essential for leishmania infantum
Free Radical Biology and Medicine, 2014Co-Authors: Andre F Sousa, Diego Benítez, Marcelo A. Comini, Timo Jaeger, Leopold Flohé, Ana M. Tomás, Ana Georgina Gomesalves, Joana Passos, Friedrich StuhlmannAbstract:Abstract Trypanothione is a unique and essential redox metabolite of trypanosomatid parasites, the biosynthetic pathway of which is regarded as a promising target for antiparasitic drugs. Synthesis of Trypanothione occurs by the consecutive conjugation of two glutathione molecules to spermidine. Both reaction steps are catalyzed by Trypanothione synthetase (TRYS), a molecule known to be essential in Trypanosoma brucei. However, other trypanosomatids (including some Leishmania species and Trypanosoma cruzi) potentially express one additional enzyme, glutathionylspermidine synthetase (GSPS), capable of driving the first step of Trypanothione synthesis yielding glutathionylspermidine. Because this monothiol can substitute for Trypanothione in some reactions, the possibility existed that TRYS was redundant in parasites harboring GSPS. To clarify this issue, the functional relevance of both GSPS and TRYS was investigated in Leishmania infantum (Li). Employing a gene-targeting approach, we generated a gsps−/− knockout line, which was viable and capable of replicating in both life cycle stages of the parasite, thus demonstrating the superfluous role of LiGSPS. In contrast, elimination of both LiTRYS alleles was not possible unless parasites were previously complemented with an episomal copy of the gene. Retention of extrachromosomal LiTRYS in the trys−/−/+TRYS line after several passages in culture further supported the essentiality of this gene for survival of L. infantum (including its clinically relevant stage), hence ruling out the hypothesis of functional complementation by LiGSPS. Chemical targeting of LiTRYS with a drug-like compound was shown to also lead to parasite death. Overall, this study disqualifies GSPS as a target for drug development campaigns and, by genetic and chemical evidence, validates TRYS as a chemotherapeutic target in a parasite endowed with GSPS and, thus, probably along the entire trypanosomatid lineage.
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Preparative enzymatic synthesis of Trypanothione and Trypanothione analogues
International journal for parasitology, 2009Co-Authors: Marcelo A. Comini, Natalie Dirdjaja, Mariel Kaschel, R. Luise Krauth-siegelAbstract:Abstract Trypanosomatids, the causative agents of several tropical diseases, have a unique thiol metabolism based on Trypanothione [bis(glutathionyl)spermidine]. Enzymes of the pathway are attractive drug target molecules but the availability of Trypanothione remains an obstacle. Here, we present a convenient method for the production of Trypanothione and Trypanothione disulfide in >200 mg quantities using a mutant of Crithidia fasciculata Trypanothione synthetase in which Cys59 has been replaced by an alanine residue. The reagent costs less than 1% of the commercial price of Trypanothione disulfide. The protocol also allows the synthesis of related glutathione conjugates. It will greatly facilitate the thorough analysis of this parasite’s metabolism and drug screening approaches against Trypanothione-dependent enzymes.
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redox control in trypanosomatids parasitic protozoa with Trypanothione based thiol metabolism
Biochimica et Biophysica Acta, 2008Co-Authors: Luise R Krauthsiegel, Marcelo A. CominiAbstract:Trypanosomes and leishmania, the causative agents of several tropical diseases, possess a unique redox metabolism which is based on Trypanothione. The bis(glutathionyl)spermidine is the central thiol that delivers electrons for the synthesis of DNA precursors, the detoxification of hydroperoxides and other Trypanothione-dependent pathways. Many of the reactions are mediated by tryparedoxin, a distant member of the thioredoxin protein family. Trypanothione is kept reduced by the parasite-specific flavoenzyme Trypanothione reductase. Since glutathione reductases and thioredoxin reductases are missing, the reaction catalyzed by Trypanothione reductase represents the only connection between the NADPH- and the thiol-based redox metabolisms. Thus, cellular thiol redox homeostasis is maintained by the biosynthesis and reduction of Trypanothione. Nearly all proteins of the parasite-specific Trypanothione metabolism have proved to be essential.
