Mechanistic and biological characterisation of novel N5-substituted paullones targeting the biosynthesis of trypanothione in Leishmania.

Trypanothione synthetase (TryS) produces N1,N8-bis(glutathionyl)spermidine (or trypanothione) at the expense of ATP. Trypanothione is a metabolite unique and essential for survival and drug-resistance of trypanosomatid parasites. In this study, we report the mechanistic and biological characterisation of optimised N5-substituted paullone analogues with anti-TryS activity. Several of the new derivatives retained submicromolar IC50 against leishmanial TryS. The binding mode to TryS of the most potent paullones has been revealed by means of kinetic, biophysical and molecular modelling approaches. A subset of analogues showed an improved potency (EC50 0.5-10 µM) and selectivity (20-35) against the clinically relevant stage of Leishmania braziliensis (mucocutaneous leishmaniasis) and L. infantum (visceral leishmaniasis). For a selected derivative, the mode of action involved intracellular depletion of trypanothione. Our findings shed light on the molecular interaction of TryS with rationally designed inhibitors and disclose a new set of compounds with on-target activity against different Leishmania species.

Gamma-glutamylcysteine synthetase and tryparedoxin 1 exert high control on the antioxidant system in Trypanosoma cruzi contributing to drug resistance and infectivity.

Trypanothione (T(SH)2) is the main antioxidant metabolite for peroxide reduction in Trypanosoma cruzi; therefore, its metabolism has attracted attention for therapeutic intervention against Chagas disease. To validate drug targets within the T(SH)2 metabolism, the strategies and methods of Metabolic Control Analysis and kinetic modeling of the metabolic pathway were used here, to identify the steps that mainly control the pathway fluxes and which could be appropriate sites for therapeutic intervention. For that purpose, gamma-glutamylcysteine synthetase (γECS), trypanothione synthetase (TryS), trypanothione reductase (TryR) and the tryparedoxin cytosolic isoform 1 (TXN1) were separately overexpressed to different levels in T. cruzi epimastigotes and their degrees of control on the pathway flux as well as their effect on drug resistance and infectivity determined. Both experimental in vivo as well as in silico analyses indicated that γECS and TryS control T(SH)2 synthesis by 60-74% and 15-31%, respectively. γECS overexpression prompted up to a 3.5-fold increase in T(SH)2 concentration, whereas TryS overexpression did not render an increase in T(SH)2 levels as a consequence of high T(SH)2 degradation. The peroxide reduction flux was controlled for 64-73% by TXN1, 17-20% by TXNPx and 11-16% by TryR. TXN1 and TryR overexpression increased H2O2 resistance, whereas TXN1 overexpression increased resistance to the benznidazole plus buthionine sulfoximine combination. γECS overexpression led to an increase in infectivity capacity whereas that of TXN increased trypomastigote bursting. The present data suggested that inhibition of high controlling enzymes such as γECS and TXN1 in the T(SH)2 antioxidant pathway may compromise the parasite's viability and infectivity.

The Thiol-polyamine Metabolism of Trypanosoma cruzi: Molecular Targets and Drug Repurposing Strategies.

Chagas´ disease continues to be a challenging and neglected public health problem in many American countries. The etiologic agent, Trypanosoma cruzi, develops intracellularly in the mammalian host, which hinders treatment efficacy. Progress in the knowledge of parasite biology and host-pathogen interaction has not been paralleled by the development of novel, safe and effective therapeutic options. It is then urgent to seek for novel therapeutic candidates and to implement drug discovery strategies that may accelerate the discovery process. The most appealing targets for pharmacological intervention are those essential for the pathogen and, whenever possible, absent or significantly different from the host homolog. The thiol-polyamine metabolism of T. cruzi offers interesting candidates for a rational design of selective drugs. In this respect, here we critically review the state of the art of the thiolpolyamine metabolism of T. cruzi and the pharmacological potential of its components. On the other hand, drug repurposing emerged as a valid strategy to identify new biological activities for drugs in clinical use, while significantly shortening the long time and high cost associated with de novo drug discovery approaches. Thus, we also discuss the different drug repurposing strategies available with a special emphasis in their applications to the identification of drug candidates targeting essential components of the thiol-polyamine metabolism of T. cruzi.

Genome sequencing and assessment of plant growth-promoting properties of a Serratia marcescens strain isolated from vermicompost.

BACKGROUND: Plant-bacteria associations have been extensively studied for their potential in increasing crop productivity in a sustainable manner. Serratia marcescens is a species of Enterobacteriaceae found in a wide range of environments, including soil. RESULTS: Here we describe the genome sequencing and assessment of plant growth-promoting abilities of S. marcescens UENF-22GI, a strain isolated from mature cattle manure vermicompost. In vitro, S. marcescens UENF-22GI is able to solubilize P and Zn, to produce indole compounds (likely IAA), to colonize hyphae and counter the growth of two phytopathogenic fungi. Inoculation of maize with this strain remarkably increased seedling growth and biomass under greenhouse conditions. The S. marcescens UENF-22GI genome has 5 Mb, assembled in 17 scaffolds comprising 4662 genes (4528 are protein-coding). No plasmids were identified. S. marcescens UENF-22GI is phylogenetically placed within a clade comprised almost exclusively of non-clinical strains. We identified genes and operons that are likely responsible for the interesting plant-growth promoting features that were experimentally described. The S. marcescens UENF-22GI genome harbors a horizontally-transferred genomic island involved in antibiotic production, antibiotic resistance, and anti-phage defense via a novel ADP-ribosyltransferase-like protein and possible modification of DNA by a deazapurine base, which likely contributes to its competitiveness against other bacteria. CONCLUSIONS: Collectively, our results suggest that S. marcescens UENF-22GI is a strong candidate to be used in the enrichment of substrates for plant growth promotion or as part of bioinoculants for agriculture.

Discovery of factors linked to antimony resistance in Leishmania panamensis through differential proteome analysis.

The rate of treatment failure to antileishmanial chemotherapy in Latin America is up to 64%. Parasite drug resistance contributes to an unknown proportion of treatment failures. Identification of clinically relevant molecular mechanisms responsible for parasite drug resistance is critical to the conservation of available drugs and to the discovery of novel targets to reverse the resistant phenotype. We conducted comparative proteomic-based analysis of Leishmania (Viannia) panamensis lines selected in vitro for resistance to trivalent antimony (Sb(III)) to identify factors associated with antimony resistance. Using 2-dimensional gel electrophoresis, two distinct sub-proteomes (soluble in NP-40/urea and Triton X-114, respectively) of promastigotes of WT and Sb(III)-resistant lines were generated. Overall, 9 differentially expressed putative Sb-resistance factors were detected and identified by mass spectrometry. These constituted two major groups: (a) proteins involved in general stress responses and (b) proteins with highly specific metabolic and transport functions, potentially directly contributing to the Sb-resistance mechanism. Notably, the sulfur amino acid-metabolizing enzymes S-adenosylmethionine synthetase (SAMS) and S-adenosylhomocysteine hydrolase (SAHH) were over-expressed in Sb(III)-resistant lines and Sb(III)-resistant clinical isolates. These enzymes play a central role in the upstream synthesis of precursors of trypanothione, a key molecule involved in Sb-resistance in Leishmania parasites, and suggest involvement of epigenetic regulation in response to drug exposure. These data re-enforce the importance of thiol metabolism in Leishmania Sb resistance, reveal previously unrecognized steps in the mechanism(s) of Sb tolerance, and suggest a cross-talk between drug resistance, metabolism and virulence.

Transgenic biosynthesis of trypanothione protects Escherichia coli from radiation-induced toxicity.

Trypanothione is a unique diglutathionyl-spermidine conjugate found in abundance in trypanosomes but not in other eukaryotes. Because trypanothione is a naturally occurring polyamine thiol reminiscent of the synthetic drug amifostine, it may be a useful protector against radiation and oxidative stress. For these reasons we hypothesized that trypanothione might serve as a radioprotective agent when produced in bacteria. To accomplish this objective, the trypanothione synthetase and reductase genes from T. cruzi were introduced into E. coli and their expression was verified by qPCR and immunoblotting. Trypanothione synthesis in bacteria, detected by HPLC, resulted in decreased intracellular levels of reactive oxygen species as determined by H(2)DCFDA oxidation. Moreover, E. coli genomic DNA was protected from radiation-induced DNA damage by 4.6-fold in the presence of trypanothione compared to control bacteria. Concordantly, the transgenic E. coli expressing trypanothione were 4.3-fold more resistant to killing by (137)Cs gamma radiation compared to E. coli devoid of trypanothione expression. Thus we have shown for the first time that E. coli can be genetically engineered to express the trypanothione biosynthetic pathway and produce trypanothione, which results in their radioresistance. These results warrant further research to explore the possibility of developing trypanothione as a novel radioprotective agent.

Polyamine biosynthesis in Phytomonas: biochemical characterisation of a very unstable ornithine decarboxylase.

The metabolism of polyamines as well as their functions as growth regulators in plants have been extensively studied for many years. However, almost nothing is known about the biosynthesis and roles of these substances in Phytomonas spp., parasites of several plants. We have used HPLC and electrophoretic analyses to investigate the presence and metabolism of polyamines in Phytomonas Jma strain, detecting both putrescine and spermidine but not spermine. Experiments carried out by incubation of intact parasites with labelled ornithine or putrescine showed the formation of radioactive putrescine or spermidine, respectively. These results indicated that Phytomonas Jma can synthesise these polyamines through the action of ornithine decarboxylase (ODC) and spermidine synthase. On the other hand, we could not detect the conversion of arginine to agmatine, suggesting the absence of arginine decarboxylase (ADC) in Phytomonas. However, we cannot ensure the complete absence of this enzymatic activity in the parasite. Phytomonas ODC required pyridoxal 5'-phosphate for maximum activity and was specifically inhibited by α-difluoromethylornithine. The metabolic turnover of the enzyme was very high, with a half-life of 10-15 min, one of the shortest found among all ODC enzymes studied to date. The parasite proteasome seems to be involved in degradation of the enzyme, since Phytomonas ODC can be markedly stabilized by MG-132, a well known proteasome inhibitor. The addition of polyamines to Phytomonas cultures did not decrease ODC activity, strongly suggesting the possible absence of antizyme in this parasite.

Insights into the redox biology of Trypanosoma cruzi: Trypanothione metabolism and oxidant detoxification.

Trypanosoma cruzi is the etiologic agent of Chagas' disease, an infection that affects several million people in Latin America. With no immediate prospect of a vaccine and problems associated with current chemotherapies, the development of new treatments is an urgent priority. Several aspects of the redox metabolism of this parasite differ enough from those in the mammalian host to be considered targets for drug development. Here, we review the information about a trypanosomatid-specific molecule centrally involved in redox metabolism, the dithiol trypanothione, and the main effectors of cellular antioxidant defense. We focus mainly on data from T. cruzi, making comparisons with other trypanosomatids whenever possible. In these parasites trypanothione participates in crucial thiol-disulfide exchange reactions and serves as electron donor in different metabolic pathways, from synthesis of DNA precursors to oxidant detoxification. Interestingly, the levels of several enzymes involved in trypanothione metabolism and oxidant detoxification increase during the transformation of T. cruzi to its mammalian-infective form and the overexpression of some of them has been associated with increased resistance to macrophage-dependent oxidative killing. Together, the evidence suggests a central role of the trypanothione-dependent antioxidant systems in the infection process.

Drug effects on drug targets: inhibition of enzymes by neuroleptics, antimycotics, antibiotics and other drugs on human pathogenic amoebas and their anti-proliferative effects.

This paper reviews the inhibition of various enzymes by neuroleptics, anti-mycotics, antibiotics and other drugs on three species of human pathogenic amoebas, mainly Entamoeba histolytica, Acanthamoeba polyphaga and Naegleria fowleri, and their antiproliferative effects. A recent patent registered by Philip relates to the combination of an antibacterial formulation and antifungal agent for producing a therapeutically effective quantity of an antimicrobial that is suitable for suppressing or treating fungal growth. The rationale behind this patent focused on essential and valid targets with a description of the main pathogenic characteristics of these amoebas. The study of new targets, such as trypanothione and trypanothione reductase, and the drug effects of selected agents were arranged into six main groups: A) Inhibition of disulfide reducing enzymes by neuroleptics, antimycotics and antibiotics; B) Comparative evaluation of the efficacies of several drugs with antiproliferative activities; C) Inhibition of the enzymes for the synthesis of trypanothione, such as ornithine decarboxylase, spermidine synthase and trypanothione synthetase; D) Inhibition of the glycolytic enzyme PPi-dependent phosphofructokinase (PFK) from Entamoeba and Naegleria by pyrophosphate analogues, different from the host enzyme; E) Inhibition of enzymes secreted by these parasites to invade the human host, for example cysteine proteinases; and F) Inhibition of encystment pathways and cyst-wall assembly proteins.

Design, synthesis, and biological evaluation of phosphinopeptides against Trypanosoma cruzi targeting trypanothione biosynthesis.

As a part of our project aimed at the search for new safe chemotherapeutic and chemoprophylactic agents against American trypanosomiasis (Chagas's disease), a series of phosphinopeptides structurally related to glutathione was designed, synthesized, and evaluated as antiproliferative agents against the parasite responsible for this disease, the hemoflagellated protozoan Trypanosoma cruzi. The rationale for the synthesis of these compounds was supported on the basis that the presence of the phosphinic acid moiety would mimic the tetrahedral transition state of trypanothione synthase (TryS), a typical C:N ligase, and the molecular target of these drugs. Of the designed compounds, 53 and 54 were potent growth inhibitors against the clinically more relevant form of T. cruzi (amastigotes) growing in myoblasts. The efficacy for these drugs was comparable to that exhibited by the well-known antiparasitic agent WC-9. The simple phosphinopeptide structure found as a pharmacophore in the present study constitutes a starting point for the development of straightforward optimized drugs.

Effects of buthionine sulfoximine nifurtimox and benznidazole upon trypanothione and metallothionein proteins in Trypanosoma cruzi.

Proteins rich in sulfhydryl groups, such as metallothionein, are present in several strains of the parasite Trypanosoma cruzi, the etiological agent of Chagas' disease. Metallothionein-like protein concentrations ranged from 5.1 to 13.2 pmol/mg protein depending on the parasite strain and growth phase. Nifurtimox and benznidazole, used in the treatment of Chagas' disease, decreased metallothionein activity by approximately 70%. T. cruzi metallothionein was induced by ZnCl2. Metallothionein from T. cruzi was partially purified and its monobromobimane derivative showed a molecular weight of approximately 10,000 Da by SDS-PAGE analysis. The concentration of trypanothione, the major glutathione conjugate in T. cruzi, ranged from 3.8 to 10.8 nmol/mg protein, depending on the culture phase. The addition of buthionine sulfoximine to the protozoal culture considerably reduced the concentration of trypanothione and had no effect upon the metallothionein concentration. The possible contribution of metallothionein-like proteins to drug resistance in T. cruzi is discussed.

Inhibition of polyamine synthesis arrests trichomonad growth and induces destruction of hydrogenosomes.

Trichomonad parasites such as Tritrichomonas foetus produce large amounts of putrescine (1,4-diaminobutane), which is transported out of the cell via an antiport mechanism which results in the uptake of a molecule of spermine. The importance of putrescine to the survival of the parasite and its role in the biology of T. foetus was investigated by use of the putrescine analogue 1, 4-diamino-2-butanone (DAB). Growth of T. foetus in vitro was significantly inhibited by 20 mM DAB, which was reversed by the addition of exogenous 40 mM putrescine. High-performance liquid chromatography analysis of 20 mM DAB-treated T. foetus revealed that putrescine, spermidine, and spermine levels were reduced by 89, 52, and 43%, respectively, compared to those in control cells. The DAB treatment induced several ultrastructural alterations, which were primarily observed in the redox organelles termed hydrogenosomes. These organelles were progressively degraded, giving rise to large vesicles that displayed material immunoreactive with an antibody to beta-succinyl-coenzyme A synthetase, a hydrogenosomal enzyme. A protective role for polyamines as stabilizing agents in the trichomonad hydrogenosomal membrane is proposed.

Polyamines in growth and dimorphism of Paracoccidioides brasiliensis.

Putrescine and spermidine were the only polyamines found in Paracoccidioides brasiliensis, a dimorphic fungus pathogenic for humans. Free polyamines (putrescine > spermidine) increased during the first 24 h of yeast growth, with a second peak at 42 h, and also during the first 12 h of mycelium-to-yeast transition (spermidine > putrescine). Conjugated and bound polyamines were also quantified. 1, 4-Diamino-2-butanone decreased free putrescine and spermidine accumulation by inhibiting the activity of ornithine decarboxylase. The increase in free polyamines corresponds to bud emergence in yeast growth and to the mycelium-to-yeast transition of P. brasiliensis.

Polyamines in the male reproductive system.

This review covers some common aspects of the biosynthesis, interconversion pathways and biochemical functions of polyamines. A particular emphasis is given in experimental models as well as humans, to their presence in the male gonad, prostate gland, seminal vesicles, epididymis and semen. The interaction between hormones (androgens, LH, FSH and PRL) and the main enzymes involved on the polyamine biosynthesis, and the relationship of these compounds on cell growth and differentiation, are also discussed. In this regard, an attention is offered to the potential role of polyamines during early spermatogenesis stages and the use of some enzymes involved in their biosynthesis as sensitive and specific markers of the action of androgens and antiandrogens in the epididymis. Finally, a special issue is addressed to the controversial information documented on polyamines, their oxidation products and the relationship with male fertility.

Polyamines in the male reproductive system

This review covers some common aspects of the biosynthesis, interconversion pathways and biochemical functions of polyamines. A particular emphasis is given in experitemtal models as well as humans, to their presence in the male gonad, postate gland, seminal vesicles, epididymis and semen. The interaction between hormones (androgens, LH, FSH and PRL) and the main enzymes involved on the polymine biosynthesis, and the relationship of these compounds on cell growth and differentation, are also discussed. In this regard, an attention is offered to the potential role of polymines during early spermatogenesis stages and the use of some enzymed involved in their biosynthesis as sensitive and specific markers of the action of androgens and antiandrogens in the epididymis. Finally, a special issue is addressed to the controversial information documented on polymines, their oxidation products and the relationship with male fertility. (AU)

Polyamines in the male reproductive system

This review covers some common aspects of the biosynthesis, interconversion pathways and biochemical functions of polyamines. A particular emphasis is given in experitemtal models as well as humans, to their presence in the male gonad, postate gland, seminal vesicles, epididymis and semen. The interaction between hormones (androgens, LH, FSH and PRL) and the main enzymes involved on the polymine biosynthesis, and the relationship of these compounds on cell growth and differentation, are also discussed. In this regard, an attention is offered to the potential role of polymines during early spermatogenesis stages and the use of some enzymed involved in their biosynthesis as sensitive and specific markers of the action of androgens and antiandrogens in the epididymis. Finally, a special issue is addressed to the controversial information documented on polymines, their oxidation products and the relationship with male fertility.

2,4-Dichlorophenoxyacetic acid effects on polyamine biosynthesis.

2,4-Dichlorophenoxyacetic acid (2,4-D) is a herbicide extensively used in agriculture. It was considered of interest to study its toxicity on animal cells. We had previously determined that 1 mM 2,4-D can inhibit cell growth, DNA and protein synthesis of cultured Chinese hamster ovary cells (CHO) with cell accumulation in the G1/S interphase of the cell cycle. The present work examined the effects of 2,4-D on polyamine biosynthesis. The results suggest some possible mechanism of the herbicide's toxic effects on animal cells.