Giardia duodenalis: Flavohemoglobin is involved in drug biotransformation and resistance to albendazole.

Giardia duodenalis causes giardiasis, a major diarrheal disease in humans worldwide whose treatment relies mainly on metronidazole (MTZ) and albendazole (ABZ). The emergence of ABZ resistance in this parasite has prompted studies to elucidate the molecular mechanisms underlying this phenomenon. G. duodenalis trophozoites convert ABZ into its sulfoxide (ABZSO) and sulfone (ABZSOO) forms, despite lacking canonical enzymes involved in these processes, such as cytochrome P450s (CYP450s) and flavin-containing monooxygenases (FMOs). This study aims to identify the enzyme responsible for ABZ metabolism and its role in ABZ resistance in G. duodenalis. We first determined that the iron-containing cofactor heme induces higher mRNA expression levels of flavohemoglobin (gFlHb) in Giardia trophozoites. Molecular docking analyses predict favorable interactions of gFlHb with ABZ, ABZSO and ABZSOO. Spectral analyses of recombinant gFlHb in the presence of ABZ, ABZSO and ABZSOO showed high affinities for each of these compounds with Kd values of 22.7, 19.1 and 23.8 nM respectively. ABZ and ABZSO enhanced gFlHb NADH oxidase activity (turnover number 14.5 min-1), whereas LC-MS/MS analyses of the reaction products showed that gFlHb slowly oxygenates ABZ into ABZSO at a much lower rate (turnover number 0.01 min-1). Further spectroscopic analyses showed that ABZ is indirectly oxidized to ABZSO by superoxide generated from the NADH oxidase activity of gFlHb. In a similar manner, the superoxide-generating enzyme xanthine oxidase was able to produce ABZSO in the presence of xanthine and ABZ. Interestingly, we find that gFlHb mRNA expression is lower in albendazole-resistant clones compared to those that are sensitive to this drug. Furthermore, all albendazole-resistant clones transfected to overexpress gFlHb displayed higher susceptibility to the drug than the parent clones. Collectively these findings indicate a role for gFlHb in ABZ conversion to its sulfoxide and that gFlHb down-regulation acts as a passive pharmacokinetic mechanism of resistance in this parasite.

Lactate oxidation is linked to energy conservation and to oxygen detoxification via a putative terminal cytochrome oxidase in Methanosarcina acetivorans.

The marine archaeon Methanosarcina acetivorans contains a putative NAD + -independent d-lactate dehydrogenase (D-iLDH/glycolate oxidase) encoded by the MA4631 gene, belonging to the FAD-oxidase C superfamily. Nucleotide sequences similar to MA4631 gene, were identified in other methanogens and Firmicutes with >90 and 35-40% identity, respectively. Therefore, the lactate metabolism in M. acetivorans is reported here. Cells subjected to intermittent pulses of oxygen (air-adapted; AA-Ma cells) consumed lactate only in combination with acetate, increasing methane production and biomass yield. In AA-Ma cells incubated with d-lactate plus [14C]-l-lactate, the radioactive label was found in methane, CO2 and glycogen, indicating that lactate metabolism fed both methanogenesis and gluconeogenesis. Moreover, d-lactate oxidation was coupled to O2-consumption which was sensitive to HQNO; also, AA-Ma cells showed high transcript levels of gene dld and those encoding subunits A (MA1006) and B (MA1007) of a putative cytochrome bd quinol oxidase, compared to anaerobic control cells. An E. coli mutant deficient in dld complemented with the MA4631 gene, grew with d-lactate as carbon source and showed membrane-bound d-lactate:quinone oxidoreductase activity. The product of the MA4631 gene is a FAD-containing monomer showing activity of iLDH with preference to d-lactate. The results suggested that air adapted M. acetivorans is able to co-metabolize lactate and acetate with associated oxygen consumption by triggering the transcription and synthesis of the D-iLDH and a putative cytochrome bd: methanophenazine (quinol) oxidoreductase. Biomass generation and O2 consumption, suggest a potentially new oxygen detoxification mechanism coupled to energy conservation in this methanogen.

Protein acetylation effects on enzyme activity and metabolic pathway fluxes.

Acetylation of proteins seems a widespread process found in the three domains of life. Several studies have shown that besides histones, acetylation of lysine residues also occurs in non-nuclear proteins. Hence, it has been suggested that this covalent modification is a mechanism that might regulate diverse metabolic pathways by modulating enzyme activity, stability, and/or subcellular localization or interaction with other proteins. However, protein acetylation levels seem to have low correlation with modification of enzyme activity and pathway fluxes. In addition, the results obtained with mutant enzymes that presumably mimic acetylation have frequently been over-interpreted. Moreover, there is a generalized lack of rigorous enzyme kinetic analysis in parallel to acetylation level determinations. The purpose of this review is to analyze the current findings on the impact of acetylation on metabolic enzymes and its repercussion on metabolic pathways function/regulation.

In Vitro and In Silico Analysis of New n-Butyl and Isobutyl Quinoxaline-7-carboxylate 1,4-di-N-oxide Derivatives against Trypanosoma cruzi as Trypanothione Reductase Inhibitors.

American trypanosomiasis is a worldwide health problem that requires attention due to ineffective treatment options. We evaluated n-butyl and isobutyl quinoxaline-7-carboxylate 1,4-di-N-oxide derivatives against trypomastigotes of the Trypanosoma cruzi strains NINOA and INC-5. An in silico analysis of the interactions of 1,4-di-N-oxide on the active site of trypanothione reductase (TR) and an enzyme inhibition study was carried out. The n-butyl series compound identified as T-150 had the best trypanocidal activity against T. cruzi trypomastigotes, with a 13% TR inhibition at 44 µM. The derivative T-147 behaved as a mixed inhibitor with Ki and Ki' inhibition constants of 11.4 and 60.8 µM, respectively. This finding is comparable to the TR inhibitor mepacrine (Ki = 19 µM).

Functional characterization and subcellular distribution of two recombinant cytosolic HSP70 isoforms from Entamoeba histolytica under normal and stress conditions.

Amoebiasis is a human intestinal disease caused by the parasite Entamoeba histolytica. It has been previously demonstrated that E. histolytica heat shock protein 70 (EhHSP70) plays an important role in amoebic pathogenicity by protecting the parasite from the dangerous effects of oxidative and nitrosative stresses. Despite its relevance, this protein has not yet been characterized. In this study, the EhHSP70 genes were cloned, and the two recombinant EhHSP70 proteins were expressed, purifying and biochemically characterized. Additionally, after being subjected to some host stressors, the intracellular distribution of the proteins in the parasite was documented. Two amoebic HSP70 isoforms, EhHSP70-A and EhHSP70-B, with 637 and 656 amino acids, respectively, were identified. Kinetic parameters of ATP hydrolysis showed low rates, which were in accordance with those of the HSP70 family members. Circular dichroism analysis showed differences in their secondary structures but similarities in their thermal stability. Immunocytochemistry in trophozoites detected EhHSP70 in the nuclei and cytoplasm as well as a slight overexpression when the parasites were subjected to oxidants and heat. The structural differences of amoebic HSP70s with their human counterparts may be used to design specific inhibitors to treat human amoebiasis.

Rabeprazole inhibits several functions of Entamoeba histolytica related with its virulence.

Amoebiasis is a human parasitic disease caused by Entamoeba histolytica. The parasite can invade the large intestine and other organs such as liver; resistance to the host tissue oxygen is a condition for parasite invasion and survival. Thioredoxin reductase of E. histolytica (EhTrxR) is a critical enzyme mainly involved in maintaining reduced the redox system and detoxifying the intracellular oxygen; therefore, it is necessary for E. histolytica survival under both aerobic in vitro and in vivo conditions. In the present work, it is reported that rabeprazole (Rb), a drug widely used to treat heartburn, was able to inhibit the EhTrxR recombinant enzyme. Moreover, Rb affected amoebic proliferation and several functions required for parasite virulence such as cytotoxicity, oxygen reduction to hydrogen peroxide, erythrophagocytosis, proteolysis, and oxygen and complement resistances. In addition, amoebic pre-incubation with sublethal Rb concentration (600 µM) promoted amoebic death during early liver infection in hamsters. Despite the high Rb concentration used to inhibit amoebic virulence, the wide E. histolytica pathogenic-related functions affected by Rb strongly suggest that its molecular structure can be used as scaffold to design new antiamoebic compounds with lower IC50 values.

Computational Drug Repositioning for Chagas Disease Using Protein-Ligand Interaction Profiling.

Chagas disease, caused by Trypanosoma cruzi (T. cruzi), affects nearly eight million people worldwide. There are currently only limited treatment options, which cause several side effects and have drug resistance. Thus, there is a great need for a novel, improved Chagas treatment. Bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS) has emerged as a promising pharmacological target. Moreover, some human dihydrofolate reductase (HsDHFR) inhibitors such as trimetrexate also inhibit T. cruzi DHFR-TS (TcDHFR-TS). These compounds serve as a starting point and a reference in a screening campaign to search for new TcDHFR-TS inhibitors. In this paper, a novel virtual screening approach was developed that combines classical docking with protein-ligand interaction profiling to identify drug repositioning opportunities against T. cruzi infection. In this approach, some food and drug administration (FDA)-approved drugs that were predicted to bind with high affinity to TcDHFR-TS and whose predicted molecular interactions are conserved among known inhibitors were selected. Overall, ten putative TcDHFR-TS inhibitors were identified. These exhibited a similar interaction profile and a higher computed binding affinity, compared to trimetrexate. Nilotinib, glipizide, glyburide and gliquidone were tested on T. cruzi epimastigotes and showed growth inhibitory activity in the micromolar range. Therefore, these compounds could lead to the development of new treatment options for Chagas disease.

Mutant p53R248Q downregulates oxidative phosphorylation and upregulates glycolysis under normoxia and hypoxia in human cervix cancer cells.

Mutations in p53 are strongly associated with several highly malignant cancer phenotypes but its role in regulating energy metabolism has not been completely elucidated. The effect on glycolysis and oxidative phosphorylation (OxPhos) of mutant p53R248Q overexpression in HeLa cells (HeLa-M) was analyzed and compared with cells overexpressing wild-type p53 (HeLa-H) and nontransfected cells containing negligible p53 levels (HeLa-L). p53 R248Q overexpression induced early cell detachment during in vitro growth; however, detached HeLa-M cells showed high viability, shorter generation time and significant diminution in the adhesion proteins E-cadherin and ß-catenin versus HeLa-H and HeLa-L cells. Under normoxia, a lower growth rate of attached HeLa-M cells correlated with decreased levels of proliferating cell nuclear antigen (PCNA), peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α), adenosine monophosphate-activated protein kinase (AMPK), mitochondrial proteins (20-80%) and OxPhos flux (69 ± 12%). On the contrary, HeLa-M also showed increased contents of CDKN1A, nuclear factor κB (NF-κB), c-MYC, hypoxia-inducible factor 1-α (HIF-1α; 1-4 times), glycolytic HIF-1α targets (2-4 times), and glycolysis flux (2-fold) versus HeLa-H. In consequence, glycolysis provided ~70% of the cellular adenosine triphosphate (ATP) in HeLa-M cells under normoxia whereas, OxPhos predominated (65-82%) in HeLa-H and HeLa-L cells. Pifithrin-α, a specific p53 inhibitor, did not alter the p53 R248Q target protein contents and OxPhos and glycolytic fluxes, and a poor HIF-1α-p53 R248Q interaction was attained, in HeLa-M cells. These observations suggested that p53 R248Q deficiently interacted with pifithrin-α and HIF-1α. Therefore, lower mitochondrial biogenesis, deficient HIF-1α/mutant p53 interaction, and development of a pseudohypoxic state under normoxia were the apparent biochemical mechanisms underlying glycolysis activation and OxPhos downregulation in HeLa-M cells.

Roles of acetyl-CoA synthetase (ADP-forming) and acetate kinase (PPi-forming) in ATP and PPi supply in Entamoeba histolytica.

BACKGROUND: Acetate is an end-product of the PPi-dependent fermentative glycolysis in Entamoeba histolytica; it is synthesized from acetyl-CoA by ADP-forming acetyl-CoA synthetase (ACS) with net ATP synthesis or from acetyl-phosphate by a unique PPi-forming acetate kinase (AcK). The relevance of these enzymes to the parasite ATP and PPi supply, respectively, are analyzed here. METHODS: The recombinant enzymes were kinetically characterized and their physiological roles were analyzed by transcriptional gene silencing and further metabolic analyses in amoebae. RESULTS: Recombinant ACS showed higher catalytic efficiencies (Vmax/Km) for acetate formation than for acetyl-CoA formation and high acetyl-CoA levels were found in trophozoites. Gradual ACS gene silencing (49-93%) significantly decreased the acetate flux without affecting the levels of glycolytic metabolites and ATP in trophozoites. However, amoebae lacking ACS activity were unable to reestablish the acetyl-CoA/CoA ratio after an oxidative stress challenge. Recombinant AcK showed activity only in the acetate formation direction; however, its substrate acetyl-phosphate was undetected in axenic parasites. AcK gene silencing did not affect acetate production in the parasites but promoted a slight decrease (10-20%) in the hexose phosphates and PPi levels. CONCLUSIONS: These results indicated that the main role of ACS in the parasite energy metabolism is not ATP production but to recycle CoA for glycolysis to proceed under aerobic conditions. AcK does not contribute to acetate production but might be marginally involved in PPi and hexosephosphate homeostasis. SIGNIFICANCE: The previous, long-standing hypothesis that these enzymes importantly contribute to ATP and PPi supply in amoebae can now be ruled out.

Assessment of the low inhibitory specificity of oxamate, aminooxyacetate and dichloroacetate on cancer energy metabolism.

BACKGROUND: Exceedingly high therapeutic/experimental doses of metabolic drugs such as oxamate, aminooxyacetate (AOA) and dichloroacetate (DCA) are required to diminish growth, glycolysis and oxidative phosphorylation (OxPhos) of different cancer cells. To identify the mechanisms of action of these drugs on cancer energy metabolism, a systematic analysis of their specificities was undertaken. METHODS: Hepatocarcinoma AS-30D cells were treated with the inhibitors and glycolysis and OxPhos enzyme activities, metabolites and fluxes were analyzed. Kinetic modeling of glycolysis was used to identify the regulatory mechanisms. RESULTS: Oxamate (i) not only inhibited LDH, but also PYK and ENO activities inducing an increase in the cytosolic NAD(P)H, Fru1,6BP and DHAP levels in AS-30D cells; (ii) it slightly inhibited HPI, ALD and Glc6PDH; and (iii) it inhibited pyruvate-driven OxPhos in isolated heart mitochondria. AOA (i) strongly inhibited both AAT and AlaT, and 2-OGDH and glutamate-driven OxPhos; and (ii) moderately affected GAPDH and TPI. DCA slightly affected pyruvate-driven OxPhos and Glc6PDH. Kinetic modeling of cancer glycolysis revealed that oxamate inhibition of LDH, PYK and ENO was insufficient to achieve glycolysis flux inhibition. To do so, HK, HPI, TPI and GAPDH have to be also inhibited by the accumulated Fru1,6BP and DHAP induced by oxamate. CONCLUSION: Oxamate, AOA, and DCA are not specific drugs since they inhibit several enzymes/transporters of the glycolytic and OxPhos pathways through direct interaction or indirect mechanisms. GENERAL SIGNIFICANCE: These data explain why oxamate or AOA, through their multisite inhibitory actions on glycolysis or OxPhos, may be able to decrease the proliferation of cancer cells.

Synthesis and biological evaluation of 2-methyl-1H-benzimidazole-5-carbohydrazides derivatives as modifiers of redox homeostasis of Trypanosoma cruzi.

Twelve novel benzimidazole derivatives were synthesized and their in vitro activities against epimastigotes of Trypanosoma cruzi were evaluated. Two derivatives (6 and 7), which have 4-hydroxy-3-methoxyphenyl moiety in their structures, proved to be the most active in inhibiting the parasite growth. Compound 6 showed a trypanocidal activity higher than benznidazole (IC50=5µM and 7.5µM, respectively) and less than nifurtimox (IC50=3.6µM). In addition, the ability of 6 and 7 to modify the redox homeostasis in T cruzi epimastigote was studied; cysteine and glutathione increased in parasites exposed to both compounds, whereas trypanothione only increased with 7 treatment. These results suggest that the decrease in viability of T. cruzi may be attributed to the change in cellular redox balance caused by compound 6 or 7. Furthermore, compounds 6 and 7 showed CC50 values of 160.64 and 160.66µM when tested in mouse macrophage cell line J774 and selectivity indexes (macrophage/parasite) of 32 and 20.1, respectively.

Biochemistry and Physiology of Heavy Metal Resistance and Accumulation in Euglena.

Free-living microorganisms may become suitable models for removal of heavy metals from polluted water bodies, sediments, and soils by using and enhancing their metal accumulating abilities. The available research data indicate that protists of the genus Euglena are a highly promising group of microorganisms to be used in bio-remediation of heavy metal-polluted aerobic and anaerobic acidic aquatic environments. This chapter analyzes the variety of biochemical mechanisms evolved in E. gracilis to resist, accumulate and remove heavy metals from the environment, being the most relevant those involving (1) adsorption to the external cell pellicle; (2) intracellular binding by glutathione and glutathione polymers, and their further compartmentalization as heavy metal-complexes into chloroplasts and mitochondria; (3) polyphosphate biosynthesis; and (4) secretion of organic acids. The available data at the transcriptional, kinetic and metabolic levels on these metabolic/cellular processes are herein reviewed and analyzed to provide mechanistic basis for developing genetically engineered Euglena cells that may have a greater removal and accumulating capacity for bioremediation and recycling of heavy metals.

Metabolic control analysis of the Trypanosoma cruzi peroxide detoxification pathway identifies tryparedoxin as a suitable drug target.

BACKGROUND: The principal oxidative-stress defense in the human parasite Trypanosoma cruzi is the tryparedoxin-dependent peroxide detoxification pathway, constituted by trypanothione reductase (TryR), tryparedoxin (TXN), tryparedoxin peroxidase (TXNPx) and tryparedoxin-dependent glutathione peroxidase A (GPxA). Here, Metabolic Control Analysis (MCA) was applied to quantitatively prioritize drug target(s) within the pathway by identifying its flux-controlling enzymes. METHODS: The recombinant enzymes were kinetically characterized at physiological pH/temperature. Further, the pathway was in vitro reconstituted using enzyme activity ratios and fluxes similar to those observed in the parasites; then, enzyme and substrate titrations were performed to determine their degree of control on flux. Also, kinetic characterization of the whole pathway was performed. RESULTS: Analyses of the kinetic properties indicated that TXN is the less efficient pathway enzyme derived from its high Kmapp for trypanothione and low Vmax values within the cell. MCA established that the TXN-TXNPx and TXN-GPxA redox pairs controlled by 90-100% the pathway flux, whereas 10% control was attained by TryR. The Kmapp values of the complete pathway for substrates suggested that the pathway flux was determined by the peroxide availability, whereas at high peroxide concentrations, flux may be limited by NADPH. CONCLUSION: These quantitative kinetic and metabolic analyses pointed out to TXN as a convenient drug target due to its low catalytic efficiency, high control on the flux of peroxide detoxification and role as provider of reducing equivalents to the two main peroxidases in the parasite. GENERAL SIGNIFICANCE: MCA studies provide rational and quantitative criteria to select enzymes for drug-target development.

Dual regulation of energy metabolism by p53 in human cervix and breast cancer cells.

The role of p53 as modulator of OxPhos and glycolysis was analyzed in HeLa-L (cells containing negligible p53 protein levels) and HeLa-H (p53-overexpressing) human cervix cancer cells under normoxia and hypoxia. In normoxia, functional p53, mitochondrial enzyme contents, mitochondrial electrical potential (ΔΨm) and OxPhos flux increased in HeLa-H vs. HeLa-L cells; whereas their glycolytic enzyme contents and glycolysis flux were unchanged. OxPhos provided more than 70% of the cellular ATP and proliferation was abolished by anti-mitochondrial drugs in HeLa-H cells. In hypoxia, both cell proliferations were suppressed, but HeLa-H cells exhibited a significant decrease in OxPhos protein contents, ΔΨm and OxPhos flux. Although glycolytic function was also diminished vs. HeLa-L cells in hypoxia, glycolysis provided more than 60% of cellular ATP in HeLa-H cells. The energy metabolism phenotype of HeLa-H cells was reverted to that of HeLa-L cells by incubating with pifithrin-α, a p53-inhibitor. In normoxia, the energy metabolism phenotype of breast cancer MCF-7 cells was similar to that of HeLa-H cells, whereas p53shRNAMCF-7 cells resembled the HeLa-L cell phenotype. In hypoxia, autophagy proteins and lysosomes contents increased 2-5 times in HeLa-H cells suggesting mitophagy activation. These results indicated that under normoxia p53 up-regulated OxPhos without affecting glycolysis, whereas under hypoxia, p53 down-regulated both OxPhos (severely) and glycolysis (weakly). These p53 effects appeared mediated by the formation of p53-HIF-1α complexes. Therefore, p53 exerts a dual and contrasting regulatory role on cancer energy metabolism, depending on the O2level.

The oxygen reduction pathway and heat shock stress response are both required for Entamoeba histolytica pathogenicity.

Several species belonging to the genus Entamoeba can colonize the mouth or the human gut; however, only Entamoeba histolytica is pathogenic to the host, causing the disease amoebiasis. This illness is responsible for one hundred thousand human deaths per year worldwide, affecting mainly underdeveloped countries. Throughout its entire life cycle and invasion of human tissues, the parasite is constantly subjected to stress conditions. Under in vitro culture, this microaerophilic parasite can tolerate up to 5 % oxygen concentrations; however, during tissue invasion the parasite has to cope with the higher oxygen content found in well-perfused tissues (4-14 %) and with reactive oxygen and nitrogen species derived from both host and parasite. In this work, the role of the amoebic oxygen reduction pathway (ORP) and heat shock response (HSP) are analyzed in relation to E. histolytica pathogenicity. The data suggest that in contrast with non-pathogenic E. dispar, the higher level of ORP and HSPs displayed by E. histolytica enables its survival in tissues by diminishing and detoxifying intracellular oxidants and repairing damaged proteins to allow metabolic fluxes, replication and immune evasion.

Maintenance of intracellular hypoxia and adequate heat shock response are essential requirements for pathogenicity and virulence of Entamoeba histolytica.

Adhesion to cells, cytotoxicity and proteolysis are functions required for virulence and pathogenicity of Entamoeba histolytica. However, there was no correlation between these in vitro functions and the early elimination of non-pathogenic E. dispar and non-virulent E. histolytica (nvEh) in experimental amoebic liver abscesses developed in hamsters. Thus, additional functions may be involved in amoebic pathogenicity and virulence. In the present study, an integral experimental assessment, including innovative technologies for analyses of amoebal pathophysiology, cell biology, biochemistry and transcriptomics, was carried out to elucidate whether other cellular processes are involved in amoebal pathogenicity and virulence. In comparison with virulent E. histolytica, the data indicated that the main reasons for the early clearance of nvEh from hamster liver are decreased intracellular H2 O2 detoxification rate and deficient heat shock protein expression, whereas for E. dispar, it is a relatively lower capacity for O2 reduction. Therefore, maintenance of an intracellular hypoxic environment combined with the induction of an adequate parasite response to oxidative stress are essential requirements for Entamoeba survival in the liver, and therefore for pathogenicity.

Sulfate uptake in photosynthetic Euglena gracilis. Mechanisms of regulation and contribution to cysteine homeostasis.

BACKGROUND: Sulfate uptake was analyzed in photosynthetic Euglena gracilis grown in sulfate sufficient or sulfate deficient media, or under Cd(2+) exposure or Cys overload, to determine its regulatory mechanisms and contribution to Cys homeostasis. RESULTS: In control and sulfate deficient or Cd(2+)-stressed cells, one high affinity and two low affinity sulfate transporters were revealed, which were partially inhibited by photophosphorylation and oxidative phosphorylation inhibitors and ionophores, as well as by chromate and molybdate; H(+) efflux also diminished in presence of sulfate. In both sulfate deficient and Cd(2+)-exposed cells, the activity of the sulfate transporters was significantly increased. However, the content of thiol-metabolites was lower in sulfate-deficient cells, and higher in Cd(2+)-exposed cells, in comparison to control cells. In cells incubated with external Cys, sulfate uptake was strongly inhibited correlating with 5-times increased intracellular Cys. Re-supply of sulfate to sulfate deficient cells increased the Cys, γ-glutamylcysteine and GSH pools, and to Cys-overloaded cells resulted in the consumption of previously accumulated Cys. In contrast, in Cd(2+) exposed cells none of the already elevated thiol-metabolites changed. CONCLUSIONS: (i) Sulfate transport is an energy-dependent process; (ii) sulfate transporters are over-expressed under sulfate deficiency or Cd(2+) stress and their activity can be inhibited by high internal Cys; and (iii) sulfate uptake exerts homeostatic control of the Cys pool.

Metabolic control analysis as a strategy to identify therapeutic targets, the case of cancer glycolysis.

The use of kinetic modeling and metabolic control analysis (MCA) to identify possible therapeutic targets and to investigate the controlling and regulatory mechanisms in cancer glycolysis is here reviewed. The glycolytic pathway has been considered a target to decrease cancer cell growth; however, its occurrence in normal cells makes it difficult to design therapeutic strategies that target this pathway in pathological cells. Notwithstanding, the over-expression of all enzymes and transporters, as well as the expression of isoenzymes with different kinetic and regulatory properties in cancer cells, suggested a different distribution of the control of glycolytic flux than that observed in normal cells. Kinetic models of glycolysis are constructed with enzyme kinetics experimental data, validated with the steady-state metabolite concentrations and glycolytic fluxes; applying MCA, permitted us to identify the steps with the highest control of glycolysis in cancer cells, but low control in normal cells. The cancer glycolysis main controlling steps under several metabolic conditions were: glucose transport, hexokinase and hexose-6-phosphate isomerase (HPI); whereas in normal cells were: the first two and phosphofructokinase-1. HPI is the best therapeutic target because it exerts high control in cancer glycolytic flux, but not in normal cells. Furthermore, kinetic modeling also contributed to identifying new feed-back and feed-forward regulatory loops in cancer cells glycolysis, and to understanding the mode of metabolic action of glycolytic inhibitors. Thus, MCA and metabolic modeling allowed to propose new strategies for inhibiting glycolysis in cancer cells.

Metabolic control analysis of the transsulfuration pathway and the compensatory role of the cysteine transport in Trypanosoma cruzi.

Trypanosoma cruzi is the causal agent of American Trypanosomiasis or Chagas Disease in humans. The current drugs for its treatment benznidazole and nifurtimox have inconveniences of toxicity and efficacy; therefore, the search for new therapies continues. Validation through genetic strategies of new drug targets against the parasite metabolism have identified numerous essential genes. Target validation can be further narrowed by applying Metabolic Control Analysis (MCA) to determine the flux control coefficients of the pathway enzymes. That coefficient is a quantitative value that represents the degree in which an enzyme/transporter determines the flux of a metabolic pathway; those with the highest coefficients can be promising drug targets. Previous studies have demonstrated that cysteine (Cys) is a key precursor for the synthesis of trypanothione, the main antioxidant metabolite in the parasite. In this research, MCA was applied in an ex vivo system to the enzymes of the reverse transsulfuration pathway (RTP) for Cys synthesis composed by cystathionine beta synthase (CBS) and cystathionine gamma lyase (CGL). The results indicated that CGL has 90% of the control of the pathway flux. Inhibition of CGL with propargylglycine (PAG) decreased the levels of Cys and trypanothione and depleted those of glutathione in epimastigotes (proliferative stage in the insect vector); these metabolite changes were prevented by supplementing with Cys, suggesting a compensatory role of the Cys transport (CysT). Indeed, Cys supplementation (but not PAG treatment) increased the activity of the CysT in epimastigotes whereas in trypomastigotes (infective stage in mammals) CysT was increased when they were incubated with PAG. Our results suggested that CGL could be a potential drug target given its high control on the RTP flux and its effects on the parasite antioxidant defense. However, the redundant Cys supply pathways in the parasite may require inhibition of the CysT as well. Our findings also suggest differential responses of the Cys supply pathways in different parasite stages.

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.