RESUMEN
Malaria parasites increase their host erythrocyte's permeability to obtain essential nutrients from plasma and facilitate intracellular growth. In the human Plasmodium falciparum pathogen, this increase is mediated by the plasmodial surface anion channel (PSAC) and has been linked to CLAG3, a protein integral to the host erythrocyte membrane and encoded by a member of the conserved clag multigene family. Whether paralogs encoded by other clag genes also insert at the host membrane is unknown; their contributions to PSAC formation and other roles served are also unexplored. Here, we generated transfectant lines carrying epitope-tagged versions of each CLAG. Each paralog is colocalized with CLAG3, with concordant trafficking via merozoite rhoptries to the host erythrocyte membrane of newly invaded erythrocytes. Each also exists within infected cells in at least two forms: an alkaline-extractable soluble form and a form integral to the host membrane. Like CLAG3, CLAG2 has a variant region cleaved by extracellular proteases, but CLAG8 and CLAG9 are protease resistant. Paralog knockout lines, generated through CRISPR/Cas9 transfection, exhibited uncompromised growth in PGIM, a modified medium with higher physiological nutrient levels; this finding is in marked contrast to a recently reported CLAG3 knockout parasite. CLAG2 and CLAG8 knockout lines exhibited compensatory increases in the transcription of the remaining clags and associated rhoph genes, yielding increased PSAC-mediated uptake for specific solutes. We also report on the distinct transport properties of these knockout lines. Similar membrane topologies at the host membrane are consistent with each CLAG paralog contributing to PSAC, but other roles require further examination.
RESUMEN
To validate therapeutic targets in metabolic pathways of trypanosomatids, the criterion of enzyme essentiality determined by gene knockout or knockdown is usually being applied. Since, it is often found that most of the enzymes/proteins analyzed are essential, additional criteria have to be implemented for drug target prioritization. Metabolic control analysis (MCA), often in conjunction with kinetic pathway modeling, offers such possibility for prioritization. MCA is a theoretical and experimental approach to analyze how metabolic pathways are controlled. It involves strategies to perform quantitative analyses to determine the degree in which an enzyme controls a pathway flux, a value called flux control coefficient ([Formula: see text]). By determining the [Formula: see text] of individual steps in a metabolic pathway, the distribution of control of the pathway is established, that is, the identification of the main flux-controlling steps. Therefore, MCA can help in ranking pathway enzymes as drug targets from a metabolic perspective. In this chapter, three approaches to determine [Formula: see text] are reviewed: (1) In vitro pathway reconstitution, (2) manipulation of enzyme activities within parasites, and (3) in silico kinetic modeling of the metabolic pathway. To perform these methods, accurate experimental data of enzyme activities, metabolite concentrations and pathway fluxes are necessary. The methodology is illustrated with the example of trypanothione metabolism of Trypanosoma cruzi and protocols to determine such experimental data for this metabolic process are also described. However, the MCA strategy can be applied to any metabolic pathway in the parasite and general directions to perform it are provided in this chapter.
Asunto(s)
Desarrollo de Medicamentos/métodos , Metabolómica/métodos , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/metabolismo , Extractos Celulares/aislamiento & purificación , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/parasitología , Simulación por Computador , Glutatión/análogos & derivados , Glutatión/metabolismo , Humanos , Cinética , Redes y Vías Metabólicas/efectos de los fármacos , Modelos Biológicos , Terapia Molecular Dirigida/métodos , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Espermidina/análogos & derivados , Espermidina/metabolismo , Tripanocidas/farmacología , Tripanocidas/uso terapéutico , Trypanosoma cruzi/efectos de los fármacosRESUMEN
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.
Asunto(s)
Antioxidantes/metabolismo , Glutamato-Cisteína Ligasa/genética , Glutatión/análogos & derivados , Proteínas Protozoarias/genética , Espermidina/análogos & derivados , Tiorredoxinas/genética , Trypanosoma cruzi/efectos de los fármacos , Amida Sintasas/genética , Amida Sintasas/metabolismo , Butionina Sulfoximina/farmacología , Línea Celular , Combinación de Medicamentos , Resistencia a Medicamentos/genética , Fibroblastos/parasitología , Regulación de la Expresión Génica , Glutamato-Cisteína Ligasa/metabolismo , Glutatión/antagonistas & inhibidores , Glutatión/biosíntesis , Humanos , Peróxido de Hidrógeno/farmacología , NADH NADPH Oxidorreductasas/genética , NADH NADPH Oxidorreductasas/metabolismo , Nitroimidazoles/farmacología , Oxidación-Reducción , Estrés Oxidativo , Peroxidasas/genética , Peroxidasas/metabolismo , Proteínas Protozoarias/metabolismo , Transducción de Señal , Espermidina/antagonistas & inhibidores , Espermidina/biosíntesis , Tiorredoxinas/metabolismo , Tripanocidas/farmacología , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/genéticaRESUMEN
In the search for therapeutic targets in the intermediary metabolism of trypanosomatids the gene essentiality criterion as determined by using knock-out and knock-down genetic strategies is commonly applied. As most of the evaluated enzymes/transporters have turned out to be essential for parasite survival, additional criteria and approaches are clearly required for suitable drug target prioritization. The fundamentals of Metabolic Control Analysis (MCA; an approach in the study of control and regulation of metabolism) and kinetic modeling of metabolic pathways (a bottom-up systems biology approach) allow quantification of the degree of control that each enzyme exerts on the pathway flux (flux control coefficient) and metabolic intermediate concentrations (concentration control coefficient). MCA studies have demonstrated that metabolic pathways usually have two or three enzymes with the highest control of flux; their inhibition has more negative effects on the pathway function than inhibition of enzymes exerting low flux control. Therefore, the enzymes with the highest pathway control are the most convenient targets for therapeutic intervention. In this review, the fundamentals of MCA as well as experimental strategies to determine the flux control coefficients and metabolic modeling are analyzed. MCA and kinetic modeling have been applied to trypanothione metabolism in Trypanosoma cruzi and the model predictions subsequently validated in vivo. The results showed that three out of ten enzyme reactions analyzed in the T. cruzi anti-oxidant metabolism were the most controlling enzymes. Hence, MCA and metabolic modeling allow a further step in target prioritization for drug development against trypanosomatids and other parasites.
Asunto(s)
Desarrollo de Medicamentos/métodos , Enzimas/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/enzimología , Glutatión/análogos & derivados , Glutatión/metabolismo , Glucólisis/fisiología , Cinética , Modelos Biológicos , Espermidina/análogos & derivados , Espermidina/metabolismoRESUMEN
Buthionine sulfoximine (BSO) induces decreased glutathione (GSH) and trypanothione [T(SH)2 ] pools in trypanosomatids, presumably because only gamma-glutamylcysteine synthetase (γECS) is blocked. However, some BSO effects cannot be explained by exclusive γECS inhibition; therefore, its effect on the T(SH)2 metabolism pathway in Trypanosoma cruzi was re-examined. Parasites exposed to BSO did not synthesize T(SH)2 even when supplemented with cysteine or GSH, suggesting trypanothione synthetase (TryS) inhibition by BSO. Indeed, recombinant γECS and TryS, but not GSH synthetase, were inhibited by BSO and kinetics and docking analyses on a TcTryS 3D model suggested BSO binding at the GSH site. Furthermore, parasites overexpressing γECS and TryS showed ~ 50% decreased activities after BSO treatment. These results indicated that BSO is also an inhibitor of TryS.
Asunto(s)
Butionina Sulfoximina/farmacología , Glutatión/análogos & derivados , Espermidina/análogos & derivados , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/metabolismo , Amida Sintasas/antagonistas & inhibidores , Amida Sintasas/química , Amida Sintasas/genética , Animales , Inhibidores Enzimáticos/farmacología , Glutamato-Cisteína Ligasa/antagonistas & inhibidores , Glutamato-Cisteína Ligasa/genética , Glutatión/biosíntesis , Glutatión/metabolismo , Glutatión Sintasa/antagonistas & inhibidores , Glutatión Sintasa/genética , Humanos , Cinética , Redes y Vías Metabólicas/efectos de los fármacos , Simulación del Acoplamiento Molecular , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espermidina/biosíntesis , Trypanosoma cruzi/genéticaRESUMEN
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.
Asunto(s)
Bencimidazoles/farmacología , Homeostasis/efectos de los fármacos , Hidrazinas/farmacología , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacos , Animales , Bencimidazoles/síntesis química , Bencimidazoles/química , Relación Dosis-Respuesta a Droga , Hidrazinas/síntesis química , Hidrazinas/química , Ratones , Estructura Molecular , Oxidación-Reducción , Relación Estructura-Actividad , Tripanocidas/síntesis química , Tripanocidas/química , Trypanosoma cruzi/crecimiento & desarrollo , Trypanosoma cruzi/metabolismoRESUMEN
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.
Asunto(s)
Oxidorreductasas/metabolismo , Peróxidos/metabolismo , Proteínas Protozoarias/metabolismo , Tiorredoxinas/metabolismo , Trypanosoma cruzi/metabolismo , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/genética , Enfermedad de Chagas/metabolismo , Sistemas de Liberación de Medicamentos , Humanos , Cinética , Oxidorreductasas/química , Oxidorreductasas/genética , Peróxidos/química , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tiorredoxinas/química , Tiorredoxinas/genética , Trypanosoma cruzi/química , Trypanosoma cruzi/genéticaRESUMEN
A kinetic model of trypanothione [T(SH)(2)] metabolism in Trypanosoma cruzi was constructed based on enzyme kinetic parameters determined under near-physiological conditions (including glutathione synthetase), and the enzyme activities, metabolite concentrations and fluxes determined in the parasite under control and oxidizing conditions. The pathway structure is characterized by a T(SH)(2) synthetic module of low flux and low catalytic capacity, and another more catalytically efficient T(SH)(2) -dependent antioxidant/regenerating module. The model allowed quantification of the contribution of each enzyme to the control of T(SH)(2) synthesis and concentration (flux control and concentration control coefficients, respectively). The main control of flux was exerted by γ-glutamylcysteine synthetase (γECS) and trypanothione synthetase (TryS) (control coefficients of 0.58-0.7 and 0.49-0.58, respectively), followed by spermidine transport (0.24); negligible flux controls by trypantothione reductase (TryR) and the T(SH)(2)-dependent antioxidant machinery were determined. The concentration of reduced T(SH)(2) was controlled by TryR (0.98) and oxidative stress (-0.99); however, γECS and TryS also exerted control on the cellular level of T(SH(2)) when they were inhibited by more than 70%. The model predicted that in order to diminish the T(SH)(2) synthesis flux by 50%, it is necessary to inhibit γECS or TryS by 58 or 63%, respectively, or both by 50%, whereas more than 98% inhibition was required for TryR. Hence, simultaneous and moderate inhibition of γECS and TryS appears to be a promising multi-target therapeutic strategy. In contrast, use of highly potent and specific inhibitors for TryR and the antioxidant machinery is necessary to affect the antioxidant capabilities of the parasites.