RESUMEN

Cruzipain (CZP), the major cysteine protease present in T. cruzi, the ethiological agent of Chagas disease, has attracted particular attention as a therapeutic target for the development of targeted covalent inhibitors (TCI). The vast chemical space associated with the enormous molecular diversity feasible to explore by means of modern synthetic approaches allows the design of CZP inhibitors capable of exhibiting not only an efficient enzyme inhibition but also an adequate translation to anti-T. cruzi activity. In this work, a computer-aided design strategy was developed to combinatorially construct and screen large libraries of 1,4-disubstituted 1,2,3-triazole analogues, further identifying a selected set of candidates for advancement towards synthetic and biological activity evaluation stages. In this way, a virtual molecular library comprising more than 75 thousand diverse and synthetically feasible analogues was studied by means of molecular docking and molecular dynamic simulations in the search of potential TCI of CZP, guiding the synthetic efforts towards a subset of 48 candidates. These were synthesized by applying a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) centered synthetic scheme, resulting in moderate to good yields and leading to the identification of 12 hits selectively inhibiting CZP activity with IC50 in the low micromolar range. Furthermore, four triazole derivatives showed good anti-T. cruzi inhibition when studied at 50 µM; and Ald-6 excelled for its high antitrypanocidal activity and low cytotoxicity, exhibiting complete in vitro biological activity translation from CZP to T. cruzi. Overall, not only Ald-6 merits further advancement to preclinical in vivo studies, but these findings also shed light on a valuable chemical space where molecular diversity might be explored in the search for efficient triazole-based antichagasic agents.

Asunto(s)

Cisteína Endopeptidasas , Simulación del Acoplamiento Molecular , Proteínas Protozoarias , Triazoles , Trypanosoma cruzi , Triazoles/química , Triazoles/farmacología , Triazoles/síntesis química , Cisteína Endopeptidasas/química , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/química , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/enzimología , Inhibidores de Cisteína Proteinasa/química , Inhibidores de Cisteína Proteinasa/farmacología , Inhibidores de Cisteína Proteinasa/síntesis química , Simulación de Dinámica Molecular , Relación Estructura-Actividad , Diseño Asistido por Computadora , Diseño de Fármacos , Humanos , Estructura Molecular , Tripanocidas/farmacología , Tripanocidas/química , Tripanocidas/síntesis química , Enfermedad de Chagas/tratamiento farmacológico

RESUMEN

American trypanosomiasis or Chagas disease, caused by Trypanosoma cruzi (T. cruzi), affects approximately 6-7 million people worldwide. However, its pharmacological treatment causes several uncomfortable side effects, causing patients' treatment abandonment. Therefore, there is a need for new and better treatments. In this work, the molecular docking of nine hundred twenty-four FDA-approved drugs on three different sites of trypanothione reductase of T. cruzi (TcTR) was carried out to find potential trypanocidal agents. Finally, biological evaluations in vitro and in vivo were conducted with the selected FDA-approved drugs. Digoxin, alendronate, flucytosine, and dihydroergotamine showed better trypanocidal activity than the reference drugs benznidazole and nifurtimox in the in vitro evaluation against the trypomastigotes form. Further, these FDA-approved drugs were able to reduce 20-50% parasitemia in a short time in an in vivo model, although with less efficiency than benznidazole. Therefore, the results suggest a combined therapy of repurposed and canonical drugs against T. cruzi infection.

Asunto(s)

Enfermedad de Chagas , Simulación del Acoplamiento Molecular , NADH NADPH Oxidorreductasas , Tripanocidas , Trypanosoma cruzi , Tripanocidas/farmacología , Tripanocidas/química , NADH NADPH Oxidorreductasas/antagonistas & inhibidores , NADH NADPH Oxidorreductasas/química , NADH NADPH Oxidorreductasas/metabolismo , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/enzimología , Enfermedad de Chagas/tratamiento farmacológico , Animales , Humanos , United States Food and Drug Administration , Aprobación de Drogas , Evaluación Preclínica de Medicamentos , Estados Unidos , Ratones

RESUMEN

There is an urgent need to develop new, safer, and more effective drugs against Chagas disease (CD) as well as related kinetoplastid diseases. Targeting and inhibiting the Trypanosoma cruzi proteasome has emerged as a promising therapeutic approach in this context. To expand the chemical space for this class of inhibitors, we performed virtual screening campaigns with emphasis on shape-based similarity and ADMET prioritization. We describe the ideation and application of robustly validated shape queries for these campaigns, which furnished 44 compounds for biological evaluation. Five hit compounds demonstrated in vitro antitrypanosomal activity by potential inhibition of T. cruzi proteasome and notable chemical diversities, particularly, LCQFTC11. Structural insights were achieved by homology modeling, sequence/structure alignment, proteasome-species comparison, docking, molecular dynamics, and MMGBSA binding affinity estimations. These methods confirmed key interactions as well as the stability of LCQFTC11 at the ß4/ß5 subunits' binding site of the T. cruzi proteasome, consistent with known inhibitors. Our results warrant future assay confirmation of our hit as a T. cruzi proteasome inhibitor. Importantly, we also shed light into dynamic details for a proteasome inhibition mechanism that shall be further investigated. We expect to contribute to the development of viable CD drug candidates through such a relevant approach.

Asunto(s)

Simulación del Acoplamiento Molecular , Complejo de la Endopetidasa Proteasomal , Inhibidores de Proteasoma , Trypanosoma cruzi , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/enzimología , Complejo de la Endopetidasa Proteasomal/metabolismo , Complejo de la Endopetidasa Proteasomal/química , Inhibidores de Proteasoma/farmacología , Inhibidores de Proteasoma/química , Simulación de Dinámica Molecular , Tripanocidas/farmacología , Tripanocidas/química , Sitios de Unión , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/parasitología , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Relación Estructura-Actividad , Unión Proteica

RESUMEN

The protozoan parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. are responsible for continued propagation of neglected tropical diseases such as African sleeping sickness, Chagas disease and leishmaniasis respectively. Following a report that captopril targets Leishmania donovani dipeptidyl carboxypeptidase, a series of simple proline amides and captopril analogues were synthesized and found to exhibit 1-2 µM in vitro inhibition and selectivity against Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. The results were corroborated with computational docking studies. Arguably, the synthetic proline amides represent the structurally simplest examples of in vitro pan antiprotozoal compounds.

Asunto(s)

Captopril , Trypanosoma brucei brucei , Trypanosoma cruzi , Captopril/farmacología , Captopril/química , Captopril/síntesis química , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/enzimología , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/enzimología , Relación Estructura-Actividad , Simulación del Acoplamiento Molecular , Tripanocidas/farmacología , Tripanocidas/química , Tripanocidas/síntesis química , Estructura Molecular , Leishmania/efectos de los fármacos , Leishmania/enzimología , Antiprotozoarios/farmacología , Antiprotozoarios/química , Antiprotozoarios/síntesis química , Humanos

RESUMEN

Chagas disease is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. It bears a significant global health burden with limited treatment options, thus calling for the development of new and effective drugs. Certain trypanosomal metabolic enzymes have been suggested to be druggable and valid for subsequent inhibition. In this study, the crystal structure of glycerol kinase from T. cruzi, a key enzyme in glycerol metabolism in this parasite, is presented. Structural analysis allowed a detailed description of the glycerol binding pocket, while comparative assessment pinpointed a potential regulatory site which may serve as a target for selective inhibition. These findings advance the understanding of glycerol metabolism in eukaryotes and provide a solid basis for the future treatment of Chagas disease.

Asunto(s)

Enfermedad de Chagas , Glicerol Quinasa , Trypanosoma cruzi , Trypanosoma cruzi/enzimología , Glicerol Quinasa/química , Glicerol Quinasa/metabolismo , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/parasitología , Cristalografía por Rayos X , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Modelos Moleculares , Humanos , Sitios de Unión , Glicerol/química , Conformación Proteica

RESUMEN

Phospholipases A2 (PLA2) comprise a superfamily of enzymes that specifically catalyze hydrolysis of the ester bond at the sn-2 position of glycerophospholipids, generating lysophospholipids and fatty acids. In Rhodnius prolixus, one of the main vectors of the Chagas's disease etiologic agent Trypanosoma cruzi, it was previously shown that lysophosphatidylcholine, a bioactive lipid, found in the insect's saliva, contributes to the inhibition of platelet aggregation, and increases the production of nitric oxide, an important vasodilator. Due to its role in potentially generating LPC, here we studied the PLA2 present in the salivary glands of R. prolixus. PLA2 activity is approximately 100 times greater in the epithelium than in the contents of salivary glands. Our study reveals the role of the RpPLA2XIIA gene in the insect feeding performance and in the fatty acids composition of phospholipids extracted from the salivary glands. Knockdown of RpPLA2XIIA significantly altered the relative amounts of palmitic, palmitoleic, oleic and linoleic acids. A short-term decrease in the expression of RpPLA2III and RpPLA2XIIA in the salivary glands of R. prolixus was evident on the third day after infection by T. cruzi. Taken together, our results contribute to the understanding of the role of PLA2 in the salivary glands of hematophagous insects and show that the parasite is capable of modulating even tissues that are not colonized by it.

Asunto(s)

Fosfolipasas A2 , Rhodnius , Glándulas Salivales , Trypanosoma cruzi , Animales , Rhodnius/parasitología , Rhodnius/enzimología , Rhodnius/genética , Glándulas Salivales/parasitología , Glándulas Salivales/enzimología , Glándulas Salivales/metabolismo , Trypanosoma cruzi/genética , Trypanosoma cruzi/enzimología , Fosfolipasas A2/metabolismo , Fosfolipasas A2/genética , Ácidos Grasos/metabolismo , Enfermedad de Chagas/parasitología , Insectos Vectores/parasitología , Insectos Vectores/enzimología

RESUMEN

According to the World Health Organization, Chagas disease (CD) is the most prevalent poverty-promoting neglected tropical disease. Alarmingly, climate change is accelerating the geographical spreading of CD causative parasite, Trypanosoma cruzi, which additionally increases infection rates. Still, CD treatment remains challenging due to a lack of safe and efficient drugs. In this work, we analyze the viability of T. cruzi Akt-like kinase (TcAkt) as drug target against CD including primary structural and functional information about a parasitic Akt protein. Nuclear Magnetic Resonance derived information in combination with Molecular Dynamics simulations offer detailed insights into structural properties of the pleckstrin homology (PH) domain of TcAkt and its binding to phosphatidylinositol phosphate ligands (PIP). Experimental data combined with Alpha Fold proposes a model for the mechanism of action of TcAkt involving a PIP-induced disruption of the intramolecular interface between the kinase and the PH domain resulting in an open conformation enabling TcAkt kinase activity. Further docking experiments reveal that TcAkt is recognized by human inhibitors PIT-1 and capivasertib, and TcAkt inhibition by UBMC-4 and UBMC-6 is achieved via binding to TcAkt kinase domain. Our in-depth structural analysis of TcAkt reveals potential sites for drug development against CD, located at activity essential regions.

Asunto(s)

Enfermedad de Chagas , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Trypanosoma cruzi , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/efectos de los fármacos , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/parasitología , Humanos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Unión Proteica

RESUMEN

Chagas disease is one of the world's neglected tropical diseases, caused by the human pathogenic protozoan parasite Trypanosoma cruzi. There is currently a lack of effective and tolerable clinically available therapeutics to treat this life-threatening illness and the discovery of modern alternative options is an urgent matter. T. cruzi glucokinase (TcGlcK) is a potential drug target because its product, d-glucose-6-phosphate, serves as a key metabolite in the pentose phosphate pathway, glycolysis, and gluconeogenesis. In 2019, we identified a novel cluster of TcGlcK inhibitors that also exhibited anti-T. cruzi efficacy called the 3-nitro-2-phenyl-2H-chromene analogues. This was achieved by performing a target-based high-throughput screening (HTS) campaign of 13,040 compounds. The selection criteria were based on first determining which compounds strongly inhibited TcGlcK in a primary screen, followed by establishing on-target confirmed hits from a confirmatory assay. Compounds that exhibited notable in vitro trypanocidal activity over the T. cruzi infective form (trypomastigotes and intracellular amastigotes) co-cultured in NIH-3T3 mammalian host cells, as well as having revealed low NIH-3T3 cytotoxicity, were further considered. Compounds GLK2-003 and GLK2-004 were determined to inhibit TcGlcK quite well with IC50 values of 6.1 µM and 4.8 µM, respectively. Illuminated by these findings, we herein screened a small compound library consisting of thirteen commercially available 3-nitro-2-phenyl-2H-chromene analogues, two of which were GLK2-003 and GLK2-004 (compounds 1 and 9, respectively). Twelve of these compounds had a one-point change from the chemical structure of GLK2-003. The analogues were run through a similar primary screening and confirmatory assay protocol to our previous HTS campaign. Subsequently, three in vitro biological assays were performed where compounds were screened against (a) T. cruzi (Tulahuen strain) infective form co-cultured within NIH-3T3 cells, (b) T. brucei brucei (427 strain) bloodstream form, and (c) NIH-3T3 host cells alone. We report on the TcGlcK inhibitor constant determinations, mode of enzyme inhibition, in vitro antitrypanosomal IC50 determinations, and an assessment of structure-activity relationships. Our results reveal that the 3-nitro-2-phenyl-2H-chromene scaffold holds promise and can be further optimized for both Chagas disease and human African trypanosomiasis early-stage drug discovery research.

Asunto(s)

Benzopiranos , Glucoquinasa , Tripanocidas , Trypanosoma cruzi , Animales , Humanos , Ratones , Benzopiranos/farmacología , Benzopiranos/química , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/parasitología , Descubrimiento de Drogas/métodos , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Glucoquinasa/metabolismo , Glucoquinasa/antagonistas & inhibidores , Ensayos Analíticos de Alto Rendimiento , Simulación del Acoplamiento Molecular , Células 3T3 NIH , Relación Estructura-Actividad , Tripanocidas/farmacología , Tripanocidas/química , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/enzimología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología

RESUMEN

The protozoan parasite Trypanosoma cruzi is the causative agent of American trypanosomiasis, otherwise known as Chagas disease. To survive in the host, the T. cruzi parasite needs antioxidant defense systems. One of these is a hybrid heme peroxidase, the T. cruzi ascorbate peroxidase-cytochrome c peroxidase enzyme (TcAPx-CcP). TcAPx-CcP has high sequence identity to members of the class I peroxidase family, notably ascorbate peroxidase (APX) and cytochrome c peroxidase (CcP), as well as a mitochondrial peroxidase from Leishmania major (LmP). The aim of this work was to solve the structure and examine the reactivity of the TcAPx-CcP enzyme. Low temperature electron paramagnetic resonance spectra support the formation of an exchange-coupled [Fe(IV)=O Trp233•+] compound I radical species, analogous to that used in CcP and LmP. We demonstrate that TcAPx-CcP is similar in overall structure to APX and CcP, but there are differences in the substrate-binding regions. Furthermore, the electron transfer pathway from cytochrome c to the heme in CcP and LmP is preserved in the TcAPx-CcP structure. Integration of steady state kinetic experiments, molecular dynamic simulations, and bioinformatic analyses indicates that TcAPx-CcP preferentially oxidizes cytochrome c but is still competent for oxidization of ascorbate. The results reveal that TcAPx-CcP is a credible cytochrome c peroxidase, which can also bind and use ascorbate in host cells, where concentrations are in the millimolar range. Thus, kinetically and functionally TcAPx-CcP can be considered a hybrid peroxidase.

Asunto(s)

Citocromo-c Peroxidasa , Trypanosoma cruzi , Antioxidantes , Ascorbato Peroxidasas/genética , Ascorbato Peroxidasas/metabolismo , Ácido Ascórbico/metabolismo , Enfermedad de Chagas/parasitología , Citocromo-c Peroxidasa/química , Citocromo-c Peroxidasa/genética , Citocromo-c Peroxidasa/metabolismo , Citocromos c/metabolismo , Hemo/metabolismo , Humanos , Peroxidasa/metabolismo , Peroxidasas/metabolismo , Especificidad por Sustrato , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/metabolismo

RESUMEN

Trypanothione synthetase (TryS) catalyses the synthesis of N1,N8-bis(glutathionyl)spermidine (trypanothione), which is the main low molecular mass thiol supporting several redox functions in trypanosomatids. TryS attracts attention as molecular target for drug development against pathogens causing severe and fatal diseases in mammals. A drug discovery campaign aimed to identify and characterise new inhibitors of TryS with promising biological activity was conducted. A large compound library (n = 51,624), most of them bearing drug-like properties, was primarily screened against TryS from Trypanosoma brucei (TbTryS). With a true-hit rate of 0.056%, several of the TbTryS hits (IC50 from 1.2 to 36 µM) also targeted the homologue enzyme from Leishmania infantum and Trypanosoma cruzi (IC50 values from 2.6 to 40 µM). Calmidazolium chloride and Ebselen stand out for their multi-species anti-TryS activity at low µM concentrations (IC50 from 2.6 to 13.8 µM). The moieties carboxy piperidine amide and amide methyl thiazole phenyl were identified as novel TbTryS inhibitor scaffolds. Several of the TryS hits presented one-digit µM EC50 against T. cruzi and L. donovani amastigotes but proved cytotoxic against the human osteosarcoma and macrophage host cells (selectivity index ≤ 3). In contrast, seven hits showed a significantly higher selectivity against T. b. brucei (selectivity index from 11 to 182). Non-invasive redox assays confirmed that Ebselen, a multi-TryS inhibitor, induces an intracellular oxidative milieu in bloodstream T. b. brucei. Kinetic and mass spectrometry analysis revealed that Ebselen is a slow-binding inhibitor that modifies irreversible a highly conserved cysteine residue from the TryS's synthetase domain. The most potent TbTryS inhibitor (a singleton containing an adamantine moiety) exerted a non-covalent, non-competitive (with any of the substrates) inhibition of the enzyme. These data feed the drug discovery pipeline for trypanosomatids with novel and valuable information on chemical entities with drug potential.

Asunto(s)

Amida Sintasas/antagonistas & inhibidores , Antineoplásicos/farmacología , Antiprotozoarios/farmacología , Leishmania infantum/efectos de los fármacos , Trypanosoma cruzi/efectos de los fármacos , Amida Sintasas/metabolismo , Antineoplásicos/síntesis química , Antineoplásicos/química , Antiprotozoarios/síntesis química , Antiprotozoarios/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Leishmania infantum/enzimología , Macrófagos/efectos de los fármacos , Estructura Molecular , Relación Estructura-Actividad , Trypanosoma cruzi/enzimología

RESUMEN

Metacaspases are known to have a fundamental role in apoptosis-like, a programmed cellular death (PCD) in plants, fungi, and protozoans. The last includes several parasites that cause diseases of great interest to public health, mostly without adequate treatment and included in the neglected tropical diseases category. One of them is Trypanosoma cruzi which causes Chagas disease and has two metacaspases involved in its PCD: TcMCA3 and TcMCA5. Their roles seemed different in PCD, TcMCA5 appears as a proapoptotic protein negatively regulated by its C-terminal sequence, while TcMCA3 is described as a cell cycle regulator. Despite this, the precise role of TcMCA3 and TcMCA5 and their atomic structures remain elusive. Therefore, developing methodologies to allow investigations of those metacaspases is relevant. Herein, we produced full-length and truncated versions of TcMCA5 and applied different strategies for their folded recombinant production from E. coli inclusion bodies. Biophysical assays probed the efficacy of the production method in providing a high yield of folded recombinant TcMCA5. Moreover, we modeled the TcMCA5 protein structure using experimental restraints obtained by XLMS. The experimental design for novel methods and the final protocol provided here can guide studies with other metacaspases. The production of TcMCA5 allows further investigations as protein crystallography, HTS drug discovery to create potential therapeutic in the treatment of Chagas' disease and in the way to clarify how the PCD works in the parasite.

Asunto(s)

Caspasas/química , Replegamiento Proteico , Proteínas Protozoarias/química , Trypanosoma cruzi/enzimología , Caspasas/genética , Dominios Proteicos , Proteínas Protozoarias/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Trypanosoma cruzi/genética

RESUMEN

Cyclic nucleotide phosphodiesterases have been implicated in the proliferation, differentiation and osmotic regulation of trypanosomatids; in some trypanosomatid species, they have been validated as molecular targets for the development of new therapeutic agents. Because the experimental structure of Trypanosoma cruzi PDEb1 (TcrPDEb1) has not been solved so far, an homology model of the target was created using the structure of Trypanosoma brucei PDEb1 (TbrPDEb1) as a template. The model was refined by extensive enhanced sampling molecular dynamics simulations, and representative snapshots were extracted from the trajectory by combined clustering analysis. This structural ensemble was used to develop a structure-based docking model of the target. The docking accuracy of the model was validated by redocking and cross-docking experiments using all available crystal structures of TbrPDEb1, whereas the scoring accuracy was validated through a retrospective screen, using a carefully curated dataset of compounds assayed against TbrPDEb1 and/or TcrPDEb1. Considering the results from in silico validations, the model may be applied in prospective virtual screening campaigns to identify novel hits, as well as to guide the rational design of potent and selective inhibitors targeting this enzyme.

Asunto(s)

3',5'-AMP Cíclico Fosfodiesterasas/química , Proteínas Protozoarias/química , Bibliotecas de Moléculas Pequeñas/química , Trypanosoma cruzi/enzimología , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Secuencia de Aminoácidos , Área Bajo la Curva , Sitios de Unión , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Estructura Terciaria de Proteína , Proteínas Protozoarias/metabolismo , Curva ROC , Alineación de Secuencia , Bibliotecas de Moléculas Pequeñas/metabolismo , Trypanosoma brucei brucei/enzimología

RESUMEN

Writing and erasing of posttranslational modifications are crucial to phenotypic plasticity and antigenic variation of eukaryotic pathogens. Targeting pathogens' modification machineries, thus, represents a valid approach to fighting parasitic diseases. However, identification of parasitic targets and the development of selective anti-parasitic drugs still represent major bottlenecks. Here, we show that the zinc-dependent histone deacetylases (HDACs) of the protozoan parasite Trypanosoma cruzi are key regulators that have significantly diverged from their human counterparts. Depletion of T. cruzi class I HDACs tcDAC1 and tcDAC2 compromises cell-cycle progression and division, leading to cell death. Notably, tcDAC2 displays a deacetylase activity essential to the parasite and shows major structural differences with human HDACs. Specifically, tcDAC2 harbors a modular active site with a unique subpocket targeted by inhibitors showing substantial anti-parasitic effects in cellulo and in vivo. Thus, the targeting of the many atypical HDACs in pathogens can enable anti-parasitic selective chemical impairment.

Asunto(s)

Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/genética , Trypanosoma cruzi/metabolismo , Animales , Dominio Catalítico , Ciclo Celular , División Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/parasitología , Chlorocebus aethiops , ADN Protozoario , Femenino , Prueba de Complementación Genética , Inhibidores de Histona Desacetilasas/química , Histona Desacetilasas/química , Interacciones Huésped-Parásitos , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Filogenia , Conformación Proteica , Procesamiento Proteico-Postraduccional , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Eliminación de Secuencia , Trypanosoma cruzi/efectos de los fármacos , Células Vero

RESUMEN

Trypanosoma cruzi faces a variety of environmental scenarios during its life cycle, which include changes in the redox environment that requires a fine regulation of a complex antioxidant arsenal of enzymes. Reversible posttranslational modifications, as lysine acetylation, are a fast and economical way for cells to react to environmental conditions. Recently, we found that the main antioxidant enzymes, including the mitochondrial superoxide dismutase A (TcSODA) are acetylated in T. cruzi, suggesting that protein acetylation could participate in the oxidative stress response in T. cruzi. Therefore, we investigated whether mitochondrial lysine deacetylase TcSir2rp3 was involved in the activity control of TcSODA. We observed an increased resistance to hydrogen peroxide and menadione in parasites overexpressing TcSir2rp3. Increased resistance was also found for benznidazole and nifurtimox, known to induce reactive oxidative and nitrosactive species in the parasite, associated to that a reduction in the ROS levels was observed. To better understand the way TcSir2rp3 could contributes to oxidative stress response, we analyzed the expression of TcSODA in the TcSir2rp3 overexpressing parasites and did not detect any increase in protein levels of this enzyme. However, we found that these parasites presented higher levels of superoxide dismutase activity, and also that TcSir2rp3 and TcSODA interacts in vivo. Knowing that TcSODA is acetylated at lysine residues K44 and K97, and that K97 is located at a similar region in the protein structure as K68 in human manganese superoxide dismutase (MnSOD), responsible for regulating MnSOD activity, we generated mutated versions of TcSODA at K44 and K97 and found that replacing K97 by glutamine, which mimics an acetylated lysine, negatively affects the enzyme activity in vitro. By using molecular dynamics approaches, we revealed that acetylation of K97 induces specific conformational changes in TcSODA with respect to hydrogen-bonding pattern to neighbor residues, suggesting a key participation of this residue to modulate the affinity to O2- . Taken together, our results showed for the first time the involvement of lysine acetylation in the maintenance of homeostatic redox state in trypanosomatids, contributing to the understanding of mechanisms used by T. cruzi to progress during the infection.

Asunto(s)

Mitocondrias/enzimología , Estrés Oxidativo , Sirtuinas , Trypanosoma cruzi , Oxidación-Reducción , Sirtuinas/genética , Sirtuinas/metabolismo , Superóxido Dismutasa/genética , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/genética

RESUMEN

Trypanosoma cruzi, the agent of Chagas disease, accumulates polyphosphate (polyP) and Ca2+ inside acidocalcisomes. The alkalinization of this organelle stimulates polyP hydrolysis and Ca2+ release. Here, we report that histidine ammonia lyase (HAL), an enzyme that catalyzes histidine deamination with production of ammonia (NH3) and urocanate, is responsible for acidocalcisome alkalinization. Histidine addition to live parasites expressing HAL fused to the pH-sensitive emission biosensor green fluorescent protein (GFP) variant pHluorin induced alkalinization of acidocalcisomes. PolyP decreased HAL activity of epimastigote lysates or the recombinant protein but did not cause its polyphosphorylation, as determined by the lack of HAL electrophoretic shift on NuPAGE gels using both in vitro and in vivo conditions. We demonstrate that HAL binds strongly to polyP and localizes to the acidocalcisomes and cytosol of the parasite. Four lysine residues localized in the HAL C-terminal region are instrumental for its polyP binding, its inhibition by polyP, its function inside acidocalcisomes, and parasite survival under starvation conditions. Expression of HAL in yeast deficient in polyP degradation decreased cell fitness. This effect was enhanced by histidine and decreased when the lysine-rich C-terminal region was deleted. In conclusion, this study highlights a mechanism for stimulation of acidocalcisome alkalinization linked to amino acid metabolism. IMPORTANCE Trypanosoma cruzi is the etiologic agent of Chagas disease and is characterized by the presence of acidocalcisomes, organelles rich in phosphate and calcium. Release of these molecules, which are necessary for growth and cell signaling, is induced by alkalinization, but a physiological mechanism for acidocalcisome alkalinization was unknown. In this work, we demonstrate that a histidine ammonia lyase localizes to acidocalcisomes and is responsible for their alkalinization.

Asunto(s)

Histidina Amoníaco-Liasa/metabolismo , Orgánulos/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/enzimología , Álcalis/metabolismo , Secuencias de Aminoácidos , Calcio/metabolismo , Enfermedad de Chagas/parasitología , Histidina/metabolismo , Histidina Amoníaco-Liasa/química , Histidina Amoníaco-Liasa/genética , Humanos , Orgánulos/química , Polifosfatos/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Trypanosoma cruzi/genética , Trypanosoma cruzi/crecimiento & desarrollo , Trypanosoma cruzi/metabolismo

RESUMEN

Dihydroxyacetone (DHA) can be used as an energy source by many cell types; however, it is toxic at high concentrations. The enzyme dihydroxyacetone kinase (DAK) has shown to be involved in DHA detoxification and osmoregulation. Among protozoa of the genus Trypanosoma, T. brucei, which causes sleeping sickness, is highly sensitive to DHA and does not have orthologous genes to DAK. Conversely, T. cruzi, the etiological agent of Chagas Disease, has two putative ATP-dependent DAK (TcDAKs) sequences in its genome. Here we show that T. cruzi epimastigote lysates present a DAK specific activity of 27.1 nmol/min/mg of protein and that this form of the parasite is able to grow in the presence of 2 mM DHA. TcDAK gene was cloned and the recombinant enzyme (recTcDAK) was expressed in Escherichia coli. An anti-recTcDAK serum reacted with a protein of the expected molecular mass of 61 kDa in epimastigotes. recTcDAK presented maximal activity using Mg+2, showing a Km of 6.5 µM for DHA and a K0.5 of 124.7 µM for ATP. As it was reported for other DAKs, recTcDAK activity was inhibited by FAD with an IC50 value of 0.33 mM. In conclusion, TcDAK is the first DAK described in trypanosomatids confirming another divergent metabolism between T. brucei and T. cruzi.

Asunto(s)

Fosfotransferasas (Aceptor de Grupo Alcohol)/aislamiento & purificación , Trypanosoma cruzi/enzimología , Secuencia de Aminoácidos , Animales , Western Blotting , Chlorocebus aethiops , Dihidroxiacetona/metabolismo , Dihidroxiacetona/toxicidad , Electroforesis en Gel de Poliacrilamida , Técnica del Anticuerpo Fluorescente , Masculino , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Osmorregulación , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/clasificación , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma cruzi/efectos de los fármacos , Células Vero

RESUMEN

Trypanosoma rangeli is a non-virulent hemoflagellate parasite infecting humans, wild and domestic mammals in Central and Latin America. The share of genotypic, phenotypic, and biological similarities with the virulent, human-infective T. cruzi and T. brucei, allows comparative studies on mechanisms of pathogenesis. In this study, investigation of the T. rangeli Arginine Kinase (TrAK) revealed two highly similar copies of the AK gene in this taxon, and a distinct expression profile and activity between replicative and infective forms. Although TrAK expression seems stable during epimastigotes growth, the enzymatic activity increases during the exponential growth phase and decreases from the stationary phase onwards. No differences were observed in activity or expression levels of TrAK during in vitro differentiation from epimastigotes to infective forms, and no detectable AK expression was observed for blood trypomastigotes. Overexpression of TrAK by T. rangeli showed no effects on the in vitro growth pattern, differentiation to infective forms, or infectivity to mice and triatomines. Although differences in TrAK expression and activity were observed among T. rangeli strains from distinct genetic lineages, our results indicate an up-regulation during parasite replication and putative post-translational myristoylation of this enzyme. We conclude that up-regulation of TrAK activity in epimastigotes appears to improve proliferation fitness, while reduced TrAK expression in blood trypomastigotes may be related to short-term and subpatent parasitemia in mammalian hosts.

Asunto(s)

Arginina Quinasa/metabolismo , Procesamiento Proteico-Postraduccional , Trypanosoma cruzi/enzimología , Trypanosoma rangeli/enzimología , Secuencia de Aminoácidos , Animales , Arginina Quinasa/biosíntesis , Arginina Quinasa/clasificación , Arginina Quinasa/genética , Western Blotting , ADN Protozoario/aislamiento & purificación , Electroforesis en Gel Bidimensional , Femenino , Flagelos/enzimología , Técnica del Anticuerpo Fluorescente Indirecta , Ratones , Ratones Endogámicos BALB C , Filogenia , Alineación de Secuencia , Trypanosoma cruzi/clasificación , Trypanosoma cruzi/genética , Trypanosoma cruzi/patogenicidad , Trypanosoma rangeli/clasificación , Trypanosoma rangeli/genética , Trypanosoma rangeli/patogenicidad , Regulación hacia Arriba , Virulencia

RESUMEN

Cruzipains are the main papain-like cysteine proteases of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease. Encoded by a multigenic family, previous studies have estimated the presence of dozens of copies spread over multiple chromosomes in different parasite strains. Here, we describe the complete gene repertoire of cruzipain in three parasite strains, their genomic organization, and expression pattern throughout the parasite life cycle. Furthermore, we have analyzed primary sequence variations among distinct family members as well as structural differences between the main groups of cruzipains. Based on phylogenetic inferences and residue positions crucial for enzyme function and specificity, we propose the classification of cruzipains into two families (I and II), whose genes are distributed in two or three separate clusters in the parasite genome, according with the strain. Family I comprises nearly identical copies to the previously characterized cruzipain 1/cruzain, whereas Family II encompasses three structurally distinct sub-types, named cruzipain 2, cruzipain 3, and cruzipain 4. RNA-seq data derived from the CL Brener strain indicates that Family I genes are mainly expressed by epimastigotes, whereas trypomastigotes mainly express Family II genes. Significant differences in the active sites among the enzyme sub-types were also identified, which may play a role in their substrate selectivity and impact their inhibition by small molecules.

Asunto(s)

Dominio Catalítico , Cisteína Endopeptidasas/genética , Proteínas Protozoarias/genética , Trypanosoma cruzi/genética , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/metabolismo , Regulación del Desarrollo de la Expresión Génica , Estadios del Ciclo de Vida/genética , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/enzimología , Trypanosoma cruzi/crecimiento & desarrollo

RESUMEN

BACKGROUND: Dihydroorotate dehydrogenase (DHODH) has long been recognized as an important drug target for proliferative and parasitic diseases, including compounds that exhibit trypanocidal action and broad-spectrum antiviral activity. Despite numerous and successful efforts in structural and functional characterization of DHODHs, as well as in the development of inhibitors, DHODH hot spots remain largely unmapped and underexplored. OBJECTIVE: This review describes the tools that are currently available for the identification and characterization of hot spots in protein structures and how freely available webservers can be exploited to predict DHODH hot spots. Moreover, it provides for the first time a review of the antiviral properties of DHODH inhibitors. METHODS: X-ray structures from human (HsDHODH) and Trypanosoma cruzi DHODH (TcDHODH) had their hot spots predicted by both FTMap and Fragment Hotspot Maps web servers. RESULTS: FTMap showed that hot spot occupancy in HsDHODH is correlated with the ligand efficiency (LE) of its known inhibitors, and Fragment Hotspot Maps pointed out the contribution of selected moieties to the overall LE. The conformational flexibility of the active site loop in TcDHODH was found to have a major impact on the druggability of the orotate binding site. In addition, both FTMap and Fragment Hotspot Maps servers predict a novel pocket in TcDHODH dimer interface (S6 site). CONCLUSION: This review reports how hot spots can be exploited during hit-to-lead steps, docking studies or even to improve inhibitor binding profile and by doing so using DHODH as a model, points to new drug development opportunities.

Asunto(s)

Dihidroorotato Deshidrogenasa/antagonistas & inhibidores , Dihidroorotato Deshidrogenasa/química , Desarrollo de Medicamentos/tendencias , Antivirales , Dihidroorotato Deshidrogenasa/metabolismo , Humanos , Trypanosoma cruzi/enzimología

RESUMEN

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is the key regulatory enzyme of the purine salvage pathway present in the members of trypanosomatids. The parasite solely depends on this pathway for the synthesis of nucleotides due to the absence of the de novo pathway. This study intends to identify putative inhibitors towards Trypanosoma cruzi HGPRT (TcHGPRT). Initial virtual screening was performed with substructures of phosphoribosyl pyrophosphate (PRPP), an original substrate of HGPRT. Twenty compounds that had greater binding energy than the substrate was treated as hits and was further screened and narrowed down through induced fit docking which resulted in top five compounds which was distinguished into two groups based on the ligand occupancy within the PRPP binding site of TcHGPRT. Group-I compounds (PubChem CID 130316561 and 134978234) are analogous to PRPP structure with greater occupancy, were preferred over Group-II compounds which had lesser occupancy than the substrate. However, one compound (22404820) among Group II was chosen for further analysis considering its significant electrostatic interactions. Molecular docking studies revealed the requirement of an electronegative moiety like phosphate group to be present in the ligand due to the presence of metal ions in the substrate binding site. The three chosen compounds along with PRPP were subjected to molecular dynamics analysis, which indicated a strong presence of electrostatic interaction. Considering the dynamic stability of interactions as well as pharmacological properties of ligands based on absorption, distribution, metabolism, excretion prediction, Group-I compounds were selected as lead compounds and were subjected to molecular electrostatic potential analysis to determine the charge distribution of the compound. The overall analysis thus suggests both 130316561 and 134978234 can be used as TcHGPRT inhibitors. Furthermore, these computational results emphasize the requirement of phosphorylated ligands which are essential in mediating electrostatic interactions and to compete with the binding affinity of the original substrate.

Asunto(s)

Inhibidores Enzimáticos/farmacología , Hipoxantina Fosforribosiltransferasa/antagonistas & inhibidores , Hipoxantina Fosforribosiltransferasa/química , Proteínas Protozoarias/antagonistas & inhibidores , Trypanosoma cruzi/enzimología , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacocinética , Humanos , Hipoxantina Fosforribosiltransferasa/metabolismo , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Electricidad Estática