RESUMEN
The NAD+-dependent lysine deacylase sirtuin 2 (Sirt2) is involved in multiple pathological conditions such as cancer. Targeting Sirt2 has thus received an increased interest for therapeutic purposes. Furthermore, the orthologue from Schistosoma mansoni (SmSirt2) has been considered for the potential treatment of the neglected tropical disease schistosomiasis. We previously identified a 1,2,4-oxadiazole-based scaffold from the screening of the "Kinetobox" library as a dual inhibitor of human Sirt2 (hSirt2) and SmSirt2. Herein, we describe the structure-activity studies on 1,2,4-oxadiazole-based analogues, which are potent inhibitors of human Sirt2 deacetylation. As proposed by docking studies, a substrate-competitive and cofactor-noncompetitive binding mode of inhibition could be determined in vitro via binding assays and kinetic analysis and further confirmed by a crystal structure of an oxadiazole inhibitor in complex with hSirt2. Optimized analogues reduced cell viability and inhibited prostate cancer cell migration, in correlation with Sirt2 deacetylase inhibition both in vitro and in cells.
Asunto(s)
Oxadiazoles , Sirtuina 2 , Sirtuina 2/antagonistas & inhibidores , Sirtuina 2/metabolismo , Oxadiazoles/farmacología , Oxadiazoles/química , Oxadiazoles/síntesis química , Humanos , Relación Estructura-Actividad , Simulación del Acoplamiento Molecular , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Schistosoma mansoni/efectos de los fármacos , Schistosoma mansoni/enzimología , Movimiento Celular/efectos de los fármacosRESUMEN
Schistosomiasis, caused by a parasitic blood fluke of the genus Schistosoma, is a global health problem for which new chemotherapeutic options are needed. We explored the scaffold of gallinamide A, a natural peptidic metabolite of marine cyanobacteria that has previously been shown to inhibit cathepsin L-type proteases. We screened a library of 19 synthetic gallinamide A analogs and identified nanomolar inhibitors of the cathepsin B-type protease SmCB1, which is a drug target for the treatment of schistosomiasis mansoni. Against cultured S. mansoni schistosomula and adult worms, many of the gallinamides generated a range of deleterious phenotypic responses. Imaging with a fluorescent-activity-based probe derived from gallinamide A demonstrated that SmCB1 is the primary target for gallinamides in the parasite. Furthermore, we solved the high-resolution crystal structures of SmCB1 in complex with gallinamide A and its two analogs and describe the acrylamide covalent warhead and binding mode in the active site. Quantum chemical calculations evaluated the contribution of individual positions in the peptidomimetic scaffold to the inhibition of the target and demonstrated the importance of the P1' and P2 positions. Our study introduces gallinamides as a powerful chemotype that can be exploited for the development of novel antischistosomal chemotherapeutics.
Asunto(s)
Catepsina B , Schistosoma mansoni , Catepsina B/antagonistas & inhibidores , Catepsina B/metabolismo , Animales , Schistosoma mansoni/enzimología , Schistosoma mansoni/efectos de los fármacos , Cristalografía por Rayos X , Esquistosomicidas/farmacología , Esquistosomicidas/química , Unión Proteica , Modelos MolecularesRESUMEN
The burden of human schistosomiasis, a known but neglected tropical disease in Sub-Saharan Africa, has been worrisome in recent years. It is becoming increasingly difficult to tackle schistosomiasis with praziquantel, a drug known to be effective against all Schistosoma species, due to reports of reduced efficacy and resistance. Therefore, this study seeks to investigate the antischistosomal potential of phytochemicals from Azadirachta indica against proteins that have been implicated as druggable targets for the treatment of schistosomiasis using computational techniques. In this study, sixty-three (63) previously isolated and characterized phytochemicals from A. indica were identified from the literature and retrieved from the PubChem database. In silico screening was conducted to assess the inhibitory potential of these phytochemicals against three receptors (Schistosoma mansoni Thioredoxin glutathione reductase, dihydroorotate dehydrogenase, and Arginase) that may serve as therapeutic targets for schistosomiasis treatment. Molecular docking, ADMET prediction, ligand interaction, MMGBSA, and molecular dynamics simulation of the hit compounds were conducted using the Schrodinger molecular drug discovery suite. The results show that Andrographolide possesses a satisfactory pharmacokinetic profile, does not violate the Lipinski rule of five, binds with favourable affinity with the receptors, and interacts with key amino acids at the active site. Importantly, its interaction with dihydroorotate dehydrogenase, an enzyme responsible for the catalysis of the de novo pyrimidine nucleotide biosynthetic pathway rate-limiting step, shows a glide score and MMGBSA of -10.19 and -45.75 Kcal/mol, respectively. In addition, the MD simulation shows its stability at the active site of the receptor. Overall, this study revealed that Andrographolide from Azadirachta indica could serve as a potential lead compound for the development of an anti-schistosomal drug.
Asunto(s)
Azadirachta , Dihidroorotato Deshidrogenasa , Simulación del Acoplamiento Molecular , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Esquistosomiasis , Azadirachta/química , Animales , Esquistosomiasis/tratamiento farmacológico , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/antagonistas & inhibidores , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Humanos , Fitoquímicos/farmacología , Fitoquímicos/química , Simulación de Dinámica Molecular , Schistosoma mansoni/efectos de los fármacos , Schistosoma mansoni/enzimología , NADH NADPH Oxidorreductasas/antagonistas & inhibidores , NADH NADPH Oxidorreductasas/metabolismo , Extractos Vegetales/química , Extractos Vegetales/farmacología , Simulación por Computador , Esquistosomicidas/farmacología , Esquistosomicidas/química , Esquistosomicidas/uso terapéutico , Complejos Multienzimáticos/antagonistas & inhibidores , Complejos Multienzimáticos/metabolismo , Praziquantel/farmacología , Praziquantel/química , Praziquantel/uso terapéuticoRESUMEN
In this study, we have identified and optimized two lead structures from an in-house screening, with promising results against the parasitic flatworm Schistosoma mansoni and its target protease S. mansoni cathepsin B1 (SmCB1). Our correlation analysis highlighted the significance of physicochemical properties for the compounds' in vitro activities, resulting in a dual approach to optimize the lead structures, regarding both phenotypic effects in S. mansoni newly transformed schistosomula (NTS), adult worms, and SmCB1 inhibition. The optimized compounds from both approaches ("phenotypic" vs "SmCB1" approach) demonstrated improved efficacy against S. mansoni NTS and adult worms, with 2h from the "SmCB1" approach emerging as the most potent compound. 2h displayed nanomolar inhibition of SmCB1 (Ki = 0.050 µM) while maintaining selectivity toward human off-target cathepsins. Additionally, the greatly improved efficacy of compound 2h toward S. mansoni adults (86% dead worms at 10 µM, 68% at 1 µM, 35% at 0.1 µM) demonstrates its potential as a new therapeutic agent for schistosomiasis, underlined by its improved permeability.
Asunto(s)
Catepsina B , Schistosoma mansoni , Schistosoma mansoni/efectos de los fármacos , Schistosoma mansoni/enzimología , Schistosoma mansoni/genética , Animales , Catepsina B/antagonistas & inhibidores , Catepsina B/metabolismo , Esquistosomiasis mansoni/tratamiento farmacológico , Diseño de Fármacos , Humanos , Fenotipo , Relación Estructura-Actividad , Antihelmínticos/farmacología , Antihelmínticos/química , Proteínas del Helminto/antagonistas & inhibidoresRESUMEN
Lactate dehydrogenase (LDH) from Schistosoma mansoni has peculiar properties for a eukaryotic LDH. Schistosomal LDH (SmLDH) isolated from schistosomes, and the recombinantly expressed protein, are strongly inhibited by ATP, which is neutralized by fructose-1,6-bisphosphate (FBP). In the conserved FBP/anion binding site we identified two residues in SmLDH (Val187 and Tyr190) that differ from the conserved residues in LDHs of other eukaryotes, but are identical to conserved residues in FBP-sensitive prokaryotic LDHs. Three-dimensional (3D) models were generated to compare the structure of SmLDH with other LDHs. These models indicated that residues Val187, and especially Tyr190, play a crucial role in the interaction of FBP with the anion pocket of SmLDH. These 3D models of SmLDH are also consistent with a competitive model of SmLDH inhibition in which ATP (inhibitor) and FBP (activator) compete for binding in a well-defined anion pocket. The model of bound ATP predicts a distortion of the nearby key catalytic residue His195, resulting in enzyme inhibition. To investigate a possible physiological role of this allosteric regulation of LDH in schistosomes we made a kinetic model in which the allosteric regulation of the glycolytic enzymes can be varied. The model showed that inhibition of LDH by ATP prevents fermentation to lactate in the free-living stages in water and ensures complete oxidation via the Krebs cycle of the endogenous glycogen reserves. This mechanism of allosteric inhibition by ATP prevents the untimely depletion of these glycogen reserves, the only fuel of the free-living cercariae. Neutralization by FBP of this ATP inhibition of LDH prevents accumulation of glycolytic intermediates when S. mansoni schistosomula are confronted with the sudden large increase in glucose availability upon penetration of the final host. It appears that the LDH of S. mansoni is special and well suited to deal with the variations in glucose availability the parasite encounters during its life cycle.
Asunto(s)
Adenosina Trifosfato , L-Lactato Deshidrogenasa , Modelos Moleculares , Schistosoma mansoni , Schistosoma mansoni/enzimología , Schistosoma mansoni/metabolismo , Animales , L-Lactato Deshidrogenasa/metabolismo , L-Lactato Deshidrogenasa/genética , Cinética , Adenosina Trifosfato/metabolismo , Fructosadifosfatos/metabolismo , Ratones , Secuencia de Aminoácidos , Biomphalaria/parasitología , Sitios de UniónRESUMEN
Schistosomiasis is a debilitating parasitic disease caused by intravascular flatworms called schistosomes (blood flukes) that affects >200 million people worldwide. Proteomic analysis has revealed the surprising presence of classical glycolytic enzymes - typically cytosolic proteins - located on the extracellular surface of the parasite tegument (skin). Immunolocalization experiments show that phosphoglycerate mutase (PGM) is widely expressed in parasite tissues and is highly expressed in the tegument. We demonstrate that live Schistosoma mansoni parasites express enzymatically active PGM on their tegumental surface. Suppression of PGM using RNA interference (RNAi) diminishes S. mansoni PGM (SmPGM) gene expression, protein levels, and surface enzyme activity. Sequence comparisons place SmPGM in the cofactor (2,3-bisphosphoglycerate)-dependent PGM (dPGM) family. We have produced recombinant SmPGM (rSmPGM) in an enzymatically active form in Escherichia coli. The Michaelis-Menten constant (Km) of rSmPGM for its glycolytic substrate (3-phosphoglycerate) is 0.85 mM ± 0.02. rSmPGM activity is inhibited by the dPGM-specific inhibitor vanadate. Here, we show that rSmPGM not only binds to plasminogen but also promotes its conversion to an active form (plasmin) in vitro. This supports the hypothesis that host-interactive tegumental proteins (such as SmPGM), by enhancing plasmin formation, may help degrade blood clots around the worms in the vascular microenvironment and thus promote parasite survival in vivo.
Title: La phosphoglycérate mutase de Schistosoma mansoni une ectoenzyme glycolytique avec un potentiel thrombolytique. Abstract: La schistosomiase est une maladie parasitaire débilitante causée par des vers plats intravasculaires appelés schistosomes qui affecte plus de 200 millions de personnes dans le monde. L'analyse protéomique a révélé la présence surprenante d'enzymes glycolytiques classiques typiquement des protéines cytosoliques situées sur la surface extracellulaire du tégument du parasite. Des expériences d'immunolocalisation montrent que la phosphoglycérate mutase (PGM) est largement exprimée dans les tissus parasitaires et fortement exprimée dans le tégument. Nous démontrons que les parasites Schistosoma mansoni vivants expriment une PGM enzymatiquement active sur leur surface tégumentaire. La suppression de la PGM à l'aide de l'interférence ARN (ARNi) diminue l'expression du gène PGM de S. mansoni (SmPGM), les niveaux de protéines et l'activité enzymatique de surface. Les comparaisons de séquences placent la SmPGM dans la famille des PGM dépendantes du cofacteur (2,3-bisphosphoglycérate) (dPGM). Nous avons produit de la SmPGM recombinante (rSmPGM) sous une forme enzymatiquement active dans Escherichia coli. La constante de Michaelis-Menten (Km) de rSmPGM pour son substrat glycolytique (3-phosphoglycérate) est de 0,85 mM ± 0,02. L'activité de la rSmPGM est inhibée par le vanadate, un inhibiteur spécifique des dPGM. Ici, nous montrons que rSmPGM non seulement se lie au plasminogène mais favorise également sa conversion en une forme active (plasmine) in vitro. Cela soutient l'hypothèse selon laquelle les protéines tégumentaires interactives avec l'hôte (telles que SmPGM), en améliorant la formation de plasmine, peuvent aider à dégrader les caillots sanguins autour des vers dans le microenvironnement vasculaire et ainsi favoriser la survie du parasite in vivo.
Asunto(s)
Schistosoma mansoni/enzimología , Esquistosomiasis , Animales , Fibrinolisina/metabolismo , Interacciones Huésped-Parásitos , Humanos , Fosfoglicerato Mutasa/genética , Fosfoglicerato Mutasa/metabolismo , Proteómica , Esquistosomiasis/parasitologíaRESUMEN
Parasitic diseases cause significant global morbidity and mortality particularly in the poorest regions of the world. Schistosomiasis, one of the most widespread neglected tropical diseases, affects more than 200 million people worldwide. Histone deacetylase (HDAC) inhibitors are prominent epigenetic drugs that are being investigated in the treatment of several diseases, including cancers and parasitic diseases. Schistosoma mansoni HDAC8 (SmHDAC8) is highly expressed in all life cycle stages of the parasite, and selective inhibition is required in order to avoid undesirable off-target effects in the host. Herein, by X-ray crystal structures of SmHDAC8-inhibitor complexes, biochemical and phenotypic studies, we found two schistosomicidal spiroindoline derivatives binding a novel site, next to Trp198, on the enzyme surface. We determined that by acting on this site, either by mutation of the Trp198 or by compound binding, a decrease in the activity of the enzyme is achieved. Remarkably, this allosteric site differs from the human counterpart; rather, it is conserved in all Schistosoma species, as well as Rhabidoptera and Trematoda classes, thus paving the way for the design of HDAC8-selective allosteric inhibitors with improved properties.
Asunto(s)
Antihelmínticos , Proteínas del Helminto , Inhibidores de Histona Desacetilasas , Histona Desacetilasas , Schistosoma mansoni , Animales , Humanos , Sitios de Unión , Proteínas del Helminto/química , Proteínas del Helminto/genética , Inhibidores de Histona Desacetilasas/farmacología , Inhibidores de Histona Desacetilasas/química , Histona Desacetilasas/química , Histona Desacetilasas/genética , Schistosoma mansoni/enzimología , Schistosoma mansoni/genética , Antihelmínticos/química , Antihelmínticos/farmacología , Cristalografía por Rayos XRESUMEN
α-galactosidase (α-GAL) and α-N-acetylgalactosaminidase (α-NAGAL) are two glycosyl hydrolases responsible for maintaining cellular homeostasis by regulating glycan substrates on proteins and lipids. Mutations in the human genes encoding either enzyme lead to neurological and neuromuscular impairments seen in both Fabry- and Schindler/Kanzaki- diseases. Here, we investigate whether the parasitic blood fluke Schistosoma mansoni, responsible for the neglected tropical disease schistosomiasis, also contains functionally important α-GAL and α-NAGAL proteins. As infection, parasite maturation and host interactions are all governed by carefully-regulated glycosylation processes, inhibiting S. mansoni's α-GAL and α-NAGAL activities could lead to the development of novel chemotherapeutics. Sequence and phylogenetic analyses of putative α-GAL/α-NAGAL protein types showed Smp_089290 to be the only S. mansoni protein to contain the functional amino acid residues necessary for α-GAL/α-NAGAL substrate cleavage. Both α-GAL and α-NAGAL enzymatic activities were higher in females compared to males (p<0.05; α-NAGAL > α-GAL), which was consistent with smp_089290's female biased expression. Spatial localisation of smp_089290 revealed accumulation in parenchymal cells, neuronal cells, and the vitellaria and mature vitellocytes of the adult schistosome. siRNA-mediated knockdown (>90%) of smp_089290 in adult worms significantly inhibited α-NAGAL activity when compared to control worms (siLuc treated males, p<0.01; siLuc treated females, p<0.05). No significant reductions in α-GAL activities were observed in the same extracts. Despite this, decreases in α-NAGAL activities correlated with a significant inhibition in adult worm motility as well as in egg production. Programmed CRISPR/Cas9 editing of smp_089290 in adult worms confirmed the egg reduction phenotype. Based on these results, Smp_089290 was determined to act predominantly as an α-NAGAL (hereafter termed SmNAGAL) in schistosome parasites where it participates in coordinating movement and oviposition processes. Further characterisation of SmNAGAL and other functionally important glycosyl hydrolases may lead to the development of a novel anthelmintic class of compounds.
Asunto(s)
Proteínas del Helminto/fisiología , Movimiento/fisiología , Oviposición/fisiología , Schistosoma mansoni/enzimología , alfa-N-Acetilgalactosaminidasa/fisiología , Animales , Femenino , Masculino , Ratones , Esquistosomiasis mansoniRESUMEN
The twister ribozyme is widely distributed over numerous organisms and is especially abundant in Schistosoma mansoni, but has no confirmed biological function. Of the 17 non-LTR retrotransposons known in S. mansoni, none have thus far been associated with ribozymes. Here we report the identification of novel twister variant (T-variant) ribozymes and their function in S. mansoni non-LTR retrotransposition. We show that T-variant ribozymes are located at the 5' end of Perere-3 non-LTR retrotransposons in the S. mansoni genome. T-variant ribozymes were demonstrated to be catalytically active in vitro. In reporter constructs, T-variants were shown to cleave in vivo, and cleavage of T-variants was sufficient for the translation of downstream reporter genes. Our analysis shows that the T-variants and Perere-3 are transcribed together. Target site duplications (TSDs); markers of target-primed reverse transcription (TPRT) and footmarks of retrotransposition, are located adjacent to the T-variant cleavage site and suggest that T-variant cleavage has taken place inS. mansoni. Sequence heterogeneity in the TSDs indicates that Perere-3 retrotransposition is not site-specific. The TSD sequences contribute to the 5' end of the terminal ribozyme helix (P1 stem). Based on these results we conclude that T-variants have a functional role in Perere-3 retrotransposition.
Asunto(s)
ARN Catalítico/química , Retroelementos , Schistosoma mansoni/genética , Animales , Secuencia de Bases , Genoma de los Helmintos , ARN Catalítico/metabolismo , Schistosoma mansoni/enzimologíaRESUMEN
Several Schistosoma species cause Schistosomiasis, an endemic disease in 78 countries that is ranked second amongst the parasitic diseases in terms of its socioeconomic impact and human health importance. The drug recommended for treatment by the WHO is praziquantel (PZQ), but there are concerns associated with PZQ, such as the lack of information about its exact mechanism of action, its high price, its effectiveness - which is limited to the parasite's adult form - and reports of resistance. The parasites lack the de novo purine pathway, rendering them dependent on the purine salvage pathway or host purine bases for nucleotide synthesis. Thus, the Schistosoma purine salvage pathway is an attractive target for the development of necessary and selective new drugs. In this study, the purine nucleotide phosphorylase II (PNP2), a new isoform of PNP1, was submitted to a high-throughput fragment-based hit discovery using a crystallographic screening strategy. PNP2 was crystallized and crystals were soaked with 827 fragments, a subset of the Maybridge 1000 library. X-ray diffraction data was collected and structures were solved. Out of 827-screened fragments we have obtained a total of 19 fragments that show binding to PNP2. Fourteen of these fragments bind to the active site of PNP2, while five were observed in three other sites. Here we present the first fragment screening against PNP2.
Asunto(s)
Descubrimiento de Drogas/métodos , Purina-Nucleósido Fosforilasa/química , Purina-Nucleósido Fosforilasa/metabolismo , Piridinas/metabolismo , Pirimidinas/metabolismo , Schistosoma mansoni/enzimología , Tiazoles/metabolismo , Animales , Dominio Catalítico , Cristalización , Cristalografía por Rayos X/métodos , Dimetilsulfóxido/farmacología , Evaluación Preclínica de Medicamentos/métodos , Modelos Moleculares , Conformación Proteica en Hélice alfa , Purina-Nucleósido Fosforilasa/genética , Esquistosomiasis mansoni/tratamiento farmacológico , Esquistosomiasis mansoni/parasitologíaRESUMEN
Schistosomiasis is a major neglected parasitic disease that affects more than 265 million people worldwide and for which the control strategy consists of mass treatment with the only available drug, praziquantel. In this study, we chemically optimized our previously reported benzhydroxamate-based inhibitors of Schistosoma mansoni histone deacetylase 8 (smHDAC8). Crystallographic analysis provided insights into the inhibition mode of smHDAC8 activity by the highly potent inhibitor 5o. Structure-based optimization of the novel inhibitors was carried out using the available crystal structures as well as docking studies on smHDAC8. The compounds were evaluated in screens for inhibitory activity against schistosome and human HDACs (hHDAC). The in vitro and docking results were used for detailed structure activity relationships. The synthesized compounds were further investigated for their lethality against the schistosome larval stage using a fluorescence-based assay. The most promising inhibitor 5o showed significant dose-dependent killing of the schistosome larvae and markedly impaired egg laying of adult worm pairs maintained in culture.
Asunto(s)
Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Esquistosomiasis/tratamiento farmacológico , Animales , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Células HEK293 , Inhibidores de Histona Desacetilasas/síntesis química , Inhibidores de Histona Desacetilasas/química , Humanos , Modelos Moleculares , Estructura Molecular , Proteínas Recombinantes/metabolismo , Schistosoma mansoni/enzimología , Relación Estructura-ActividadRESUMEN
Tegumental carbonic anhydrase from the worm Schistosoma mansoni (SmCA) is considered a new anti-parasitic target because suppressing its expression interferes with schistosome metabolism and virulence. Here, we present the inhibition profiles of selenoureido compounds on recombinant SmCA and resolution of the first X-ray crystal structures of SmCA in adduct with a selection of such inhibitors. The key molecular features of such compounds in adduct with SmCA were obtained and compared to the human isoform hCA II, in order to understand the main structural factors responsible for enzymatic affinity and selectivity. Compounds that more specifically inhibited the schistosome versus human enzymes were identified. The results expand current knowledge in the field and pave the way for the development of more potent antiparasitic agents in the near future.
Asunto(s)
Derivados del Benceno/farmacología , Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/metabolismo , Compuestos de Organoselenio/farmacología , Schistosoma mansoni/enzimología , Sulfonamidas/farmacología , Animales , Derivados del Benceno/síntesis química , Derivados del Benceno/química , Inhibidores de Anhidrasa Carbónica/síntesis química , Inhibidores de Anhidrasa Carbónica/química , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Modelos Moleculares , Estructura Molecular , Compuestos de Organoselenio/síntesis química , Compuestos de Organoselenio/química , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/químicaRESUMEN
BACKGROUND: The blood flukes of genus Schistosoma are the causative agent of schistosomiasis, a parasitic disease that infects more than 200 million people worldwide. Proteases of schistosomes are involved in critical steps of host-parasite interactions and are promising therapeutic targets. We recently identified and characterized a group of S1 family Schistosoma mansoni serine proteases, including SmSP1 to SmSP5. Expression levels of some SmSPs in S. mansoni are low, and by standard genome sequencing technologies they are marginally detectable at the method threshold levels. Here, we report their spatial gene expression patterns in adult S. mansoni by the high-sensitivity localization assay. METHODOLOGY: Highly sensitive fluorescence in situ RNA hybridization (FISH) was modified and used for the localization of mRNAs encoding individual SmSP proteases (including low-expressed SmSPs) in tissues of adult worms. High sensitivity was obtained due to specifically prepared tissue and probes in combination with the employment of a signal amplification approach. The assay method was validated by detecting the expression patterns of a set of relevant reference genes including SmCB1, SmPOP, SmTSP-2, and Sm29 with localization formerly determined by other techniques. RESULTS: FISH analysis revealed interesting expression patterns of SmSPs distributed in multiple tissues of S. mansoni adults. The expression patterns of individual SmSPs were distinct but in part overlapping and were consistent with existing transcriptome sequencing data. The exception were genes with significantly low expression, which were also localized in tissues where they had not previously been detected by RNA sequencing methods. In general, SmSPs were found in various tissues including reproductive organs, parenchymal cells, esophagus, and the tegumental surface. CONCLUSIONS: The FISH-based assay provided spatial information about the expression of five SmSPs in adult S. mansoni females and males. This highly sensitive method allowed visualization of low-abundantly expressed genes that are below the detection limits of standard in situ hybridization or by RNA sequencing. Thus, this technical approach turned out to be suitable for sensitive localization studies and may also be applicable for other trematodes. The results suggest that SmSPs may play roles in diverse processes of the parasite. Certain SmSPs expressed at the surface may be involved in host-parasite interactions.
Asunto(s)
Expresión Génica , Proteínas del Helminto/genética , Hibridación Fluorescente in Situ/métodos , ARN/metabolismo , Schistosoma mansoni/enzimología , Schistosoma mansoni/genética , Serina Proteasas/genética , Animales , Femenino , Perfilación de la Expresión Génica , Hibridación Fluorescente in Situ/normas , MasculinoRESUMEN
Histone-modifying proteins have been identified as promising targets to treat several diseases including cancer and parasitic ailments. In silico methods have been incorporated within a variety of drug discovery programs to facilitate the identification and development of novel lead compounds. In this study, we explore the binding modes of a series of benzhydroxamates derivatives developed as histone deacetylase inhibitors of Schistosoma mansoni histone deacetylase (smHDAC) using molecular docking and binding free energy (BFE) calculations. The developed docking protocol was able to correctly reproduce the experimentally established binding modes of resolved smHDAC8-inhibitor complexes. However, as has been reported in former studies, the obtained docking scores weakly correlate with the experimentally determined activity of the studied inhibitors. Thus, the obtained docking poses were refined and rescored using the Amber software. From the computed protein-inhibitor BFE, different quantitative structure-activity relationship (QSAR) models could be developed and validated using several cross-validation techniques. Some of the generated QSAR models with good correlation could explain up to ~73% variance in activity within the studied training set molecules. The best performing models were subsequently tested on an external test set of newly designed and synthesized analogs. In vitro testing showed a good correlation between the predicted and experimentally observed IC50 values. Thus, the generated models can be considered as interesting tools for the identification of novel smHDAC8 inhibitors.
Asunto(s)
Proteínas del Helminto/química , Inhibidores de Histona Desacetilasas/química , Histona Desacetilasas/química , Relación Estructura-Actividad Cuantitativa , Schistosoma mansoni/enzimología , Animales , Relación Dosis-Respuesta a Droga , Proteínas del Helminto/antagonistas & inhibidores , Inhibidores de Histona Desacetilasas/farmacología , Conformación Molecular , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Unión Proteica , Reproducibilidad de los ResultadosRESUMEN
The enzyme Glyceraldehyde-3-Phosphate Dehydrogenase from Schistosoma mansoni (SmGAPDH) is characterized as a therapeutical target for schistosomiasis. In this context, we report here the experimental structure, structural analyses and comparisons of SmGAPDH, the first one from a Platyhelminth. The enzyme was expressed, purified and assayed for crystallization, what allowed the obtainment of crystals of sufficient quality to collect X-ray diffraction data up to 2.51 Å resolution. SmGAPDH is the only GAPDH to present the sequence NNR (its residues 114-116) which leads to (especially R116) a hydrogen bond network that possibly reflects on the flexibility of residues to interact with the adenine part of NAD+, speculated to be important for differential drug design.
Asunto(s)
Gliceraldehído-3-Fosfato Deshidrogenasas/química , Proteínas del Helminto/química , Modelos Moleculares , Schistosoma mansoni/enzimología , Animales , Cristalografía por Rayos XRESUMEN
Schistosomiasis is a neglected tropical disease that affects more than 250 million people worldwide. The only drug available for its treatment undergoes first-pass hepatic metabolism and is not capable of preventing reinfection, which makes the search of new therapies urgently needed. Due to the essential role of fumarases in metabolism, these enzymes represent potential targets for developing novel schistosomiasis treatments. Here, we evaluate the expression profiles for class I and class II fumarases from Schistosoma mansoni (SmFHI and SmFHII, respectively), and report the complete characterization of SmFHII. The first SmFHII structure in complex with L-malate was determined at 1.85 Å resolution. The significant thermoshift observed for SmFHII in the presence of identified ligands makes the differential scanning fluorimetry an adequate technique for ligand screening. A complete kinetic characterization of SmFHII was performed, and comparison with the human fumarase (HsFH) revealed differences regarding the turnover number (kcat). Structural characterization allowed us to identify differences between SmFHII and HsFH that could be explored to design new selective inhibitors. This work represents the very first step towards validate the fumarases as drug targets to treat schistosomiasis. Our results provide the structural basis to rational search for selective ligands.
Asunto(s)
Fumarato Hidratasa/farmacología , Schistosoma mansoni/enzimología , Esquistosomiasis mansoni/tratamiento farmacológico , Animales , Femenino , Fumarato Hidratasa/metabolismo , Cinética , Ligandos , Masculino , Ratones , Schistosoma mansoni/metabolismo , Esquistosomiasis/tratamiento farmacológico , Esquistosomiasis/metabolismo , Esquistosomiasis mansoni/metabolismoRESUMEN
Type 2 immunity plays an essential role in the maintenance of metabolic homeostasis and its disruption during obesity promotes meta-inflammation and insulin resistance. Infection with the helminth parasite Schistosoma mansoni and treatment with its soluble egg antigens (SEA) induce a type 2 immune response in metabolic organs and improve insulin sensitivity and glucose tolerance in obese mice, yet, a causal relationship remains unproven. Here, we investigated the effects and underlying mechanisms of the T2 ribonuclease omega-1 (ω1), one of the major S mansoni immunomodulatory glycoproteins, on metabolic homeostasis. We show that treatment of obese mice with plant-produced recombinant ω1, harboring similar glycan motifs as present on the native molecule, decreased body fat mass, and improved systemic insulin sensitivity and glucose tolerance in a time- and dose-dependent manner. This effect was associated with an increase in white adipose tissue (WAT) type 2 T helper cells, eosinophils, and alternatively activated macrophages, without affecting type 2 innate lymphoid cells. In contrast to SEA, the metabolic effects of ω1 were still observed in obese STAT6-deficient mice with impaired type 2 immunity, indicating that its metabolic effects are independent of the type 2 immune response. Instead, we found that ω1 inhibited food intake, without affecting locomotor activity, WAT thermogenic capacity or whole-body energy expenditure, an effect also occurring in leptin receptor-deficient obese and hyperphagic db/db mice. Altogether, we demonstrate that while the helminth glycoprotein ω1 can induce type 2 immunity, it improves whole-body metabolic homeostasis in obese mice by inhibiting food intake via a STAT6-independent mechanism.
Asunto(s)
Ingestión de Alimentos , Endorribonucleasas/uso terapéutico , Glicoproteínas/uso terapéutico , Proteínas del Helminto/uso terapéutico , Obesidad/tratamiento farmacológico , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Animales , Células Cultivadas , Endorribonucleasas/farmacología , Glicoproteínas/farmacología , Proteínas del Helminto/farmacología , Locomoción , Macrófagos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Schistosoma mansoni/enzimología , Linfocitos T Colaboradores-Inductores/efectos de los fármacos , Termogénesis , Nicotiana/genética , Nicotiana/metabolismoRESUMEN
CRISPR/Cas9-mediated genome editing shows cogent potential for the genetic modification of helminth parasites. We report successful gene knock-in (KI) into the genome of the egg of Schistosoma mansoni by combining CRISPR/Cas9 with single-stranded oligodeoxynucleotides (ssODNs). We edited the acetylcholinesterase (AChE) gene of S. mansoni targeting two guide RNAs (gRNAs), X5 and X7, located on exon 5 and exon 7 of Smp_154600, respectively. Eggs recovered from livers of experimentally infected mice were transfected by electroporation with a CRISPR/Cas9-vector encoding gRNA X5 or X7 combining with/ without a ssODN donor. Next generation sequencing analysis of reads of amplicon libraries spanning targeted regions revealed that the major modifications induced by CRISPR/Cas9 in the eggs were generated by homology directed repair (HDR). Furthermore, soluble egg antigen from AChE-edited eggs exhibited markedly reduced AChE activity, indicative that programed Cas9 cleavage mutated the AChE gene. Following injection of AChE-edited schistosome eggs into the tail veins of mice, an significantly enhanced Th2 response involving IL-4, -5, -10, and-13 was detected in lung cells and splenocytes in mice injected with X5-KI eggs in comparison to control mice injected with unmutated eggs. A Th2-predominant response, with increased levels of IL-4, -13, and GATA3, also was induced by X5 KI eggs in small intestine-draining mesenteric lymph node cells when the gene-edited eggs were introduced into the subserosa of the ileum of the mice. These findings confirmed the potential and the utility of CRISPR/Cas9-mediated genome editing for functional genomics in schistosomes.
Asunto(s)
Acetilcolinesterasa/metabolismo , Sistemas CRISPR-Cas , Edición Génica , Proteínas del Helminto/metabolismo , Schistosoma mansoni/enzimología , Esquistosomiasis mansoni/metabolismo , Acetilcolinesterasa/genética , Animales , Femenino , Proteínas del Helminto/genética , Ratones , Schistosoma mansoni/genética , Esquistosomiasis mansoni/genéticaRESUMEN
Schistosomiasis is a serious public health problem, prevalent in tropical and subtropical areas, especially in poor communities without access to safe drinking water and adequate sanitation. Transmission has been reported in 78 countries, and its control depends on a single drug, praziquantel, which has been used over the past 30 years. Our work is focused on exploiting target-based drug discovery strategies to develop new therapeutics to treat schistosomiasis. In particular, we are interested in evaluating the enzyme dihydroorotate dehydrogenase (DHODH) as a drug target. DHODH is a flavoenzyme that catalyzes the stereospecific oxidation of (S)-dihydroorotate (DHO) to orotate during the fourth and only redox step of the de novo pyrimidine nucleotide biosynthetic pathway. Previously, we identified atovaquone, used in the treatment of malaria, and its analogues, as potent and selective inhibitors against Schistosoma mansoni DHODH (SmDHODH). In the present article, we report the first crystal structure of SmDHODH in complex with the atovaquone analogue inhibitor 2-((4-fluorophenyl)amino)-3-hydroxynaphthalene-1,4-dione (QLA). We discuss three major findings: (a) the open conformation of the active site loop and the unveiling of a novel transient druggable pocket for class 2 DHODHs; (b) the presence of a protuberant domain, only present in Schistosoma spp DHODHs, that was found to control and modulate the dynamics of the inhibitor binding site; (c) a detailed description of an unexpected binding mode for the atovaquone analogue to SmDHODH. Our findings contribute to the understanding of the catalytic mechanism performed by class 2 DHODHs and provide the molecular basis for structure-guided design of SmDHODH inhibitors. DATABASE: The structural data are available in Protein Data Bank (PDB) database under the accession code number 6UY4.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Proteínas del Helminto/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Schistosoma mansoni/enzimología , Esquistosomiasis mansoni/prevención & control , Secuencia de Aminoácidos , Animales , Atovacuona/análogos & derivados , Atovacuona/farmacología , Biocatálisis/efectos de los fármacos , Dominio Catalítico , Dicroismo Circular , Cristalografía por Rayos X , Dihidroorotato Deshidrogenasa , Inhibidores Enzimáticos/química , Proteínas del Helminto/química , Proteínas del Helminto/genética , Humanos , Estructura Molecular , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Conformación Proteica , Schistosoma mansoni/efectos de los fármacos , Schistosoma mansoni/genética , Esquistosomiasis mansoni/parasitología , Homología de Secuencia de AminoácidoRESUMEN
Schistosomiasis threatens 800 million people worldwide. Chronic pathology manifests as hepatosplenomegaly, and intestinal schistosomiasis caused by Schistosoma mansoni can lead to liver fibrosis, cirrhosis, and blood in the stool. To assist the only FDA-approved drug, praziquantel, in parasite elimination, the development of a vaccine would be of high value. S. mansoni Cathepsin B (SmCB) is a well-documented vaccine target for intestinal schistosomiasis. Herein, we test the increased efficacy and immunogenicity of SmCB when combined with sulfated lactosyl archaeol (SLA) archaeosomes or AddaVax™ (a squalene based oil-in-water emulsion). Both vaccine formulations resulted in robust humoral and cell mediated immune responses. Impressively, both formulations were able to reduce parasite burden greater than 40% (WHO standard), with AddaVax™ reaching 86.8%. Additionally, SmCB with both adjuvants were able to reduce granuloma size and the amount of larval parasite hatched from feces, which would reduce transmission. Our data support SmCB as a target for S. mansoni vaccination; especially when used in an adjuvanted formulation.