RESUMEN
Cestodes are platyhelminth parasites with a wide range of hosts that cause neglected diseases. Neurotransmitter signaling is of critical importance for these parasites which lack circulatory, respiratory and digestive systems. For example, serotonin (5-HT) and serotonergic G-protein coupled receptors (5-HT GPCRs) play major roles in cestode motility, development and reproduction. In previous work, we deorphanized a group of 5-HT7 type GPCRs from cestodes. However, little is known about another type of 5-HT GPCR, the 5-HT1 clade, which has been studied in several invertebrate phyla but not in platyhelminthes. Three putative 5-HT GPCRs from Echinococcus canadensis, Mesocestoides vogae (syn. M. corti) and Hymenolepis microstoma were cloned, sequenced and bioinformatically analyzed. Evidence grouped these new sequences within the 5-HT1 clade of GPCRs but differences in highly conserved GPCR motifs were observed. Transcriptomic analysis, heterologous expression and immunolocalization studies were performed to characterize the E. canadensis receptor, called Eca-5-HT1a. Functional heterologous expression studies showed that Eca-5-HT1a is highly specific for serotonin. 5-Methoxytryptamine and α-methylserotonin, both known 5-HT GPCR agonists, give stimulatory responses whereas methysergide, a known 5-HT GPCR ligand, give an antagonist response in Eca-5-HT1a. Mutants obtained by the substitution of key predicted residues resulted in severe impairment of receptor activity, confirming that indeed, these residues have important roles in receptor function. Immunolocalization studies on the protoscolex stage from E. canadensis, showed that Eca-5-HT1a is localized in branched fibers which correspond to the nervous system of the parasite. The patterns of immunoreactive fibers for Eca-5-HT1a and for serotonin were intimately intertwined but not identical, suggesting that they are two separate groups of fibers. These data provide the first functional, pharmacological and localization report of a serotonergic receptor that putatively belongs to the 5-HT1 type of GPCRs in cestodes. The serotonergic GPCR characterized here may represent a new target for antiparasitic intervention.
Asunto(s)
Cestodos/metabolismo , Proteínas del Helminto/metabolismo , Sistema Nervioso/metabolismo , Receptores de Serotonina 5-HT1/metabolismo , Secuencia de Aminoácidos , Animales , Echinococcus/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas del Helminto/química , Proteínas del Helminto/genética , Humanos , Hymenolepis/metabolismo , Receptores de Serotonina 5-HT1/química , Receptores de Serotonina 5-HT1/genética , Alineación de Secuencia , Antagonistas de la Serotonina/farmacología , Agonistas de Receptores de Serotonina/farmacologíaRESUMEN
Cestode parasites cause neglected diseases, such as echinococcosis and cysticercosis, which represent a significant problem in human and animal health. Benzimidazoles and praziquantel are the only available drugs for chemotherapy and it is therefore important to identify new alternative drugs against cestode parasites. Histone deacetylases (HDACs) are validated drug targets for the treatment of cancer and other diseases, including neglected diseases. However, knowledge of HDACs in cestodes is very scarce. In this work, we investigated cestode HDACs as potential drug targets to develop new therapies against neglected diseases caused by cestodes. Here we showed the full repertoire of HDAC coding genes in several members of the class Cestoda. Between 6 and 7 zinc-dependent HDAC coding genes were identified in the genomes of species from Echinococcus, Taenia, Mesocestoides and Hymenolepis genera. We classified them as Class I and II HDACs and analyzed their transcriptional expression levels throughout developmental stages of Echinococcus spp. We confirmed for the first time the complete HDAC8 nucleotide sequences from Echinococcus canadensis G7 and Mesocestoides corti. Homology models for these proteins showed particular structural features which differentiate them from HDAC8 from Homo sapiens. Furthermore, we showed that Trichostatin A (TSA), a pan-HDAC inhibitor, decreases the viability of M. corti, alters its tegument and morphology and produces an increment of the total amount of acetylated proteins, including acetylated histone H4. These results suggest that HDAC from cestodes are functional and might play important roles on survival and development. The particular structural features observed in cestode HDAC8 proteins suggest that these enzymes could be selectively targeted. This report provides the basis for further studies on cestode HDAC enzymes and for discovery of new HDAC inhibitors for the treatment of neglected diseases caused by cestode parasites.
Asunto(s)
Cestodos/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Enfermedades Desatendidas/tratamiento farmacológico , Enfermedades Desatendidas/parasitología , Animales , Cestodos/enzimología , Infecciones por Cestodos/tratamiento farmacológico , Femenino , Histonas/metabolismo , Ácidos Hidroxámicos/farmacología , Ratones , Ratones Endogámicos BALB C , Ratas , Ratas WistarRESUMEN
The prenylated flavanone 2'-4'-dihidroxy-5'-(1" '-dimethylallyl)-6-prenylpinocembrin) (6PP), isolated from the roots of Dalea elegans, shows antimicrobial activity. The aim of this study was to evaluate mitochondrial toxicity and antioxidant properties of 6PP. Addition of micromolar concentrations of 6PP to rat liver mitochondria, stimulated O2 uptake in state 4 and inhibited it in state 3 when malate-glutamate was the respiratory substrate, and inhibited O2 uptake in state 3 when succinate was the substrate. Highest concentration of 6PP also inhibited O2 uptake in state 4 in the latter case; in both conditions, respiratory control index values were decreased. This flavanone collapsed the mitochondrial membrane potential in a concentration-dependent manner. 6PP also inhibited F0F1-ATPase activity in coupled mitochondria and in submitochondrial particles. In the latter, this compound also inhibited NADH oxidase and succinate dehydrogenase activities. HEp-2 cells were incubated for 24 h with 6PP in presence or absence of 0.5% albumin. As measured by reduction of the mitochondrial-related probe MTT, in the albumin-free condition, 6PP was cytotoxic in a concentration-dependent manner; on the other hand, albumin decreased 6PP effect. In addition, in rat liver microsomes 6PP: (1) inhibited the enzymatic lipid peroxidation, (2) exhibited significant scavenging activity, measured by DPPH reduction assay and (3) demonstrated significant antioxidant activity by decreasing the reduction of Mo(VI) to Mo(V). We suggest that 6PP impairs the hepatic energy metabolism by acting as mitochondrial uncoupler and by inhibiting enzymatic activities linked to the respiratory chain. 6PP also exerts both antioxidant and antiradical activities. Due to its cytotoxicity, this molecule, and its future structure developments, can be considered as a potentially promising therapeutic agent, for instance in cancer chemotherapy.
Asunto(s)
Antineoplásicos/farmacología , Antioxidantes/farmacología , Inhibidores Enzimáticos/farmacología , Fabaceae/química , Flavanonas/farmacología , Flavonoides/farmacología , Mitocondrias Hepáticas/efectos de los fármacos , Animales , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Antioxidantes/química , Antioxidantes/aislamiento & purificación , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/aislamiento & purificación , Flavanonas/química , Flavanonas/aislamiento & purificación , Flavonoides/química , Flavonoides/aislamiento & purificación , Humanos , Peroxidación de Lípido/efectos de los fármacos , Masculino , Mitocondrias Hepáticas/metabolismo , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/metabolismo , Estructura Molecular , Complejos Multienzimáticos/antagonistas & inhibidores , NADH NADPH Oxidorreductasas/antagonistas & inhibidores , Oxígeno/antagonistas & inhibidores , Oxígeno/metabolismo , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/farmacología , Raíces de Plantas/química , Prenilación , ATPasas de Translocación de Protón/antagonistas & inhibidores , Ratas , Ratas Wistar , Succinato Deshidrogenasa/antagonistas & inhibidores , Células Tumorales CultivadasRESUMEN
BACKGROUND: Cestodes are a diverse group of parasites, some of them being agents of neglected diseases. In cestodes, little is known about the functional properties of G protein coupled receptors (GPCRs) which have proved to be highly druggable targets in other organisms. Notably, serotoninergic G-protein coupled receptors (5-HT GPCRs) play major roles in key functions like movement, development and reproduction in parasites. METHODOLOGY/PRINCIPAL FINDINGS: Three 5-HT GPCRs from Echinococcus granulosus and Mesocestoides corti were cloned, sequenced, bioinformatically analyzed and functionally characterized. Multiple sequence alignment with other GPCRs showed the presence of seven transmembrane segments and conserved motifs but interesting differences were also observed. Phylogenetic analysis grouped these new sequences within the 5-HT7 clade of GPCRs. Molecular modeling showed a striking resemblance in the spatial localization of key residues with their mammalian counterparts. Expression analysis using available RNAseq data showed that both E. granulosus sequences are expressed in larval and adult stages. Localization studies performed in E. granulosus larvae with a fluorescent probe produced a punctiform pattern concentrated in suckers. E. granulosus and M. corti larvae showed an increase in motility in response to serotonin. Heterologous expression revealed elevated levels of cAMP production in response to 5-HT and two of the GPCRs showed extremely high sensitivity to 5-HT (picomolar range). While each of these GPCRs was activated by 5-HT, they exhibit distinct pharmacological properties (5-HT sensitivity, differential responsiveness to ligands). CONCLUSIONS/SIGNIFICANCE: These data provide the first functional report of GPCRs in parasitic cestodes. The serotoninergic GPCRs characterized here may represent novel druggable targets for antiparasitic intervention.
Asunto(s)
Cestodos/fisiología , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Secuencias de Aminoácidos , Animales , Cestodos/genética , Cestodos/crecimiento & desarrollo , Infecciones por Cestodos/tratamiento farmacológico , Clonación Molecular , Biología Computacional , Echinococcus granulosus/genética , Echinococcus granulosus/fisiología , Larva/fisiología , Mesocestoides/genética , Mesocestoides/crecimiento & desarrollo , Mesocestoides/fisiología , Modelos Moleculares , Filogenia , Conformación Proteica , Receptores Acoplados a Proteínas G/genética , Alineación de Secuencia , Serotonina/farmacologíaRESUMEN
In this work, the synthesis of a series of 2-arylazoimidazole derivatives 6-20 has been achieved through the reaction of imidazole with aryldiazonium salts, followed by ultrasound-assisted alkylation. This approach has important advantages including higher yield, shorter reaction times and milder reaction conditions. The structures of the compounds obtained were determined by MS, IR; and 1H and 13C NMR. The anti-Trypanosoma cruzi activity of the 15 compounds obtained was evaluated. Two compounds with piperidino substituents in the carboxamide moiety proved to be effective inhibitors of epimastigote proliferation, obtaining inhibition values comparable to those achieved with the reference drug Benznidazole. Besides, these compounds displayed low cytotoxicity on mammalian cells. In vivo, both compounds protected mice against a challenge with a lethal Trypanosoma cruzi strain. These results allow us to propose 2-arylazoimidazoles as lead compounds for the design of novel drugs to treat Chagas' disease.
Asunto(s)
Enfermedad de Chagas/tratamiento farmacológico , Imidazoles/química , Imidazoles/uso terapéutico , Tripanocidas/química , Tripanocidas/uso terapéutico , Trypanosoma cruzi/efectos de los fármacos , Alquilación , Animales , Línea Celular , Enfermedad de Chagas/parasitología , Humanos , Imidazoles/farmacología , Masculino , Ratones , Ratones Endogámicos BALB C , Sonicación , Tripanocidas/farmacología , Trypanosoma cruzi/citologíaRESUMEN
BACKGROUND: Cells of monocyte/macrophage lineage are one of the major targets of HIV-1 infection and serve as reservoirs for viral persistence in vivo. These cells are also the target of the protozoa Trypanosoma cruzi, the causative agent of Chagas disease, being one of the most important endemic protozoonoses in Latin America. It has been demonstrated in vitro that co-infection with other pathogens can modulate HIV replication. However, no studies at cellular level have suggested an interaction between T. cruzi and HIV-1 to date. METHODOLOGY/PRINCIPAL FINDINGS: By using a fully replicative wild-type virus, our study showed that T. cruzi inhibits HIV-1 antigen production by nearly 100% (p<0.001) in monocyte-derived macrophages (MDM). In different infection schemes with luciferase-reporter VSV-G or BaL pseudotyped HIV-1 and trypomastigotes, T. cruzi induced a significant reduction of luciferase level for both pseudotypes in all the infection schemes (p<0.001), T. cruzi-HIV (>99%) being stronger than HIV-T. cruzi (approximately 90% for BaL and approximately 85% for VSV-G) infection. In MDM with established HIV-1 infection, T. cruzi significantly inhibited luciferate activity (p<0.01). By quantifying R-U5 and U5-gag transcripts by real time PCR, our study showed the expression of both transcripts significantly diminished in the presence of trypomastigotes (p<0.05). Thus, T. cruzi inhibits viral post-integration steps, early post-entry steps and entry into MDM. Trypomastigotes also caused a approximately 60-70% decrease of surface CCR5 expression on MDM. Multiplication of T. cruzi inside the MDM does not seem to be required for inhibiting HIV-1 replication since soluble factors secreted by trypomastigotes have shown similar effects. Moreover, the major parasite antigen cruzipain, which is secreted by the trypomastigote form, was able to inhibit viral production in MDM over 90% (p<0.01). CONCLUSIONS/SIGNIFICANCE: Our study showed that T. cruzi inhibits HIV-1 replication at several replication stages in macrophages, a major cell target for both pathogens.
Asunto(s)
VIH-1/fisiología , Macrófagos/parasitología , Macrófagos/virología , Monocitos/citología , Parásitos/fisiología , Trypanosoma cruzi/fisiología , Replicación Viral/fisiología , Animales , Antígenos de Protozoos/metabolismo , Antígenos CD4/metabolismo , Cisteína Endopeptidasas/metabolismo , Humanos , Ratones , Especificidad de Órganos , Parásitos/inmunología , Proteínas Protozoarias , Receptores CCR5/metabolismo , Solubilidad , Trypanosoma cruzi/inmunología , Ensamble de Virus/fisiología , Integración Viral/fisiología , Internalización del VirusRESUMEN
Se evaluó por inoculación en ratón la actividad tripanocida del Clorhidrati de Maprotilina comparativamente con la presentada por el Violeta de Genciana cuando estos compuestos se adicionaron a muestras de sangre conteniendo bajas concentraciones de parásitos. Ambas drogas presentaon actividad tripanocida cuando se las utilizó en concentraciónes del orden 10**-3M. Sin embargo, aun empleando esta concentración pudo detectarse esporádicamente infección en alguno de los animales inyectados con muestras de sangre conteniendo 10 o 100 tripomastigotes, despues de haber sido incubadas 24 h a 4-C con uno de ambos compuestos. Debido a la baja solubilidad del Clorhidrato de Maprotilina el presente estudio se realizó con muestras de sangre diluidas al medio siendo imposible evitar esta condición para la concentración 10**-3M de este compuesto. Estos resultados descartan el uso de clorhidrato de Maprotilina en bancos de sangre y previenen sobre la posibilidad eventual de transmitir infección por Trypanosoma cruzi aun con sangre tratada con Violeta de Genciana. De los 3 métodos utilizados para evaluar viabilidad parasitaria remanente en las muestras de sangre químicamente tratadas, el microhematocrito fue el más sensible