RESUMEN
In the midst of numerous setbacks that beclouds the fight against leishmaniasis; a neglected tropical disease, the search for new chemotherapeutics against this disease is of utmost importance. Leishmaniasis is a disease closely associated with poverty and endemic in Africa, Asia, southern Europe and the Americas. It is caused by parasites of the genus Leishmania and transmitted by a sandfly vector. In this study, we evaluated the antileishmanial potency of eighteen pathogen box compounds and elucidated their biosafety and possible mechanisms of action against Leishmania donovani promastigotes and amastigotes in vitro. IC50s range of 0.12±0.15 to >6.25 µg/ml and 0.13±0.004 to >6.25µg/ml were observed for the promastigotes and amastigotes, respectively. We demonstrated the ability of some of the compounds to cause cytocidal effect on the parasites, induce increased production of reactive oxygen species (ROS), disrupt the normal parasite morphology and cause the accumulation of parasites at the DNA synthesis phase of the cell cycle. We recommend a further in vivo study on these compounds to validate the findings.
Asunto(s)
Antiprotozoarios/farmacología , Ciclo Celular , Leishmania donovani/citología , Ciclo Celular/efectos de los fármacos , ADN de Cinetoplasto/metabolismo , Humanos , Concentración 50 Inhibidora , Cinética , Leishmania donovani/efectos de los fármacos , Leishmania donovani/crecimiento & desarrollo , Macrófagos/efectos de los fármacos , Macrófagos/parasitología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Oxidación-Reducción/efectos de los fármacos , Fosfatidilserinas/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The huge burden of leishmaniasis caused by the trypanosomatid protozoan parasite Leishmania is well known. This illness was included in the list of neglected tropical diseases targeted for elimination by the World Health Organization. However, the increasing evidence of resistance to existing antimonial drugs has made the eradication of the disease difficult to achieve, thus warranting the search for new drug targets. We report here studies that used computational methods to identify inhibitors of receptors from natural products. The cell division cycle-2-related kinase 12 (CRK12) receptor is a plausible drug target against Leishmania donovani. This study modelled the 3D molecular structure of the L. donovani CRK12 (LdCRK12) and screened for small molecules with potential inhibitory activity from African flora. An integrated library of 7722 African natural product-derived compounds and known inhibitors were screened against the LdCRK12 using AutoDock Vina after performing energy minimization with GROMACS 2018. Four natural products, namely sesamin (NANPDB1649), methyl ellagic acid (NANPDB1406), stylopine (NANPDB2581), and sennecicannabine (NANPDB6446) were found to be potential LdCRK12 inhibitory molecules. The molecular docking studies revealed two compounds NANPDB1406 and NANPDB2581 with binding affinities of -9.5 and -9.2 kcal/mol, respectively, against LdCRK12 which were higher than those of the known inhibitors and drugs, including GSK3186899, amphotericin B, miltefosine, and paromomycin. All the four compounds were predicted to have inhibitory constant (Ki) values ranging from 0.108 to 0.587 µM. NANPDB2581, NANPDB1649 and NANPDB1406 were also predicted as antileishmanial with Pa and Pi values of 0.415 and 0.043, 0.391 and 0.052, and 0.351 and 0.071, respectively. Molecular dynamics simulations coupled with molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) computations reinforced their good binding mechanisms. Most compounds were observed to bind in the ATP binding pocket of the kinase domain. Lys488 was predicted as a key residue critical for ligand binding in the ATP binding pocket of the LdCRK12. The molecules were pharmacologically profiled as druglike with inconsequential toxicity. The identified molecules have scaffolds that could form the backbone for fragment-based drug design of novel leishmanicides but warrant further studies to evaluate their therapeutic potential.
Asunto(s)
Antiprotozoarios/farmacología , Leishmania donovani/citología , Leishmania donovani/efectos de los fármacos , Anfotericina B/farmacología , Sitios de Unión , Ciclo Celular/efectos de los fármacos , Quinasa 9 Dependiente de la Ciclina/metabolismo , Humanos , Simulación de Dinámica Molecular , Morfolinas/farmacología , Paromomicina/farmacología , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacología , Estructura Secundaria de Proteína , Pirazoles/farmacología , Pirimidinas/farmacologíaRESUMEN
Molecular hybridization is a ligand based drug design approach is well known recent medicinal chemistry to design anti-parasitic agents. In the present study, we have designed a series of (1-phenyl-9H-pyrido [3,4-b]indol-3-yl) (4-phenylpiperazin-1-yl)methanone derivatives using molecular hybridization approach. Designed analogues were evaluated for cytotoxicity and inhibition activity against Leishmania infantum and Leishmania donovani. Among these reported analogues 7b, 7d, 7e, 7f and 7m displayed potent inhibition of both L. infantum and L. donovani. Compounds 7i and 7k exhibited selective potent inhibition of L. donovani. Especially, compounds 7e and 7k showed most potent anti-leishmanial activity against L. infantum and L. donovani respectively. Anti-leishmanial activity of these compounds is comparable with standard drugs miltefosine and pentamidine. SAR studies revealed that, electron donating group substitution on phenyl ring recommended for potent anti-leishmanial activity.
Asunto(s)
Antineoplásicos/farmacología , Antiprotozoarios/farmacología , Carbolinas/farmacología , Diseño de Fármacos , Leishmania donovani/efectos de los fármacos , Leishmania infantum/efectos de los fármacos , Piperazinas/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Antiprotozoarios/síntesis química , Antiprotozoarios/química , Carbolinas/síntesis química , Carbolinas/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Células HeLa , Humanos , Leishmania donovani/citología , Leishmania infantum/citología , Estructura Molecular , Pruebas de Sensibilidad Parasitaria , Piperazinas/síntesis química , Piperazinas/química , Relación Estructura-ActividadRESUMEN
Visceral leishmaniasis (VL) is a life threatening infectious disease caused by Leishmania donovani. It leads to the severe immune suppression in the host defense system. Higher cytotoxicity, rigorous side effects and lower therapeutic indexes (TI) of current antileishmanial drugs have created a necessity to develop new molecules with better antileishmanial activity and high TI value. In this study, we have synthesized 36 derivatives of eugenol and screened them for their activity against promastigote and amastigote forms of L. donovani. Among the synthesized derivatives, comp.35 showed better antileishmanial activity against extra cellular promastigotes (IC50- 20.13 ± 0.91 µM) and intracellular amastigotes (EC50-4.25 ± 0.26 µM). The TI value (82.24 ± 3.77) was found to improve by 10-13 fold compared to Amphotericin B and Miltefosine respectively. Treatment with comp.35 (5 µg/ml) enhanced the nitric oxide (NO) generation, iNOS2 mRNA expression (â¼8 folds increase) and decreased the arginase-1 activity (â¼4 folds) in L. donovani infected peritoneal macrophages. Comp.35 had also increased the IL-12 (â¼6 folds) and decreased the IL-10 (â¼3 folds) mRNA expression and release in vitro. Results of in vivo studies revealed that comp.35 treatment at 25 mg/kg body weight efficiently cleared the hepatic and splenic parasite burden with enhanced Th1 response in L. donovani infected BALB/c mice. Hence, this study clearly represents comp.35, as an immunomodulatory molecule, can induce host protective immune response against visceral leishmaniasis through enhanced NO generation and Th1 response, which are essentials against this deadly disease.
Asunto(s)
Antiprotozoarios/farmacología , Eugenol/farmacología , Inmunomodulación , Leishmania donovani/efectos de los fármacos , Leishmaniasis Visceral/tratamiento farmacológico , Animales , Antiprotozoarios/síntesis química , Antiprotozoarios/química , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Eugenol/síntesis química , Eugenol/química , Leishmania donovani/citología , Macrófagos/efectos de los fármacos , Macrófagos/parasitología , Masculino , Ratones , Ratones Endogámicos BALB C , Estructura Molecular , Pruebas de Sensibilidad Parasitaria , Células RAW 264.7 , Relación Estructura-ActividadRESUMEN
Metalloprotease gp63 (Leishmania donovani gp63 (Ldgp63)) is a critical virulence factor secreted by Leishmania However, how newly synthesized Ldgp63 exits the endoplasmic reticulum (ER) and is secreted by this parasite is unknown. Here, we cloned, expressed, and characterized the GTPase LdSar1 and other COPII components like LdSec23, LdSec24, LdSec13, and LdSec31 from Leishmania to understand their role in ER exit of Ldgp63. Using dominant-positive (LdSar1:H74L) and dominant-negative (LdSar1:T34N) mutants of LdSar1, we found that GTP-bound LdSar1 specifically binds to LdSec23, which binds, in turn, with LdSec24(1-702) to form a prebudding complex. Moreover, LdSec13 specifically interacted with His6-LdSec31(1-603), and LdSec31 bound the prebudding complex via LdSec23. Interestingly, dileucine 594/595 and valine 597 residues present in the Ldgp63 C-terminal domain were critical for binding with LdSec24(703-966), and GFP-Ldgp63L594A/L595A or GFP-Ldgp63V597S mutants failed to exit from the ER. Moreover, Ldgp63-containing COPII vesicle budding from the ER was inhibited by LdSar1:T34N in an in vitro budding assay, indicating that GTP-bound LdSar1 is required for budding of Ldgp63-containing COPII vesicles. To directly demonstrate the function of LdSar1 in Ldgp63 trafficking, we coexpressed RFP-Ldgp63 along with LdSar1:WT-GFP or LdSar1:T34N-GFP and found that LdSar1:T34N overexpression blocks Ldgp63 trafficking and secretion in Leishmania Finally, we noted significantly compromised survival of LdSar1:T34N-GFP-overexpressing transgenic parasites in macrophages. Taken together, these results indicated that Ldgp63 interacts with the COPII complex via LdSec24 for Ldgp63 ER exit and subsequent secretion.
Asunto(s)
Vesículas Cubiertas por Proteínas de Revestimiento/enzimología , GTP Fosfohidrolasas/metabolismo , Leishmania donovani/metabolismo , Macrófagos/parasitología , Metaloendopeptidasas/metabolismo , Proteínas Protozoarias/metabolismo , Factores de Virulencia/metabolismo , Sustitución de Aminoácidos , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Línea Celular Tumoral , Citosol/enzimología , Citosol/metabolismo , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/genética , Humanos , Membranas Intracelulares/enzimología , Membranas Intracelulares/metabolismo , Leishmania donovani/citología , Leishmania donovani/genética , Leishmania donovani/crecimiento & desarrollo , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Metaloendopeptidasas/química , Metaloendopeptidasas/genética , Mutagénesis Sitio-Dirigida , Mutación , Organismos Modificados Genéticamente , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Factores de Virulencia/química , Factores de Virulencia/genéticaRESUMEN
We have previously reported that the hypericin treatment caused spermidine starvation and death of Leishmania parasite. Here, we report different molecular events under spermidine starvation and potential role of spermidine in processes other than redox homeostasis of the parasite. We have analyzed changes in expression of several genes by using quantitative gene expression analysis. Further, these changes at molecular level were also confirmed by using biochemical and cellular studies. Altered expression of several genes involved in redox metabolism, hypusine modification of eIF5A, DNA repair pathway and autophagy was observed. There was decrease in Sir2RP expression after hypericin treatment and this decrease has been found to be associated with induced ROS due to hypericin treatment as it has been rescued by either trypanothione or spermidine supplementation. Translation initiation in the parasite was decreased upon spermidine starvation. We also observed increased AMPK expression upon hypericin treatment. The increase in intracellular ATP and NAD+ levels as well as decrease in Sir2RP expression of the parasite are cytoprotective mechanism towards generated ROS due to hypericin treatment possibly by inducing autophagy as indicated by increase in autophagy related gene expression and acridine orange staining. However, the autophagy needs to be established using more rigorous methodologies.
Asunto(s)
Leishmania donovani/efectos de los fármacos , Perileno/análogos & derivados , Espermidina/metabolismo , Tripanocidas/farmacología , Adenosina Trifosfato/metabolismo , Antracenos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Leishmania donovani/citología , Leishmania donovani/genética , Leishmania donovani/metabolismo , Leishmaniasis Visceral/tratamiento farmacológico , NAD/metabolismo , Perileno/farmacologíaRESUMEN
Exposure of Leishmania donovani to macrophage phagolysosome conditions (PC) (37°C and pH 5.5) led to increased intracellular cAMP and cAMP-mediated responses, which help in intra-macrophage survival pre-requisite for infectivity. In the absence of typical orthologs for G-proteins and G-protein coupled receptors, we sought to study the precise mechanisms for positive modulation of cAMP production during exposure to PC. Amongst two promastigote-stage specific membrane bound receptor adenylate cyclases (LdRAC-A and LdRAC-B), LdRAC-A appeared to function as a major cAMP generator following PC exposure. Pyrophosphate (PPi), an energy storage compound as well as a by-product of cAMP biosynthesis by adenylate cyclise, was found to be decreased following PC exposure. This may be due to microtubule and microfilament-driven translocation of acidocalcisomes near plasma membrane vicinity with concomitant increase of acidocalcisome membrane pyrophosphatase (LdV-H+PPase) and acidocalcisomal soluble pyrophosphatase (LdVSP1). Episomal over-expression and conditional silencing demonstrated regulatory role of V-H+PPase on cAMP trigger and consequent induction of resistance to macrophage-derived pro-oxidants and parasite killing. Furthermore, immunofluorescence analysis revealed possible co-localization of LdV-H+PPase and LdRAC-A during PC exposure. Collectively, these results suggest that translocation of acidocalcisome in membrane vicinity functions as a trigger for LdRAC-A-driven cAMP generation through depletion of PPi pool by LdV-H+PPase.
Asunto(s)
AMP Cíclico/metabolismo , Homeostasis , Leishmania donovani/citología , Leishmania donovani/enzimología , Macrófagos/citología , Fagosomas/metabolismo , Pirofosfatasas/metabolismo , Adenilil Ciclasas/metabolismo , Concentración de Iones de Hidrógeno , Espacio Intracelular/metabolismo , Macrófagos/metabolismo , Peróxidos/metabolismo , Transporte de Proteínas , TemperaturaRESUMEN
Histone acetyltransferases impact multiple processes. This study investigates the role of histone acetyltransferase HAT4 in Leishmania donovani. Though HAT4 was dispensable for survival, its elimination decreased cell viability and caused cell cycle defects, with HAT4-nulls experiencing an unusually long G2/M. Survival of HAT4-nulls in macrophages was also substantially compromised. DNA microarray analysis revealed that HAT4 modestly regulated the expression of only a select number of genes, thus not being a major modulator of global gene expression. Significantly, cdc20 was among the downregulated genes. To ascertain if decreased expression of cdc20 was responsible for HAT4-null growth and cell cycle defects we expressed LdCdc20 ectopically in HAT4-nulls. We found this to alleviate the aberrant growth and cell cycle progression patterns displayed by HAT4-nulls, with cells navigating G2/M phase and re-entering G1 phase smoothly. HAT4-nulls expressing LdCdc20 ectopically showed survival rates comparable to wild type within macrophages, suggesting that G2/M defects were responsible for poor survival of HAT4-nulls within host cells also. These are the first data analyzing the in vivo functional role of HAT4 in any trypanosomatid. Our results directly demonstrate for the first time a role for Cdc20 in regulating trypanosomatid G2/M events, opening avenues for further research in this area.
Asunto(s)
Ciclo Celular , Histona Acetiltransferasas/metabolismo , Leishmania donovani/enzimología , Animales , Supervivencia Celular , Histona Acetiltransferasas/genética , Leishmania donovani/citología , Macrófagos/citología , MutaciónRESUMEN
Diverse cellular processes are driven by motor proteins that are recruited to and generate force on lipid membranes. Surprisingly little is known about how membranes control the force from motors and how this may impact specific cellular functions. Here, we show that dynein motors physically cluster into microdomains on the membrane of a phagosome as it matures inside cells. Such geometrical reorganization allows many dyneins within a cluster to generate cooperative force on a single microtubule. This results in rapid directed transport of the phagosome toward microtubule minus ends, likely promoting phagolysosome fusion and pathogen degradation. We show that lipophosphoglycan, the major molecule implicated in immune evasion of Leishmania donovani, inhibits phagosome motion by disrupting the clustering and therefore the cooperative force generation of dynein. These findings appear relevant to several pathogens that prevent phagosome-lysosome fusion by targeting lipid microdomains on phagosomes.
Asunto(s)
Leishmania donovani/citología , Leishmania donovani/metabolismo , Lisosomas/metabolismo , Fagosomas/metabolismo , Animales , Transporte Biológico , Línea Celular , Dictyostelium/citología , Dineínas/metabolismo , Glicoesfingolípidos/metabolismo , Microdominios de Membrana/metabolismo , RatonesRESUMEN
Parasitic protozoa of the genus Leishmania are obligatory intracellular parasites that cycle between the phagolysosome of mammalian macrophages, where they proliferate as intracellular amastigotes, and the midgut of female sand flies, where they proliferate as extracellular promastigotes. Shifting between the two environments induces signaling pathway-mediated developmental processes that enable adaptation to both host and vector. Developmentally regulated expression and phosphorylation of protein kinase A subunits in Leishmania and in Trypanosoma brucei point to an involvement of protein kinase A in parasite development. To assess this hypothesis in Leishmania donovani, we determined proteome-wide changes in phosphorylation of the conserved protein kinase A phosphorylation motifs RXXS and RXXT, using a phospho-specific antibody. Rapid dephosphorylation of these motifs was observed upon initiation of promastigote to amastigote differentiation in culture. No phosphorylated sites were detected in axenic amastigotes. To analyse the kinetics of (re)phosphorylation during axenic reverse differentiation from L. donovani amastigotes to promastigotes, we first established a map of this process with morphological and molecular markers. Upon initiation, the parasites rested for 6-12 h before proliferation of an asynchronous population resumed. After early changes in cell shape, the major changes in molecular marker expression and flagella biogenesis occurred between 24 and 33 h after initiation. RXXS/T re-phosphorylation and expression of the regulatory subunit PKAR1 correlated with promastigote maturation, indicating a promastigote-specific function of protein kinase A signaling. This is supported by the localization of PKAR1 to the flagellum, an organelle reduced to a remnant in amastigote forms. We conclude that a significant increase in protein kinase A-mediated phosphorylation is part of the ordered changes that characterise the amastigote to promastigote differentiation.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Leishmania donovani/metabolismo , Estadios del Ciclo de Vida , Proteínas Protozoarias/metabolismo , Transducción de Señal , Animales , Flagelos/metabolismo , Leishmania donovani/citología , Leishmania donovani/enzimología , Fosforilación , ProteomaRESUMEN
Histone modifications impact various processes. In examining histone acetyltranferase HAT3 of Leishmania donovani, we find elimination of HAT3 causes decreased cell viability due to defects in histone deposition, and aberrant cell cycle progression pattern. HAT3 associates with proliferating cell nuclear antigen (PCNA), helping load PCNA onto chromatin in proliferating cells. HAT3-nulls show heightened sensitivity to UV radiation. Following UV exposure, PCNA cycles off/on chromatin only in cells expressing HAT3. Inhibition of the ubiquitin-proteasome pathway prior to UV exposure allows accumulation of chromatin-bound PCNA, and reveals that HAT3-nulls are deficient in PCNA monoubiquitination as well as polyubiquitination. While poor monoubiquitination of PCNA may adversely affect translesion DNA synthesis-based repair processes, polyubiquitination deficiencies may result in continued retention of chromatin-bound PCNA, leading to genomic instability. On suppressing the proteasome pathway we also find that HAT3 mediates PCNA acetylation in response to UV. HAT3-mediated PCNA acetylation may serve as a flag for PCNA ubiquitination, thus aiding DNA repair. While PCNA acetylation has previously been linked to its degradation following UV exposure, this is the first report linking a HAT-mediated PCNA acetylation to PCNA monoubiquitination. These findings add a new dimension to our knowledge of the mechanisms regulating PCNA ubiquitination post-UV exposure in eukaryotes.
Asunto(s)
Histona Acetiltransferasas/metabolismo , Leishmania donovani/enzimología , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas Protozoarias/metabolismo , Rayos Ultravioleta , Acetilación , Ciclo Celular , Núcleo Celular/enzimología , Cromatina/metabolismo , Histona Acetiltransferasas/química , Histona Acetiltransferasas/genética , Histonas/química , Histonas/metabolismo , Leishmania donovani/citología , Leishmania donovani/efectos de la radiación , Lisina/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , UbiquitinaciónRESUMEN
The emergence of resistance against antileishmanial drugs in current use necessitates the search for new classes of antileishmanial compounds. Herein we report the design, synthesis, and evaluation of a novel ferrocenylquinoline for activity against Leishmania donovani. 7-Chloro-N-[2-(1H-5-ferrocenyl-1,2,3-triazol-1-yl)ethyl]quinolin-4-amine (1) was generated by coupling an iron(II) ethynylferrocene species with 4-(2-ethylazido)amino-7-chloroquinoline using click chemistry. The synthesized compound 1 was tested for its antileishmanial activity using both promastigote and amastigote stages of L.â donovani. Compound 1 showed promising anti-promastigote activity, with an IC50 value of 15.26â µM and no cytotoxicity toward host splenocytes. From the battery of tests conducted in this study, it appears that this compound induces parasite death by promoting oxidative stress and depolarizing the mitochondrial membrane potential, thereby triggering apoptosis. These results suggest that ferrocenylquinoline 1 is a suitable lead for the development of new antileishmanial drugs.
Asunto(s)
Antiprotozoarios/química , Antiprotozoarios/farmacología , Compuestos Ferrosos/química , Compuestos Ferrosos/farmacología , Leishmania donovani/efectos de los fármacos , Antiprotozoarios/síntesis química , Apoptosis/efectos de los fármacos , Química Clic , Compuestos Ferrosos/síntesis química , Humanos , Concentración 50 Inhibidora , Leishmania donovani/citología , Leishmania major/citología , Leishmania major/efectos de los fármacos , Leishmaniasis Cutánea/tratamiento farmacológico , Leishmaniasis Visceral/tratamiento farmacológico , Metalocenos , Quinolinas/síntesis química , Quinolinas/química , Quinolinas/farmacología , Relación Estructura-ActividadRESUMEN
Persistence of liver injury alters the internal milieu, promotes deregulation of inflammatory factors, and leads to dysplastic lesions like fibrosis, cirrhosis to hepatocellular carcinoma. Our previous study revealed that leishmanial lipid (pLLD) exerts potential anti-inflammatory activity in sepsis associated hepatic injury. We now show that pLLD gives protection against chemical induced hepatotoxicity in murine system. The beneficial effect of treatment with pLLD on such hepatic injury in mice was analyzed using different assays including ELISA, FACS, western blot and immunohistochemical analysis. pLLD significantly suppressed serum enzymes and rectified the histopathological alteration to induce the antioxidant level in CCl4 intoxicated liver. Levels of several growth factors including TGF-ß, HGF, and EGF were significantly improved in serum and hepatic tissue with consequent reduction of caspase activities and expressions of Bad, Bax, p53, and NF-κBp65. Moreover, pLLD modulated inflammatory responses by decreasing the production of several cytokines and chemokines, thus preventing the infiltration of immune cells to the damaged area. It accelerated the repair process in liver damage with modulation of signalling cascade via alteration of apoptotic factors. Our experimental approaches suggest that pLLD effectively prevents liver injury mainly through down regulation of oxidative stress and inflammatory response towards anti-apoptotic changes.
Asunto(s)
Apoptosis/efectos de los fármacos , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Mediadores de Inflamación/metabolismo , Leishmania donovani/química , Leishmania donovani/citología , Lípidos/uso terapéutico , Estrés Oxidativo/efectos de los fármacos , Animales , Intoxicación por Tetracloruro de Carbono/prevención & control , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Citocinas/sangre , Hepatocitos/efectos de los fármacos , Lípidos/química , Pruebas de Función Hepática , Regeneración Hepática/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB CRESUMEN
One new flavonol methyl ether (1), along with four known compounds from the leaves of methanol extract of Vitex peduncularis Wall and three known compounds from the leaves of methanol extract of Vitex pinnata Linn (Verbenaceae) were isolated. The chemical structure of the new compound was established by detailed spectroscopic studies. The in vitro antileishmanial activities of 1 against both Leishmania donovani promastigote and amastigote forms were evaluated. To characterize the effector mechanism of compound 1 against Leishmania parasite infected THP-1 macrophage cells, RT-PCR analysis of inducible nitric oxide synthase 2 (iNOS2) was done followed by measurement of nitric oxide generation by Griess reaction. Pentostam (sodium antimonygluconate) was used as reference drug. Compound 1 exhibited better antileishmanial activity than sodium antimonygluconate (SAG) (having IC50 values for promastigote, 2.4 and 58.5 µM and for amastigotes, 0.93 and 36.2 µM, respectively). Compound 1 was less toxic than SAG towards THP-1 having CC50 of 123.7 µM and 364.3 µM, respectively. Moreover, compound 1 was found to induce a potent host-protective response by enhancing NO generation and iNOS2 expression in infected macrophages to prevent the progression of Leishmania parasite.
Asunto(s)
Flavonoles/química , Flavonoles/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Leishmania donovani/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo II/genética , Hojas de la Planta/química , Vitex/química , Línea Celular , Humanos , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/parasitología , Leishmania donovani/citología , Leishmania donovani/fisiología , Macrófagos/efectos de los fármacos , Macrófagos/parasitología , Éteres Metílicos/química , Nitritos/metabolismoRESUMEN
Leishmania are obligatory intracellular parasitic protozoa that cause a wide range of diseases in humans, cycling between extracellular promastigotes in the mid-gut of sand flies and intracellular amastigotes in the phagolysosomes of mammalian macrophages. Although many of the molecular mechanisms of development inside macrophages remain a mystery, the development of a host-free system that simulates phagolysosome conditions (37 °C and pH 5.5) has provided new insights into these processes. The time course of promastigote-to-amastigote differentiation can be divided into four morphologically distinct phases: I, signal perception (0-5 h after exposure); II, movement cessation and aggregation (5-10 h); III, amastigote morphogenesis (10-24 h); and IV, maturation (24-120 h). Transcriptomic and proteomic analyses have indicated that differentiation is a coordinated process that results in adaptation to life inside phagolysosomes. Recent phosphoproteomic analysis revealed extensive differences in phosphorylation between promastigotes and amastigotes and identified stage-specific phosphorylation motifs. We hypothesized that the differentiation signal activates a phosphorylation pathway that initiates Leishmania transformation, and here we used isobaric tags for relative and absolute quantitation to interrogate the dynamics of changes in the phosphorylation profile during Leishmania donovani promastigote-to-amastigote differentiation. Analysis of 163 phosphopeptides (from 106 proteins) revealed six distinct kinetic profiles; with increases in phosphorylation predominated during phases I and III, whereas phases II and IV were characterized by greater dephosphorylation. Several proteins (including a protein kinase) were phosphorylated in phase I after exposure to the complete differentiation signal (i.e. signal-specific; 37 °C and pH 5.5), but not after either of the physical parameters separately. Several other protein kinases (including regulatory subunits) and phosphatases also showed changes in phosphorylation during differentiation. This work constitutes the first genome-scale interrogation of phosphorylation dynamics in a parasitic protozoa, revealing the outline of a signaling pathway during Leishmania differentiation. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (identifier PXD000671). Data can be viewed using ProteinPilot™ software.
Asunto(s)
Diferenciación Celular/fisiología , Leishmania donovani/citología , Leishmania donovani/metabolismo , Proteínas Protozoarias/metabolismo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Fosforilación , Proteómica , Transducción de SeñalRESUMEN
Although leishmanial infections of humans occur globally, the major health impact lies in developing nations, thus, leishmaniases remain "neglected" diseases for new drugs development. Multidrug resistance has been documented in most countries where leishmaniases is endemic. Betulin is a widely available and affordable natural product exerting leishmanicidal activity at micromolar concentration. In this study, the molecular mechanisms of death that contribute to the anti-leishmanial activity of betulin are investigated. In promastigotes, betulin stimulated reactive oxygen species generation at micromolar concentrations in Leishmania. Apoptosis was observed in betulin-treated promastigotes using flow cytometric analysis of treated cells stained with annexin V-FITC and propidium iodide. Furthermore, betulin treatment of promastigotes led to mitochondrial membrane damage, activation of caspase-like proteases, and DNA fragmentation in Leishmania donovani promastigotes. Betulin treatment of amastigotes cultured within macrophages, resulted in a reduced number of amastigotes, with no substantive cytotoxic damage to the host macrophage cells at leishmanicidal drug concentrations.
Asunto(s)
Antiprotozoarios/uso terapéutico , Apoptosis/efectos de los fármacos , Leishmania donovani/efectos de los fármacos , Triterpenos/uso terapéutico , Fragmentación del ADN/efectos de los fármacos , ADN Protozoario/efectos de los fármacos , Electroforesis en Gel de Agar , Humanos , Leishmania donovani/citología , Macrófagos/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Estrés Oxidativo , Especies Reactivas de OxígenoRESUMEN
The aim of this study was to isolate and evaluate the withanolides in inducing apoptotic like death in Leishmania donovani in vitro. Withanolides were fractionated and isolated from the leaves of Withania somnifera and LC-MS/MS analysis of two fractions namely, F5 and F6 of ethanolic extracts, obtained through column chromatography with silica gel, was performed. The antileishmanial effect of withanolides on L. donovani promastigotes was assessed in vitro using PI dye exclusion test. The effect of withanolides on promastigote morphology was determined by scanning electron microscopy. To understand their mode of action against L. donovani, DNA fragmentation, quantification of parasites at sub G0/G1 phase, determination of phosphatidylserine externalization, measurement of reactive oxygen species (ROS) and mitochondrial membrane potential (Ψm) were done. Results showed that LC-MS/MS analysis confirmed the presence of withanolides in isolated fractions. Treatment with withanolides resulted in morphological alterations from spindle to round shape and loss of flagella/cell integrity in promastigotes. Moreover, it induced DNA nicks, cell cycle arrest at sub G0/G1 phase and externalization of phosphatidylserine in dose and time dependent manner via increase in ROS and decrease in Ψm. Results of this study indicate that withanolides induce apoptotic like death through the production of ROS from mitochondria and disruption of Ψm in promastigotes of L donovani.
Asunto(s)
Antiprotozoarios/farmacología , Apoptosis/efectos de los fármacos , Leishmania donovani/efectos de los fármacos , Withania/química , Witanólidos/farmacología , Antiprotozoarios/aislamiento & purificación , Puntos de Control del Ciclo Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Fragmentación del ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Concentración 50 Inhibidora , Leishmania donovani/citología , Leishmania donovani/genética , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Microscopía Electrónica de Rastreo , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Mitocondrias/metabolismo , Fosfatidilserinas/genética , Fosfatidilserinas/metabolismo , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/farmacología , Especies Reactivas de Oxígeno/análisis , Especies Reactivas de Oxígeno/metabolismo , Espectrometría de Masas en Tándem , Witanólidos/aislamiento & purificaciónRESUMEN
Leishmaniasis is one of the major tropical parasitic diseases, and the condition ranges in severity from self-healing cutaneous lesions to fatal visceral manifestations. There is no vaccine available against visceral leishmaniasis (VL) (also known as kala-azar in India), and current antileishmanial drugs face major drawbacks, including drug resistance, variable efficacy, toxicity and parenteral administration. We report here that n-hexane fractions of Artemisia annua leaves (AAL) and seeds (AAS) possess significant antileishmanial activity against Leishmania donovani promastigotes, with GI(50) of 14.4 and 14.6 µg ml(-1), respectively, and the IC(50) against intracellular amastigotes was found to be 6.6 and 5.05 µg ml(-1), respectively. Changes in the morphology of promastigotes and growth reversibility analysis following treatment confirmed the leishmanicidal effect of the active fractions, which presented no cytotoxic effect on mammalian cells. The antileishmanial activity was mediated via apoptosis, as evidenced by externalization of phosphatidylserine, in situ labelling of DNA fragments by terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) and cell-cycle arrest at the sub-G(0)/G(1) phase. High-performance thin-layer chromatography (HPTLC) fingerprinting showed that the content of artemisinin in crude bioactive extracts (~1.4 µg per 100 µg n-hexane fraction) was too low to account for the observed antileishmanial activity. Characterization of the active constituents by GC-MS showed that α-amyrinyl acetate, ß-amyrine and derivatives of artemisinin were the major constituents in AAL and cetin, EINECS 211-126-2 and artemisinin derivatives in AAS. Our findings indicate the presence of antileishmanial compounds besides artemisinin in the n-hexane fractions of A. annua leaves and seeds.
Asunto(s)
Antiprotozoarios/farmacología , Artemisia annua , Leishmania donovani/efectos de los fármacos , Leishmaniasis/tratamiento farmacológico , Extractos Vegetales/farmacología , Apoptosis , Puntos de Control del Ciclo Celular/efectos de los fármacos , Leishmania donovani/citología , Leishmania donovani/fisiología , Pruebas de Sensibilidad Parasitaria , Extractos Vegetales/química , Hojas de la Planta/química , Semillas/químicaRESUMEN
Histone acetyl transferases (HATs) are important histone modifiers that affect critical cellular processes like transcription, DNA replication and repairs through highly dynamic chromatin remodelling. Our earlier studies recognized LdHAT1 as a substrate of the S-phase cell cycle kinase LdCyc1-CRK3 from Leishmania donovani. Here, we confirm through site-directed mutagenesis that RXL-like cyclin-binding (Cy) motif dependent interaction of LdHAT1 with LdCyc1 is essential for its phosphorylation at a canonical Cdk target site by the kinase complex. LdHAT1 acetylates K10 residue of a peptide derived from L. donovani histone H4 N-terminal tail. Interestingly, phosphorylation of LdHAT1 by the S-phase kinase inhibits its H4K10 acetylation activity, implicating an important mechanism of periodic regulation of histone acetylation during cell cycle progression.