Primaquine-quinoxaline 1,4-di-N-oxide hybrids with action on the exo-erythrocytic forms of Plasmodium induce their effect by the production of reactive oxygen species.

BACKGROUND: The challenge in anti-malarial chemotherapy is based on the emergence of resistance to drugs and the search for medicines against all stages of the life cycle of Plasmodium spp. as a therapeutic target. Nowadays, many molecules with anti-malarial activity are reported. However, few studies about the cellular and molecular mechanisms to understand their mode of action have been explored. Recently, new primaquine-based hybrids as new molecules with potential multi-acting anti-malarial activity were reported and two hybrids of primaquine linked to quinoxaline 1,4-di-N-oxide (PQ-QdNO) were identified as the most active against erythrocytic, exoerythrocytic and sporogonic stages. METHODS: To further understand the anti-malarial mode of action (MA) of these hybrids, hepg2-CD81 were infected with Plasmodium yoelii 17XNL and treated with PQ-QdNO hybrids during 48 h. After were evaluated the production of ROS, the mitochondrial depolarization, the total glutathione content, the DNA damage and proteins related to oxidative stress and death cell. RESULTS: In a preliminary analysis as tissue schizonticidals, these hybrids showed a mode of action dependent on peroxides production, but independent of the activation of transcription factor p53, mitochondrial depolarization and arrest cell cycle. CONCLUSIONS: Primaquine-quinoxaline 1,4-di-N-oxide hybrids exert their antiplasmodial activity in the exoerythrocytic phase by generating high levels of oxidative stress which promotes the increase of total glutathione levels, through oxidation stress sensor protein DJ-1. In addition, the role of HIF1a in the mode of action of quinoxaline 1,4-di-N-oxide is independent of biological activity.

Antigens reversibly conjugated to a polymeric glyco-adjuvant induce protective humoral and cellular immunity.

Fully effective vaccines for complex infections must elicit a diverse repertoire of antibodies (humoral immunity) and CD8+ T-cell responses (cellular immunity). Here, we present a synthetic glyco-adjuvant named p(Man-TLR7), which, when conjugated to antigens, elicits robust humoral and cellular immunity. p(Man-TLR7) is a random copolymer composed of monomers that either target dendritic cells (DCs) via mannose-binding receptors or activate DCs via Toll-like receptor 7 (TLR7). Protein antigens are conjugated to p(Man-TLR7) via a self-immolative linkage that releases chemically unmodified antigen after endocytosis, thus amplifying antigen presentation to T cells. Studies with ovalbumin (OVA)-p(Man-TLR7) conjugates demonstrate that OVA-p(Man-TLR7) generates greater humoral and cellular immunity than OVA conjugated to polymers lacking either mannose targeting or TLR7 ligand. We show significant enhancement of Plasmodium falciparum-derived circumsporozoite protein (CSP)-specific T-cell responses, expansion in the breadth of the αCSP IgG response and increased inhibition of sporozoite invasion into hepatocytes with CSP-p(Man-TLR7) when compared with CSP formulated with MPLA/QS-21-loaded liposomes-the adjuvant used in the most clinically advanced malaria vaccine. We conclude that our antigen-p(Man-TLR7) platform offers a strategy to enhance the immunogenicity of protein subunit vaccines.

Novel antimalarial chloroquine- and primaquine-quinoxaline 1,4-di-N-oxide hybrids: Design, synthesis, Plasmodium life cycle stage profile, and preliminary toxicity studies.

Emergence of drug resistance and targeting all stages of the parasite life cycle are currently the major challenges in antimalarial chemotherapy. Molecular hybridization combining two scaffolds in a single molecule is an innovative strategy for achieving these goals. In this work, a series of novel quinoxaline 1,4-di-N-oxide hybrids containing either chloroquine or primaquine pharmacophores was designed, synthesized and tested against both chloroquine sensitive and multidrug resistant strains of Plasmodium falciparum. Only chloroquine-based compounds exhibited potent blood stage activity with compounds 4b and 4e being the most active and selective hybrids at this parasite stage. Based on their intraerythrocytic activity and selectivity or their chemical nature, seven hybrids were then evaluated against the liver stage of Plasmodium yoelii, Plasmodium berghei and Plasmodium falciparum infections. Compound 4b was the only chloroquine-quinoxaline 1,4-di-N-oxide hybrid with a moderate liver activity, whereas compound 6a and 6b were identified as the most active primaquine-based hybrids against exoerythrocytic stages, displaying enhanced liver activity against P. yoelii and P. berghei, respectively, and better SI values than primaquine. Although both primaquine-quinoxaline 1,4-di-N-oxide hybrids slightly reduced the infection of mosquitoes, they inhibited sporogony of P. berghei and compound 6a showed 92% blocking of transmission. In vivo liver efficacy assays revealed that compound 6a showed causal prophylactic activity affording parasitaemia reduction of up to 95% on day 4. Absence of genotoxicity and in vivo acute toxicity were also determined. These results suggest the approach of primaquine-quinoxaline 1,4-di-N-oxide hybrids as new potential dual-acting antimalarials for further investigation.

Structure-activity relationship of new antimalarial 1-aryl-3-susbtituted propanol derivatives: Synthesis, preliminary toxicity profiling, parasite life cycle stage studies, target exploration, and targeted delivery.

Design, synthesis, structure-activity relationship, cytotoxicity studies, in silico drug-likeness, genotoxicity screening, and in vivo studies of new 1-aryl-3-substituted propanol derivatives led to the identification of nine compounds with promising in vitro (55, 56, 61, 64, 66, and 70-73) and in vivo (66 and 72) antimalarial profiles against Plasmodium falciparum and Plasmodium berghei. Compounds 55, 56, 61, 64, 66 and 70-73 exhibited potent antiplasmodial activity against chloroquine-resistant strain FCR-3 (IC50s < 0.28 µM), and compounds 55, 56, 64, 70, 71, and 72 showed potent biological activity in chloroquine-sensitive and multidrug-resistant strains (IC50s < 0.7 µM for 3D7, D6, FCR-3 and C235). All of these compounds share appropriate drug-likeness profiles and adequate selectivity indexes (77 < SI < 184) as well as lack genotoxicity. In vivo efficacy tests in a mouse model showed compounds 66 and 72 to be promising candidates as they exhibited significant parasitemia reductions of 96.4% and 80.4%, respectively. Additional studies such as liver stage and sporogony inhibition, target exploration of heat shock protein 90 of P. falciparum, targeted delivery by immunoliposomes, and enantiomer characterization were performed and strongly reinforce the hypothesis of 1-aryl-3-substituted propanol derivatives as promising antimalarial compounds.

Glucose promotes resistance in lymphocytes against oxidative stress-induced apoptosis through signaling and metabolic pathways. Implications for Parkinson's disease / La glucosa promueve la resistencia en linfocitos contra el estrés oxidativo que induce apoptosis a través de rutas de señalización y metabólica. Impacto en la enfermedad de Parkinson / A glucose promove a resistência em linfócitos contra o estresse oxidativo que induz apoptose a través de rotas de sinalização e metabólica. Impacto na doença de Parkinson

SUMMARY Introduction: Parkinson's disease (PD) is a neurological disorder associated with the selective loss of dopaminergic (DAergic) neurons. Clinical data suggest that oxidative stress (OS) and dysregulation of glucose (G) metabolism are early events in PD. However, no data are available to explain the molecular connection between glucose metabolism, OS, and neuronal demise in PD. Human lymphocytes share a similar dopaminergic signaling mechanism with DAergic neurons. Further, rotenone (ROT) is a mitochondrial complex I inhibitor that selectively induces apoptosis through OS in dopaminergic neurons and lymphocytes. Thus, to test the hypothesis that G metabolism and OS are linked in dopaminergic system toxicity and PD, human lymphocytes were cultured with ROT in the presence or absence of various concentrations of glucose. Objective: This study examines the response of human lymphocytes to glucose (11, 55, 166, 277, 555 mM G) in the absence or presence of ROT (250 microM). Methods: Light and fluorescence microscopy and immunocytochemistry techniques were used to evaluate morphological and biochemical changes in human lymphocytes. Results: 55 mM G was effective in suppressing ROT-induced apoptosis in lymphocytes via 5 pathways: (i) pentose phosphate pathway (PPP), (ii) glutathione (GSH) pathway, (iii) superoxide dismutase (SOD) and catalase (CAT) antioxidant systems, and (iv) Phosphoinositide 3-kinase (PI3-K) signaling. Additionally, we report for the first time that G rescued lymphocytes from ROT-induced apoptosis by (v) activating NF-kB and down-regulating p53 and caspase-3. Signaling (e.g., LY294002) and metabolic inhibitors of these pathways (e.g., dehydroepiandrosterone (DHEA), L-buthioninesulfoximine (BSO), 1,3-Bis (2-chloroethyl)-1-nitrosourea (BCNU), mercaptosuccinic acid (MS), 3-amino-1,2,4- triazole (AT), sodium diethyldithiocarbamate (DDC)) only partially reversed the protective effect of 55 mM G on lymphocytes exposed to ROT. Conclusion: These data suggest that high G simultaneously triggers cellular signaling and antioxidant systems to ensure global cell protection against stressful conditions in DAergic.

RESUMEN Introducción: la enfermedad de Parkinson (EP) es un trastorno neurológico asociado con la pérdida selectiva de neuronas dopaminérgicas (DAérgicas). Datos clínicos sugieren que el estrés oxidativo (EO) y la desregulación del metabolismo de la glucosa (G) son eventos tempranos en la EP. Sin embargo, no existe información que explique la posible asociación molecular entre el metabolismo de la glucosa, el EO y la muerte neuronal. Los linfocitos humanos comparten mecanismos de señalización DAérgicos comunes. Más aún, la rotenona (ROT) es un inhibidor que selectivamente induce apoptosis vía EO en neuronas DAérgicas y linfocitos. Para evaluar la hipótesis que el metabolismo de la G y el EO están asociados con la toxicidad del sistema DAérgicas y EP, se cultivaron linfocitos humanos con ROT en presencia o ausencia de varias concentraciones de G. Objetivo: este estudio examina la respuesta de los linfocitos a G (11, 55, 166, 277, 555 mM) en ausencia o presencia de ROT (250 microM). Métodos: se utilizaron técnicas de microscopía de luz y fluorescencia e inmunocitoquímica para evaluar los cambios morfológicos y bioquímicos de los linfocitos. Resultados: la G 55 mM fue eficaz en suprimir la apoptosis en linfocitos inducida por ROT vía activación de 5 rutas metabólicas: (i) la vía pentosa fosfato, (ii) la vía glutatión; (iii) los sistemas antioxidantes superóxido dismutasa (SOD) y catalasa (CAT); (iv) fosfoinositol 3 cinasa (PI3-K). Además, se observó por primera vez que la G rescata linfocitos de la apoptosis inducida por ROT vía (v) activación del factor nuclear kappa-B (NFkB) y por regulación a la baja de p53 y de la caspasa-3. Se demostró que los inhibidores de señalización (v.gr. LY294002) e inhibidores metabólicos (v.gr. DHEA, BSO, BCNU, MS, DCC) revierten parcialmente los efectos citoprotectores de la G 55 mM en linfocitos expuestos a ROT. Conclusión: estos hallazgos sugieren que la alta concentración de G induce simultáneamente sistemas de señalización y antioxidantes para asegurar la protección global de la célula contra condiciones estresantes en células DAérgicas.

RESUMO Introdução: a doença de Parkinson (PD) é uma desordem neurológica associada a perda seletiva de neurônios dopaminérgicos (Daergicas). Dados clínicos sugerem que o estresse oxidativo (EO) e a desregulação do metabolismo da glicose (G) são eventos iniciais no PD. No entanto, não há nenhuma informação que explica a possível associação molecular entre o metabolismo da glicose, EO e morte neuronal. Linfócitos humanos compartilham comum Daergicos mecanismos de sinalização. Além disso, a rotenona (ROT) é um inibidor que seletivamente induz apoptose através de EO nos neurônios Daergicas e linfócitos. Para avaliar a hipótese de que o metabolismo do G e EO estão associadas com a toxicidade do Daergicas e sistema de EP, linfócitos humanos com ROT foram cultivados na presença ou ausência de várias concentrações de G. Objetivo: este estudo analisa a resposta dos linfócitos a G (55 11, 166, 277, 555 mM) na ausência ou presença de podridão (250 microM). Métodos: luz e imunocitoquímica, microscopia de fluorescência foram utilizadas técnicas para avaliar as alterações morfológicas e bioquímicas de linfócitos. Resultados: a 55mM G foi eficaz em suprimir a apoptose em linfócitos induzidos por (ROT) a través da ativação de 5 vias metabólicas: (i) a través do fosfato de pentose, (ii) a través de glutationa; (iii) a superóxido dismutase de antioxidante (SOD) e catalase (CAT) sistemas; (iv) Phosphoinositide 3 cinase (PI3-K). Além disso, foi observada pela primeira vez que o G resgata a apoptose de linfócitos induzida por (ROT) via ativação (v) por fator nuclear kappa-B, (NF-kB) e por regulação por baixo de p53 e Caspase-3. Mostrou que inibidores (por exemplo, LY294002), inibidores de sinalização e metabólicos (por exemplo, DHEA, BSO, BCNU, MS, DCC) reverteu parcialmente os efeitos dos tilacoides do G 55 mM de linfócitos expostos à (ROT). Conclusão: estes resultados sugerem que a alta concentração de G induz simultaneamente antioxidantes e sistemas de sinalização para garantir a proteção global da célula de condições estressantes nas células Daergicas.

Low doses of paraquat and polyphenols prolong life span and locomotor activity in knock-down parkin Drosophila melanogaster exposed to oxidative stress stimuli: implication in autosomal recessive juvenile parkinsonism.

Previous studies have shown that polyphenols might be potent neuroprotective agents in Drosophila melanogaster wild type Canton-S acutely or chronically treated with paraquat (PQ), a selective toxin for elimination of dopaminergic (DAergic) neurons by oxidative stress (OS), as model of Parkinson's disease (PD). This study reports for the first time that knock-down (K-D) parkin Drosophila melanogaster (TH-GAL4; UAS-RNAi-parkin) chronically exposed to PQ (0.1-0.25 mM), FeSO(4) (Fe, 0.1mM), deferoxamine (DFO, 0.01 mM) alone or (0.1mM) PQ in combination with polyphenols propyl gallate (PG, 0.1mM) and epigallocathecin gallate (EGCG, 0.1, 0.5mM) showed significantly higher life span and locomotor activity than untreated K-D flies or treated with (1, 5, 20mM) PQ alone. Whilst gallic acid (GA, 0.1, 0.5mM) alone or in the presence of PQ provoked no effect on K-D flies, epicathecin (EC, 0.5mM) only showed a positive effect on prolonging K-D flies' life span. It is shown that PG (and EGCG) protected protocerebral posterolateral 1 (PPL1) DAergic neurons against PQ. Interestingly, the protective effect of low PQ concentrations, DFO and iron might be explained by a phenomenon known as "hormesis." However, pre-fed K-D flies with (0.1mM) PQ for 7 days and then exposed to (0.25 mM) for additional 8 days affect neither survival nor climbing of K-D Drosophila compared to flies treated with (0.25 mM) PQ alone. Remarkably, K-D flies treated with 0.1mM PQ (7 days) and then with (0.25 mM) PQ plus PG (8 days) behaved almost as flies treated with (0.25 mM) PQ. Taken these data suggest that antioxidant supplements that synergistically act with low pro-oxidant stimuli to prolong and increase locomotor activity become inefficient once a threshold of OS has been reached in K-D flies. Our present findings support the notion that genetically altered Drosophila melanogaster as suitable model to study genetic and environmental factors as causal and/or modulators in the development of autosomal recessive juvenile Parkinsonism (AR-JD)/PD. Most importantly, we have shown for the first time that low amounts of stressors induce a health-promoting extending effect in K-D parkin flies. Altogether our present results open new avenues for the screening, testing and development of novel antioxidant drugs against OS stimuli in neurodegenerative disorders.

Acute and chronic metal exposure impairs locomotion activity in Drosophila melanogaster: a model to study Parkinsonism.

The biometals iron (Fe), manganese (Mn) and copper (Cu) have been associated to Parkinson's disease (PD) and Parkinsonism. In this work, we report for the first time that acute (15 mM for up to 5 days) or chronic (0.5 mM for up to 15 days) Fe, Mn and Cu exposure significantly reduced life span and locomotor activity (i.e. climbing capabilities) in Drosophila melanogaster. It is shown that the concentration of those biometals dramatically increase in Drosophila's brain acutely or chronically fed with metal. We demonstrate that the metal accumulation in the fly's head is associated with the neurodegeneration of several dopaminergic neuronal clusters. Interestingly, it is found that the PPL2ab DAergic neuronal cluster was erode by the three metals in acute and chronic metal exposure and the PPL3 DAergic cluster was also erode by the three metals but in acute metal exposure only. Furthermore, we found that the chelator desferoxamine, ethylenediaminetetraacetic acid, and D: -penicillamine were able to protect but not rescue D. melanogaster against metal intoxication. Taken together these data suggest that iron, manganese and copper are capable to destroy DAergic neurons in the fly's brain, thereby impairing their movement capabilities. This work provides for the first time metal-induced Parkinson-like symptoms in D. melanogaster. Understanding therefore the effects of biometals in the Drosophila model may provide insights into the toxic effect of metal ions and more effective therapeutic approaches to Parkinsonism.