RESUMO
Chalcones (1,3-diphenyl-2-propen-1-ones) either natural or synthetic have a plethora of biological properties including antileishmanial activities, but their development as drugs is hampered by their largely unknown mechanisms of action. We demonstrate herein that our previously described benzochalcone fluorogenic probe (HAB) could be imaged by fluorescence microscopy in live Leishmania amazonensis promastigotes where it targeted the parasite acidocalcisomes, lysosomes and the mitochondrion. As in the live zebrafish model, HAB formed yellow-emitting fluorescent complexes when associated with biological targets in Leishmania. Further, we used HAB as a reversible probe to study the binding of a portfolio of diverse chalcones and analogues in live promastigotes, using a combination of competitive flow cytometry analysis and cell microscopy. This pharmacological evaluation suggested that the binding of HAB in promastigotes was representative of chalcone pharmacology in Leishmania, with certain exogenous chalcones exhibiting competitive inhibition (ca. 20-30%) towards HAB whereas non-chalconic inhibitors showed weak capacity (ca. 3-5%) to block the probe intracellular binding. However, this methodology was restricted by the strong toxicity of several competing chalcones at high concentration, in conjunction with the limited sensitivity of the HAB fluorophore. This advocates for further optimization of this undirect target detection strategy using pharmacophore-derived reversible fluorescent probes.
Assuntos
Antiprotozoários , Chalcona , Chalconas , Leishmania , Animais , Antiprotozoários/química , Antiprotozoários/farmacologia , Sítios de Ligação , Chalcona/farmacologia , Chalconas/química , Chalconas/farmacologia , Corantes Fluorescentes , Peixe-ZebraRESUMO
Mitogen-activated protein kinases (MAPKs) are involved in environmental signal sensing. They are thus expected to play key roles in the biology of Trypanosomatid parasites, which display complex life cycles and use extracellular cues to modulate cell differentiation. Despite their relevance, structural data of Trypanosomatid MAPKs is lacking. We have now determined the crystal structure of Leishmania major LmaMPK10, a stage-specifically activated MAPK, both alone and in complex with SB203580. LmaMPK10 was observed to be more similar to p38 than to other human MAPKs. However, significant differences could be identified in the catalytic pocket, as well as in potentially regulatory sites in the N-terminal lobe. The modified pocket architecture in LmaMPK10 precludes DFG-in/DFG-out regulatory flipping as observed in mammalian MAPKs. LmaMPK10-nucleotide association was also studied, revealing a potential C-terminal autoinhibitory mechanism. Overall, these data should speed the discovery of molecules interfering with LmaMPK10 functions, with relevance for antileishmanial drug development strategies.
Assuntos
Imidazóis/química , Leishmania major/enzimologia , Proteínas Quinases Ativadas por Mitógeno/química , Inibidores de Proteínas Quinases/química , Proteínas de Protozoários/química , Piridinas/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Domínio Catalítico , Sequência Conservada , Cristalografia por Raios X , Ligação de Hidrogênio , Cinética , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Homologia de Sequência de Aminoácidos , Homologia Estrutural de Proteína , TermodinâmicaRESUMO
Epoxidation of argentilactone (1) with m-chloroperbenzoic acid gave a diasteromeric mixture of 2 and 3 in a ratio 1.8 : 1, with total yield 60%. The configuration of 7,8-oxirane ring for both diasteromers was determined by NMR analysis. Reaction of 1 with urea hydrogen peroxide gave the 3,4-epoxide (4) in 65% yield. The in vitro activity of 2, 3, 4 and argentilactone against Leishmania amazonensis was tested, only epoxides (2) and (3) showed leishmanicidal effect.
Assuntos
Antiprotozoários/farmacologia , Compostos de Epóxi/química , Compostos de Epóxi/farmacologia , Lactonas/química , Lactonas/farmacologia , Leishmania/efeitos dos fármacos , Animais , Estrutura MolecularRESUMO
Acylhydroquinone-based compounds are attractive targets for the design of new leishmanicidal drugs. We have previously described sesquiterpene quinones and hydroquinones series, which exhibit different degree of potency against Leishmania amazonensis. The present study details the preparation of acylchloroquinones and hydroquinones possessing lipophilic substituents and examines their in vitro activity against intracellular L. amazonensis amastigotes. The quinone or hydroquinone nucleus is essential for the activity of the members of the series. The lipophilicity of the cycloaliphatic systems in these members seems to attenuate the cytotoxical effect and increases the selectivity of those compounds containing the norbornene system.
Assuntos
Hidroquinonas/síntese química , Hidroquinonas/farmacologia , Leishmania mexicana/efeitos dos fármacos , Quinonas/síntese química , Quinonas/farmacologia , Animais , Antiprotozoários/síntese química , Antiprotozoários/farmacologia , Feminino , Hidroquinonas/química , Concentração Inibidora 50 , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Quinonas/química , Relação Estrutura-AtividadeRESUMO
The reaction of (+)-euryfuran 1 with several benzo-, naphtho- and benzo[b]thiophene-1,4-quinones in acetic acid yields the corresponding euryfuryl-1,4-quinones 3, 5, 7, 8, 10, 12, and 14. The structure of compounds 7, 8, 12, and 14 was assigned through 2D NMR 1H-13C HMBC experiments. The influence of the acidity of the solvent upon the reactivity and regioselectivity of the quinones to the oxidative coupling reaction, is discussed. The in vitro activity of the euryfurylquinones and their corresponding precursors against Leishmania amazonensis is described.