RESUMO
Caffeic acid and related natural compounds were previously described as Leishmania amazonensis arginase (L-ARG) inhibitors, and against the whole parasite in vitro. In this study, we tested cinnamides that were previously synthesized to target human arginase. The compound caffeic acid phenethyl amide (CAPA), a weak inhibitor of human arginase (IC50 = 60.3 ± 7.8 µM) was found to have 9-fold more potency against L-ARG (IC50 = 6.9 ± 0.7 µM). The other compounds that did not inhibit human arginase were characterized as L-ARG, showing an IC50 between 1.3-17.8 µM, and where the most active was compound 15 (IC50 = 1.3 ± 0.1 µM). All compounds were also tested against L. amazonensis promastigotes, and only the compound CAPA showed an inhibitory activity (IC50 = 80 µM). In addition, in an attempt to gain an insight into the mechanism of competitive L-ARG inhibitors, and their selectivity over mammalian enzymes, we performed an extensive computational investigation, to provide the basis for the selective inhibition of L-ARG for this series of compounds. In conclusion, our results indicated that the compounds based on cinnamoyl or 3,4-hydroxy cinnamoyl moiety could be a promising starting point for the design of potential antileishmanial drugs based on selective L-ARG inhibitors.
Assuntos
Antiprotozoários/farmacologia , Arginase/antagonistas & inibidores , Cinamatos/farmacologia , Inibidores Enzimáticos/farmacologia , Leishmania/enzimologia , Proteínas de Protozoários/antagonistas & inibidores , Animais , Sítios de Ligação , Ácidos Cafeicos/química , Bovinos , Cinamatos/química , Desenho de Fármacos , Humanos , Concentração Inibidora 50 , Cinética , Ligantes , Simulação de Dinâmica Molecular , Conformação Proteica , Proteínas Recombinantes/químicaRESUMO
This study describes the activity of five natural hydroxycinnamic acids and derived compound: caffeic (1), rosmarinic (2), chlorogenic (3), and cryptochlorogenic (4), acids and isoverbascoside (5). All compounds inhibited Leishmania amazonensis arginase with IC50 -in range of 1.5-11 µM. Compounds 2 and 5 also showed activity against promastigotes of L. amazonensis with IC50 = 61 (28-133) µM and IC50 = 14 (9-24) µM, respectively. Further computational studies applying molecular docking simulations were performed on the competitive inhibitors to gain insight into the molecular basis for arginase inhibition and could be exploited to the development of new antileishmanials drug targeting parasite arginase.
Assuntos
Antiprotozoários/química , Arginase/metabolismo , Cinamatos/química , Leishmania/enzimologia , Proteínas de Protozoários/metabolismo , Antiprotozoários/metabolismo , Antiprotozoários/farmacologia , Arginase/antagonistas & inibidores , Sítios de Ligação , Cinamatos/metabolismo , Concentração Inibidora 50 , Cinética , Leishmania/efeitos dos fármacos , Simulação de Acoplamento Molecular , Estrutura Terciária de Proteína , Proteínas de Protozoários/antagonistas & inibidoresRESUMO
Epigallocatechin-3-gallate (EGCG), a dietary polyphenol (flavanol) from green tea, possesses leishmanicidal and antitrypanosomal activity. Mitochondrial damage was observed in Leishmania treated with EGCG, and it contributed to the lethal effect. However, the molecular target has not been defined. In this study, EGCG, (+)-catechin and (-)-epicatechin were tested against recombinant arginase from Leishmania amazonensis (ARG-L) and rat liver arginase (ARG-1). The compounds inhibit ARG-L and ARG-1 but are more active against the parasite enzyme. Enzyme kinetics reveal that EGCG is a mixed inhibitor of the ARG-L while (+)-catechin and (-)-epicatechin are competitive inhibitors. The most potent arginase inhibitor is (+)-catechin (IC50â=â0.8 µM) followed by (-)-epicatechin (IC50â=â1.8 µM), gallic acid (IC50â=â2.2 µM) and EGCG (IC50â=â3.8 µM). Docking analyses showed different modes of interaction of the compounds with the active sites of ARG-L and ARG-1. Due to the low IC50 values obtained for ARG-L, flavanols can be used as a supplement for leishmaniasis treatment.
Assuntos
Arginase/antagonistas & inibidores , Arginase/química , Catequina/análogos & derivados , Catequina/química , Inibidores Enzimáticos/química , Leishmania/enzimologia , Fígado/enzimologia , Simulação de Acoplamento Molecular , Proteínas de Protozoários/antagonistas & inibidores , Animais , Anticarcinógenos/química , RatosRESUMO
Fisetin, quercetin, luteolin and 7,8-hydroxyflavone show high activity in Leishmania cultures and present low toxicity to mammalian cells. In this work, the structural aspects of 13 flavonoids were analyzed for their inhibition of the arginase enzyme from Leishmania (Leishmania) amazonensis. A higher potency of arginase inhibition was observed with fisetin, which was four and ten times greater than that of quercetin and luteolin, respectively. These data show that the hydroxyl group at position 3 contributed significantly to the inhibitory activity of arginase, while the hydroxyl group at position 5 did not. The absence of the catechol group on apigenin drastically decreased arginase inhibition. Additionally, the docking of compounds showed that the inhibitors interact with amino acids involved in the Mn(+2)-Mn(+2) metal bridge formation at the catalytic site. Due to the low IC50 values of these flavonoids, they may be used as a food supplement in leishmaniasis treatment.
Assuntos
Arginase/antagonistas & inibidores , Inibidores Enzimáticos/química , Flavonoides/química , Leishmania/enzimologia , Luteolina/química , Proteínas de Protozoários/antagonistas & inibidores , Arginase/química , Arginase/genética , Arginase/metabolismo , Domínio Catalítico , Flavonóis , Humanos , Cinética , Leishmania/genética , Leishmania/fisiologia , Leishmaniose/parasitologia , Modelos Moleculares , Estrutura Molecular , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
The plant Cecropia pachystachya Trécul is widely used in Brazilian ethnomedicine to treat hypertension, asthma, and diabetes. Arginase is an enzyme with levels that are elevated in these disorders, and it is central to Leishmania polyamine biosynthesis. The aims of this study were to evaluate antileishmanial activity and inhibition of the arginase enzyme by C. pachystachya extracts, and to study changes in cellular organization using electron microscopy. The ethanol extract of C. pachystachya was tested on Leishmania (Leishmania) amazonensis promastigote survival/proliferation and arginase activity in vitro. Qualitative ultrastructural analysis was also used to observe changes in cell organization. The major bioactive molecules of the ethanol extract were characterized using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). The ethyl acetate fraction of the ethanol extract diminished promastigote axenic growth/survival, inhibited arginase activity, and altered a mitochondrial kinetoplast DNA (K-DNA) array. The bioactive compounds of C. pachystachya were characterized as glucoside flavonoids. Orientin (9) (luteolin-8-C-glucoside) was the main component of the methanol-soluble ethyl acetate fraction obtained from the ethanol extract and is an arginase inhibitor (IC50 15.9 µM). The ethyl acetate fraction was not cytotoxic to splenocytes at a concentration of 200 µg/mL. In conclusion, C. pachystachya contains bioactive compounds that reduce the growth of L. (L.) amazonensis promastigotes, altering mitochondrial K-DNA arrangement and inhibiting arginase.
Assuntos
Antiprotozoários/farmacologia , Arginase/antagonistas & inibidores , Cecropia/química , DNA Mitocondrial/genética , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Leishmania/efeitos dos fármacos , Acetatos/química , Antiprotozoários/química , Inibidores Enzimáticos/química , Etanol/química , Flavonoides/química , Glucosídeos/farmacologia , Humanos , Leishmania/enzimologia , Leishmania/genética , Metanol/química , SolubilidadeRESUMO
Polyamine biosynthesis enzymes are promising drug targets for the treatment of leishmaniasis, Chagas' disease and African sleeping sickness. Arginase, which is a metallohydrolase, is the first enzyme involved in polyamine biosynthesis and converts arginine into ornithine and urea. Ornithine is used in the polyamine pathway that is essential for cell proliferation and ROS detoxification by trypanothione. The flavonols quercetin and quercitrin have been described as antitrypanosomal and antileishmanial compounds, and their ability to inhibit arginase was tested in this work. We characterized the inhibition of recombinant arginase from Leishmania (Leishmania) amazonensis by quercetin, quercitrin and isoquercitrin. The IC(50) values for quercetin, quercitrin and isoquercitrin were estimated to be 3.8, 10 and 4.3 µM, respectively. Quercetin is a mixed inhibitor, whereas quercitrin and isoquercitrin are uncompetitive inhibitors of L. (L.) amazonensis arginase. Quercetin interacts with the substrate l-arginine and the cofactor Mn(2+) at pH 9.6, whereas quercitrin and isoquercitrin do not interact with the enzyme's cofactor or substrate. Docking analysis of these flavonols suggests that the cathecol group of the three compounds interact with Asp129, which is involved in metal bridge formation for the cofactors Mn(A)(2+) and Mn(B)(2+) in the active site of arginase. These results help to elucidate the mechanism of action of leishmanicidal flavonols and offer new perspectives for drug design against Leishmania infection based on interactions between arginase and flavones.