Design and Synthesis of New Anthranyl Phenylhydrazides: Antileishmanial Activity and Structure-Activity Relationship.

Leishmaniasis is a neglected tropical disease affecting millions of people worldwide. A centenary approach to antimonial-based drugs was first initiated with the synthesis of urea stibamine by Upendranath Brahmachari in 1922. The need for new drug development led to resistance toward antimoniates. New drug development to treat leishmaniasis is urgently needed. In this way, searching for new substances with antileishmanial activity, we synthesized ten anthranyl phenylhydrazide and three quinazolinone derivatives and evaluated them against promastigotes and the intracellular amastigotes of Leishmania amazonensis. Three compounds showed good activity against promastigotes 1b, 1d, and 1g, with IC50 between 1 and 5 µM. These new phenylhydrazides were tested against Leishmania arginase, but they all failed to inhibit this parasite enzyme, as we have shown in a previous study. To explain the possible mechanism of action, we proposed the enzyme PTR1 as a new target for these compounds based on in silico analysis. In conclusion, the new anthranyl hydrazide derivatives can be a promising scaffold for developing new substances against the protozoa parasite.

In Vitro and In Silico Analyses of New Cinnamid and Rosmarinic Acid-Derived Compounds Biosynthesized in Escherichia coli as Leishmania amazonensis Arginase Inhibitors.

Arginase is a metalloenzyme that plays a central role in Leishmania infections. Previously, rosmarinic and caffeic acids were described as antileishmanial agents and as Leishmania amazonensis arginase inhibitors. Here, we describe the inhibition of arginase in L. amazonensis by rosmarinic acid analogs (1-7) and new caffeic acid-derived amides (8-10). Caffeic acid esters and amides were produced by means of an engineered synthesis in E. coli and tested against L. amazonensis arginase. New amides (8-10) were biosynthesized in E. coli cultured with 2 mM of different combinations of feeding substrates. The most potent arginase inhibitors showed Ki(s) ranging from 2 to 5.7 µM. Compounds 2-4 and 7 inhibited L. amazonensis arginase (L-ARG) through a noncompetitive mechanism whilst compound 9 showed a competitive inhibition. By applying an in silico protocol, we determined the binding mode of compound 9. The competitive inhibitor of L-ARG targeted the key residues within the binding site of the enzyme, establishing a metal coordination bond with the metal ions and a series of hydrophobic and polar contacts supporting its micromolar inhibition of L-ARG. These results highlight that dihydroxycinnamic-derived compounds can be used as the basis for developing new drugs using a powerful tool based on the biosynthesis of arginase inhibitors.

Cinnamides Target Leishmania amazonensis Arginase Selectively.

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.

Dietary polyphenols rutin, taxifolin and quercetin related compounds target Leishmania amazonensis arginase.

Quercetin related compounds were tested against Leishmania amazonensis arginase, a potential target for the development of new approaches in treating leishmaniasis. The IC50 and kinetic analysis were performed to determine the dissociation constant Ki and the inhibition mechanism of the parasite's arginase enzyme. The best arginase inhibition was obtained from taxifolin (dihydroquercetin) with IC50 = 1.6 ± 0.1 µM. This study showed for the first time that rutin (IC50 = 10.4 ± 0.8 µM), and human metabolite quercetin-3-O-glucuronide (IC50 = 8.2 ± 0.4 µM), target L. amazonensis arginase. In addition, computational studies applying molecular docking simulations were performed to gain insight into the molecular basis for arginase inhibition by the competitive inhibitors. Our results suggest that these compounds could be exploited to develop new approaches for treating leishmaniasis through molecular nutrition supplement in a drug-based therapy.

Cinnamic acids derived compounds with antileishmanial activity target Leishmania amazonensis arginase.

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.

Antileishmanial activity of verbascoside: Selective arginase inhibition of intracellular amastigotes of Leishmania (Leishmania) amazonensis with resistance induced by LPS plus IFN-γ.

Verbascoside is the main component of the traditional medicinal plants that were used against protozoa parasites that cause malaria and leishmaniasis. Previously, we have described verbascoside inhibition of Leishmania amazonensis arginase as well as its antileishmanial action against extracellular promastigotes. In this study, we have assessed arginase parasite inhibition in intracellular amastigotes. In addition, we verified whether verbascoside can influence the host defense against the parasite by measuring gene expression of cytokines IL-1b, IL-10, IL-18, TNF-α and murine macrophage arginase as well as nitric oxide synthase enzymes. Our results show that verbascoside acts on intracellular amastigotes of L. amazonensis (EC50=32µM) by selectively inhibiting the parasite arginase. Verbascoside did not affect the expression of cytokines or enzymes by murine macrophages. However, verbascoside was active against L. (L.) amazonensis amastigotes that were resistant to treatment with LPS and IFN-γ. Verbascoside action on L. amazonensis can be associated with reduction of the protective oxidative mechanism of the parasite leading to impaired trypanothione synthesis that is induced by the parasite arginase inhibition.

Stachytarpheta cayennensis extract inhibits promastigote and amastigote growth in Leishmania amazonensis via parasite arginase inhibition.

ETHNOPHARMACOLOGY RELEVANCE: Stachytarpheta cayennensis is a plant that is traditionally used to treat tegumentary leishmaniasis and as an anti-inflammatory agent. AIM OF THE STUDY: This study aimed to evaluate the action of S. cayennensis extracts on the Leishmania (Leishmania) amazonensis arginase enzyme. MATERIALS AND METHODS: S. cayennensis was collected from the Brazilian Amazon region. Aqueous extracts were fractionated with n-butanol. The leishmanicidal effects of the n-butanolic fraction (BUF) were evaluated in L. (L.) amazonensis promastigotes and amastigotes. BUF was tested against recombinant arginase from both L. (L.) amazonensis and macrophage arginase. Promastigote cultures and infected macrophage cultures were supplemented with L-ornithine to verify arginase inhibition. NMR analysis was used to identify the major components of BUF. RESULTS: BUF showed an EC50 of 51 and 32µg/mL against promastigotes and amastigotes of L. (L.) amazonensis, respectively. BUF contains a mixture of verbascoside and isoverbascoside (7:3 ratio) and is a potent L. (L.) amazonensis arginase inhibitor (IC50=1.2µg/mL), while macrophage arginase was weakly inhibited (IC50>1000µg/mL). The inhibition of arginase by BUF in promastigotes and amastigotes could be demonstrated by culture media supplementation with L-ornithine, a product of the hydrolysis of L-arginine by arginase. CONCLUSIONS: Leishmanicidal effects of the S. cayennensis BUF fraction on L. (L.) amazonensis are associated with selective parasite arginase inhibition.

Functional properties and stability of spray-dried pigments from Bordo grape (Vitis labrusca) winemaking pomace.

The stability of anthocyanin and phenolic compounds, the antioxidant capacity, the antimicrobial activity and the capacity to inhibit arginase from Leishmania were evaluated in spray-dried powders from Bordo grape winemaking pomace extract. The pigments were produced using maltodextrin as the carrier agent at concentrations varying from 10% to 30% and air entrance temperatures varying from 130 to 170°C. A sample of freeze-dried extract without the carrier was also evaluated. The anthocyanins in the spray-dried samples showed good stability during storage, better than the freeze-dried and liquid extracts. The samples were capable of inhibiting the growth of Staphylococcus aureus and Listeria monocytogenes and showed high inhibitory capacity against the enzyme arginase from Leishmania. These results provide evidence that Bordo grapes from the winemaking process have the potential to be used as natural pigments with functional properties.

Inhibition of Leishmania (Leishmania) amazonensis and rat arginases by green tea EGCG, (+)-catechin and (-)-epicatechin: a comparative structural analysis of enzyme-inhibitor interactions.

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 (IC50 = 0.8 µM) followed by (-)-epicatechin (IC50 = 1.8 µM), gallic acid (IC50 = 2.2 µM) and EGCG (IC50 = 3.8 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.

Dietary flavonoids fisetin, luteolin and their derived compounds inhibit arginase, a central enzyme in Leishmania (Leishmania) amazonensis infection.

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.

Leishmanicidal activity of Cecropia pachystachya flavonoids: arginase inhibition and altered mitochondrial DNA arrangement.

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.

The leishmanicidal flavonols quercetin and quercitrin target Leishmania (Leishmania) amazonensis arginase.

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.

Biochemical and biophysical properties of a highly active recombinant arginase from Leishmania (Leishmania) amazonensis and subcellular localization of native enzyme.

Arginase (L-arginine amidinohydrolase, E.C. 3.5.3.1) is a metalloenzyme that catalyses the hydrolysis of L-arginine to L-ornithine and urea. In Leishmania spp., the biological role of the enzyme may be involved in modulating NO production upon macrophage infection. Previously, we cloned and characterized the arginase gene from Leishmania (Leishmania) amazonensis. In the present work, we successfully expressed the recombinant enzyme in E. coli and performed biochemical and biophysical characterization of both the native and recombinant enzymes. We obtained K(M) and V(max) values of 23.9(+/-0.96) mM and 192.3 micromol/min mg protein (+/-14.3), respectively, for the native enzyme. For the recombinant counterpart, K(M) was 21.5(+/-0.90) mM and V(max) was 144.9(+/-8.9) micromol/min mg. Antibody against the recombinant protein confirmed a glycosomal cellular localization of the enzyme in promastigotes. Data from light scattering and small angle X-ray scattering showed that a trimeric state is the active form of the protein. We determined empirically that a manganese wash at room temperature is the best condition to purify active enzyme. The interaction of the recombinant protein with the immobilized nickel also allowed us to confirm the structural disposition of histidine at positions 3 and 324. The determined structural parameters provide substantial data to facilitate the search for selective inhibitors of parasitic sources of arginase, which could subsequently point to a candidate for leishmaniasis therapy.