Lantana camara L. induces a multi-targeted cell death process in Leishmania amazonensis.

ETNOPHARMACOLOGICAL RELEVANCE: Lantana camara L. is a species known for its broad spectrum of bioactivities and is commonly used in folk therapy to address inflammatory, dermatological, gastrointestinal, intestinal worms and protozoan diseases. It boasts a diverse array of secondary metabolites such as terpenes, flavonoids, and saponins. However, despite its rich chemical profile, there remains a scarcity of studies investigating its antileishmanial properties. AIM OF THE STUDY: This research aims to explore the antileishmanial potential of L. camara, focusing also on its mechanism of action against Leishmania amazonensis. MATERIAL AND METHODS: The ethanolic extract of L. camara leaves (LCE) was obtained through static maceration, and its phytoconstituents were identified using UFLC-QTOF-MS. The colorimetric MTT method was conducted to determine the effect of LCE on promastigotes of L. amazonensis and murine macrophages. The anti-amastigote activity was evaluated by counting intracellular parasites in macrophages after Giemsa staining. Additionally, investigations into the mechanisms underlying its action were conducted using cellular and biochemical approaches. RESULTS: LCE exhibited significant activity against both promastigotes and intracellular amastigotes of L. amazonensis, with IC50 values of 12.20 µg/mL ± 0.12 and 7.09 µg/mL ± 1.24, respectively. These IC50 values indicate very promising antileishmanial activity, comparable to those found for the positive control miltefosine (5.10 µg/mL ± 1.79 and 8.96 µg/mL ± 0.50, respectively). Notably, LCE exhibited negligible cytotoxicity on macrophages (IC50 = 223.40 µg/mL ± 47.02), demonstrating selectivity towards host cells (SI = 31.50). The antileishmanial activity of LCE involved a multi-targeted cell death process, characterized by morphological and ultrastructural alterations observed through SEM and TEM analyses, as well as oxidative effects evidenced by the inhibition of trypanothione reductase, elevation of ROS and lipid levels, and mitochondrial dysfunction evaluated using DTNB, H2DCFDA, Nile red, and JC-1 assays. Additionally, extraction of ergosterol and double labeling with annexin V and PI revealed modifications to the organization and permeability of the treated parasite's plasma membrane. LCE was found to consist predominantly of terpenes, with lantadenes A, B, and C being among the eleven compounds identified through UFLC-QTOF-MS analysis. CONCLUSIONS: The extract of L. camara presents a diverse array of chemical constituents, prominently featuring high terpene content, which may underlie its antileishmanial properties through a combination of apoptotic and non-apoptotic mechanisms of cell death induced by LCE. This study underscores the therapeutic potential of L. camara as a candidate for antileishmanial treatment, pending further validation.

4-Quinolinylhydrazone analogues kill Leishmania (Leishmania) amazonensis by inducing apoptosis and mitochondria-dependent pathway cell death.

Despite efforts, available alternatives for the treatment of leishmaniasis are still scarce. In this work we tested a class of 15 quinolinylhydrazone analogues and presented data that support the use of the most active compound in cutaneous leishmaniasis caused by Leishmania amazonensis. In general, the compounds showed activity at low concentrations for both parasitic forms (5.33-37.04 µM to promastigotes, and 14.31-61.98 µM to amastigotes). In addition, the best compound (MHZ15) is highly selective for the parasite. Biochemical studies indicate that the treatment of promastigotes with MHZ15 leads the loss of mitochondrial potential and increase in ROS levels as the primary effects, which triggers accumulation of lipid droplets, loss of plasma membrane integrity and apoptosis hallmarks, including DNA fragmentation and phosphatidylserine exposure. These effects were similar in the intracellular form of the parasite. However, in this parasitic form there is no change in plasma membrane integrity in the observed treatment time, which can be attributed to metabolic differences and the resilience of the amastigote. Also, ultrastructural changes such as vacuolization suggesting autophagy were observed. The in vivo effectiveness of MHZ15 in the experimental model of cutaneous leishmaniasis was carried out in mice of the BALB/c strain infected with L. amazonensis. The treatment by intralesional route showed that MHZ15 acted with great efficiency with significantly reduction in the parasite load in the injured paws and draining lymph nodes, without clinical signs of distress or compromise of animal welfare. In vivo toxicity was also evaluated and null alterations in the levels of hepatic enzymes aspartate aminotransferase, and alanine aminotransferase was observed. The data presented herein demonstrates that MHZ15 exhibits a range of favorable characteristics conducive to the development of an antileishmanial agent.

Subtilisin of Leishmania amazonensis as Potential Druggable Target: Subcellular Localization, In Vitro Leishmanicidal Activity and Molecular Docking of PF-429242, a Subtilisin Inhibitor.

Subtilisin proteases, found in all organisms, are enzymes important in the post-translational steps of protein processing. In Leishmania major and L. donovani, this enzyme has been described as essential to their survival; however, few compounds that target subtilisin have been investigated for their potential as an antileishmanial drug. In this study, we first show, by electron microscopy and flow cytometry, that subtilisin has broad localization throughout the cytoplasm and membrane of the parasite in the promastigote form with foci in the flagellar pocket. Through in silico analysis, the similarity between subtilisin of different Leishmania species and that of humans were determined, and based on molecular docking, we evaluated the interaction capacity of a serine protease inhibitor against both life cycle forms of Leishmania. The selected inhibitor, known as PF-429242, has already been used against the dengue virus, arenaviruses, and the hepatitis C virus. Moreover, it proved to have antilipogenic activity in a mouse model and caused hypolipidemia in human cells in vitro. Here, PF-429242 significantly inhibited the growth of L. amazonensis promastigotes of four different strains (IC50 values = 3.07 ± 0.20; 0.83 ± 0.12; 2.02 ± 0.27 and 5.83 ± 1.2 µM against LTB0016, PH8, Josefa and LV78 strains) whilst having low toxicity in the host macrophages (CC50 = 170.30 µM). We detected by flow cytometry that there is a greater expression of subtilisin in the amastigote form; however, PF-429242 had a low effect against this intracellular form with an IC50 of >100 µM for intracellular amastigotes, as well as against axenic amastigotes (94.12 ± 2.8 µM for the LV78 strain). In conclusion, even though PF-429242 does not affect the intracellular forms, this drug will serve as a tool to explore pharmacological and potentially leishmanicidal targets.

PF-429242, a Subtilisin Inhibitor, Is Effective in vitro Against Leishmania infantum.

PF-429242 is an inhibitor of subtilisin, an important protease found in Leishmania. However, studies regarding the effect of PF-429242 on Leishmania are scarce. In this work we evaluated the antileishmanial effect of PF-429242 against Leishmania infantum and the mechanism involved in the death of the parasite. PF-429242 had low toxicity against mammalian cells (peritoneal macrophages) (CC50 = 189.07 µM) and presented activity against L. infantum promastigotes (IC50 = 2.78 µM) and intracellular amastigotes (IC50 = 14.07 µM), indicating selectivity toward the parasite. Transmission electron microscopy (TEM), as well as staining of L. infantum promastigotes with MitoTracker® Red, rhodamine 123 and MitoSOX, revealed that the mitochondria was a potential target of PF-429242. In addition, PF-429242 caused an accumulation of neutral lipids in promastigotes, which was demonstrated by Nile Red staining and TEM, and induced oxidative stress (H2DCFDA staining). Furthermore the formation of autophagic vacuoles in L. infantum promastigotes was observed by MDC staining and TEM. However, the killing induced by PF-429242 in L. infantum promastigotes appeared to be unrelated to apoptosis and/or necrosis as there was no phosphatidylserine externalization, DNA fragmentation or alterations in the permeability of the parasite plasma membrane, as assessed by annexin V-FITC, TUNEL and propidium iodide staining, respectively. The morphological and ultrastructural evaluation of the promastigotes by optical microscopy, scanning electron microscopy (SEM) and TEM, revealed the presence of parasites with flagellar defects. TEM analysis of the intracellular amastigotes indicated that mitochondrial damage and autophagy could also be involved in the death of these forms after treatment with PF-429242. In addition, PF-429242 treatment stimulated NO production from infected macrophage, but only at a high concentration (100 µM), as well as an increase of TNF levels after treatment with 10 µM of PF-429242. The compound did not stimulate ROS or IL-10 production. Together, these data highlight the antileishmanial potential of PF-429242, inducing several cellular alterations in the parasite, such as mitochondrial damage, neutral lipids accumulation, oxidative stress and autophagy which culminate in the death of L. infantum, as well as modulating host cellular responses that favor the development of an immune response against the parasite.

Vitamin D increases killing of intracellular Leishmania amazonensis in vitro independently of macrophage oxidative mechanisms.

Vitamin D has been reported to activate macrophage microbicidal mechanisms by inducing the production of antimicrobial peptides and nitric oxide (NO), but conversely has been shown to contribute to a greater susceptibility to Leishmania amazonensis infection in mice. Thus, this study aimed to evaluate the role of vitamin D during intracellular infection with L. amazonensis by examining its effect on macrophage oxidative mechanisms and parasite survival in vitro. Vitamins D2 and D3 significantly inhibited promastigote and amastigote growth in vitro. Vitamin D3 was not able to induce NO and reactive oxygen species (ROS) production in uninfected macrophages or macrophages infected with L. amazonensis. In addition, vitamin D3 in combination with interferon (IFN)-γ did not enhance amastigote killing and in fact, significantly reduced NO and ROS production when compared with the effect of IFN-γ alone. In this study, we demonstrated that vitamin D directly reduces parasite growth in infected macrophages (approximately 50-60% at 50 µm) but this effect is independent of the activation of macrophage oxidative mechanisms. These findings will contribute to a better understanding of the role of vitamin D in cutaneous leishmaniasis.

Leishmanicidal therapy targeted to parasite proteases.

Leishmaniasis is considered a serious public health problem and the current available therapy has several disadvantages, which makes the search for new therapeutic targets and alternative treatments extremely necessary. In this context, this review focuses on the importance of parasite proteases as target drugs against Leishmania parasites, as a chemotherapy approach. Initially, we discuss about the current scenario for the treatment of leishmaniasis, highlighting the main drugs used and the problems related to their use. Subsequently, we describe the inhibitors of major proteases of Leishmania already discovered, such as Compound s9 (aziridine-2,3-dicarboxylate), Compound 1c (benzophenone derivative), Au2Phen (gold complex), AubipyC (gold complex), MDL 28170 (dipeptidyl aldehyde), K11777, Hirudin, diazo-acetyl norleucine methyl ester, Nelfinavir, Saquinavir, Nelfinavir, Saquinavir, Indinavir, Saquinavir, GNF5343 (azabenzoxazole), GNF6702 (azabenzoxazole), Benzamidine and TPCK. Next, we discuss the importance of the protease gene to parasite survival and the aspects of the validation of proteases as target drugs, with emphasis on gene disruption. Then, we describe novel important strategies that can be used to support the research of new antiparasitic drugs, such as molecular modeling and nanotechnology, whose main targets are parasitic proteases. And finally, we discuss possible perspectives to improve drug development. Based on all findings, proteases could be considered potential targets against leishmaniasis.

High selective antileishmanial activity of vanadium complex with stilbene derivative.

Leishmaniasis is a group of disease caused by different species of the parasite Leishmania affecting millions of people worldwide. Conventional therapy relies on multiple parenteral injections with pentavalent antimonials which exhibit high toxicity and various side effects have been reported. Hence, the research for an effective and low toxic effect drug is necessary. In the present work, the synthesis, spectroscopic and analytical characterizations of stilbene derivative (H2Salophen) and its vanadium complex (VOSalophen) are reported. Besides the chemical ancillary information, investigation of the leishmanicidal effects of these compounds were provided. The biological assays against promastigote and amastigote forms of L. amazonensis have been shown that VOSalophen exhibited a strong antiparasitic activity (IC50 of 6.65 and 3.51 µM, respectively). Furthermore, the leishmanicidal activity was concentration and time-dependent. Regarding toxicity and selectivity on mammalian cells, VOSalophen have not caused significant damage to human erythrocytes in all concentrations tested and VOSalophen was almost seven times more destructive for the intracellular parasite than for macrophages. Furthermore, the leishmanicidal activity of VOSalophen in promastigote forms of L. amazonensis could be associated to mitochondrial dysfunction and increase of the reactive oxygen species (ROS) production. In L. amazonensis-infected macrophages, VOSalophen induces ROS production and a microbicidal action NO-dependent. Our biological results indicate the effective and selective action of VOSalophen against L. amazonensis and the leishmanicidal effect can be associated to parasite disorders and immumodulatory effects.

Topical and intraluminal Carolina Rinse Solution in p44/42 and p38 MAP Kinase activation profile in rabbit jejunum after ischemia and reperfusion / Uso tópico e intraluminal da Solução de Carolina Rinse no perfil de ativação das MAP quinases p44/42 e p38 no jejuno de coelhos após isquemia e reperfusão

The effects of topical and intraluminal Carolina Rinse Solution (CRS) in p44/42 (ERK 1/2) and p38 MAPK activation profile, in rabbit jejunum after ischemia and reperfusion (I/R), were investigated in this study. Fifteen New Zealand rabbits were allocated in three groups: Sham-operated (A), Ischemia and reperfusion (B) and CRS (C). Groups B and C were subjected to one hour of ischemia and two hours of reperfusion. In group C, ten minutes prior to reperfusion, the bowel lumen was filled with CRS, and the segment was immersed in CRS until reperfusion onset. Ischemia and reperfusion stimulated the phosphorylation of the p44/42 MAPK and p38 MAPK pathways in some layers of jejunum. Progressive activation of p44/42 MAPK was chiefly localized in the crypts of Lieberkühn, circular and longitudinal muscle layers, whereas p38 MAPK was prominently activated in myenteric plexus and both muscle layers. The results of this research indicate that the chosen model of topical and intraluminal CRS does not interfere in p44/42 and p38 MAPK activation profile in rabbit jejunum subjected to I/R.

Os efeitos do uso tópico e intraluminal da Solução de Carolina Rinse (CRS), no perfil de ativação das MAP quinases p44/42 (ERK 1/2) e p38, no jejuno de coelhos após isquemia e reperfusão (I/R), foram investigados neste estudo. Quinze coelhos da raça Nova Zelândia foram alocados em três grupos: Instrumentado (A), Isquemia e Reperfusão (B) e CRS (C). Os grupos B e C foram submetidos a uma hora de isquemia e duas horas de reperfusão. No grupo C, dez minutos antes da reperfusão, o lúmen do segmento isolado foi preenchido com CRS e o segmento foi imerso em CRS até o início da reperfusão. A isquemia e reperfusão resultou em estímulo da fosforilação das MAP quinases p44/42 e p38 em algumas camadas do jejuno. A ativação progressiva de p44/42 ocorreu principalmente nas criptas de Lieberkühn e camadas musculares longitudinal e circular, enquanto p38 foi ativada principalmente no plexo mioentérico e em ambas as camadas musculares. Os resultados deste trabalho indicam que o modelo escolhido de uso tópico e intraluminal de CRS não interfere no perfil de ativação das MAP quinases p44/42 e p38 no jejuno de coelhos submetidos à I/R.