In vitro antileishmanial activity of ravuconazole, a triazole antifungal drug, as a potential treatment for leishmaniasis.

Objectives: Leishmaniasis, one of the most significant neglected diseases around the world, is caused by protozoan parasites of the Leishmania genus. Nowadays, the available aetiological treatments for leishmaniasis have variable effectiveness and several problems such as serious side effects, toxicity, high cost and an increasing number of resistance cases. Thus, there is an urgent need for safe, oral and cost-effective drugs for leishmaniases. Previously, our group has shown the effect of the ergosterol biosynthesis inhibitors on Leishmania amazonensis. Herein, we showed the effect of ravuconazole against L. amazonensis; ravuconazole is a second-generation triazole antifungal drug that has good bioavailability after oral administration and a long terminal half-life in humans, a broad activity spectrum, high effectiveness in treatment of mycosis and negligible side effects. Methods: Several methodologies were used: cell culture, fluorescence and electron microscopy, high-resolution capillary GC coupled with MS, fluorimetry and flow cytometry. Results: Our results showed that ravuconazole was able to inhibit the proliferation of L. amazonensis promastigotes and intracellular amastigotes in vitro, with single-digit to sub-micromolar IC50 values, causing several alterations in the morphology, ultrastructure, cell viability and physiology of the parasites. The mitochondrion was significantly affected by the treatment, resulting in a collapse of the mitochondrial transmembrane potential that consequently led to inhibition of ATP production, combined with an increase in reactive oxygen species and mitochondrial superoxide production; by transmission electron microscopy, the organelle displayed a completely altered ultrastructure. The treatment changed the lipid profile, showing a profound depletion of the 14-desmethyl endogenous sterol pool. Conclusions: These results suggest that ravuconazole could be an alternative option for the treatment of leishmaniasis.

Copper(I)-Phosphine Polypyridyl Complexes: Synthesis, Characterization, DNA/HSA Binding Study, and Antiproliferative Activity.

A series of copper(I)-phosphine polypyridyl complexes have been investigated as potential antitumor agents. The complexes [Cu(PPh3)2dpq]NO3 (2), [Cu(PPh3)2dppz]NO3 (3), [Cu(PPh3)2dppa]NO3 (4), and [Cu(PPh3)2dppme]NO3 (5) were synthesized by the reaction of [Cu(PPh3)2NO3] with the respective planar ligand under mild conditions. These copper complexes were fully characterized by elemental analysis, molar conductivity, FAB-MS, and NMR, UV-vis, and IR spectroscopies. Interactions between these copper(I)-phosphine polypyridyl complexes and DNA have been investigated using various spectroscopic techniques and analytical methods, such as UV-vis titrations, thermal denaturation, circular dichroism, viscosity measurements, gel electrophoresis, and competitive fluorescent intercalator displacement assays. The results of our studies suggest that these copper(I) complexes interact with DNA in an intercalative way. Furthermore, their high protein binding affinities toward human serum albumin were determined by fluorescence studies. Additionally, cytotoxicity analyses of all complexes against several tumor cell lines (human breast, MCF-7; human lung, A549; and human prostate, DU-145) and non-tumor cell lines (Chinese hamster lung, V79-4; and human lung, MRC-5) were performed. The results revealed that copper(I)-phosphine polypyridyl complexes are more cytotoxic than the corresponding planar ligand and also showed to be more active than cisplatin. A good correlation was observed between the cytostatic activity and lipophilicity of the copper(I) complexes studied here.

Anti-leishmanial evaluation of C2-aryl quinolines: mechanistic insight on bioenergetics and sterol biosynthetic pathway of Leishmania braziliensis.

A series of diverse simple C2-aryl quinolines was synthesized de novo via a straightforward synthesis based on the acid-catalyzed multicomponent imino Diels-Alder reactions. Seven selected quinolines were evaluated at different stages of Leishmania braziliensis parasite. Among them, the 6-ethyl-2-phenylquinoline 5f was able to inhibit the growth of promastigotes of this parasite without affecting the mammalian cells viability and decreasing the number of intracellular L. braziliensis amastigotes on BMDM macrophages. The mechanism of action studied for the selected compound consisted in: (1) alteration of parasite bioenergetics, by disrupting mitochondrial electrochemical potential and alkalinization of acidocalcisomes, and (2) inhibition of ergosterol biosynthetic pathway in promastigote forms. These results validate the efficiency of quinoline molecules as leishmanicide compounds.

Zinc(II)-Sterol Hydrazone Complex as a Potent Anti-Leishmania Agent: Synthesis, Characterization, and Insight into Its Mechanism of Antiparasitic Action.

Searching for new alternatives for treating leishmaniasis, we present the synthesis, characterization, and biological evaluation against Leishmania amazonensis of the new ZnCl2(H3)2 complex. H3 is 22-hydrazone-imidazoline-2-yl-chol-5-ene-3ß-ol, a well-known bioactive molecule functioning as a sterol Δ24-sterol methyl transferase (24-SMT) inhibitor. The ZnCl2(H3)2 complex was characterized by infrared, UV-vis, molar conductance measurements, elemental analysis, mass spectrometry, and NMR experiments. The biological results showed that the free ligand H3 and ZnCl2(H3)2 significantly inhibited the growth of promastigotes and intracellular amastigotes. The IC50 values found for H3 and ZnCl2(H3)2 were 5.2 µM and 2.5 µM for promastigotes, and 543 nM and 32 nM for intracellular amastigotes, respectively. Thus, the ZnCl2(H3)2 complex proved to be seventeen times more potent than the free ligand H3 against the intracellular amastigote, the clinically relevant stage. Furthermore, cytotoxicity assays and determination of selectivity index (SI) revealed that ZnCl2(H3)2 (CC50 = 5 µΜ, SI = 156) is more selective than H3 (CC50 = 10 µΜ, SI = 20). Furthermore, as H3 is a specific inhibitor of the 24-SMT, free sterol analysis was performed. The results showed that H3 was not only able to induce depletion of endogenous parasite sterols (episterol and 5-dehydroepisterol) and their replacement by 24-desalkyl sterols (cholesta-5,7,24-trien-3ß-ol and cholesta-7,24-dien-3ß-ol) but also its zinc derivative resulting in a loss of cell viability. Using electron microscopy, studies on the fine ultrastructure of the parasites showed significant differences between the control cells and parasites treated with H3 and ZnCl2(H3)2. The inhibitors induced membrane wrinkle, mitochondrial injury, and abnormal chromatin condensation changes that are more intense in the cells treated with ZnCl2(H3)2.

Benzylamines as highly potent inhibitors of the sterol biosynthesis pathway in Leishmania amazonensis leading to oxidative stress and ultrastructural alterations.

Leishmaniasis is a neglected disease caused by protozoan parasites of the Leishmania genus. Benzylamines are a class of compounds selectively designed to inhibit the squalene synthase (SQS) that catalyzes the first committed reaction on the sterol biosynthesis pathway. Herein, we studied seven new benzylamines (SBC 37-43) against Leishmania amazonensis. After the first screening of cell viability, two inhibitors (SBC 39 and SBC 40) were selected. Against intracellular amastigotes, SBC 39 and SBC 40 presented selectivity indexes of 117.7 and 180, respectively, indicating high selectivity. Analysis of the sterol composition revealed a depletion of endogenous 24-alkylated sterols such as episterol and 5-dehydroepisterol, with a concomitant accumulation of fecosterol, implying a disturbance in cellular lipid content. This result suggests a blockade of de novo sterol synthesis at the level of SQS and C-5 desaturase. Furthermore, physiological analysis and electron microscopy revealed three main alterations: (1) in the mitochondrion; (2) the presence of lipid bodies and autophagosomes; and (3) the appearance of projections in the plasma membrane. In conclusion, our results support the notion that benzylamines have a potent effect against Leishmania amazonensis and should be an exciting novel pharmaceutical lead for developing new chemotherapeutic alternatives to treat leishmaniasis.

Two squalene synthase inhibitors, E5700 and ER-119884, interfere with cellular proliferation and induce ultrastructural and lipid profile alterations in a Candida tropicalis strain resistant to fluconazole, itraconazole, and amphotericin B.

Three quinuclidine-based squalene synthase (SQS) inhibitors (BPQ-OH, E5700, and ER-119884) were evaluated against five Candida tropicalis strains with different susceptibility profiles to fluconazole (FLC), itraconazole (ITC), terbinafine (TRB), and amphotericin B (AMB). Although the quinuclidine derivatives were inactive against most C. tropicalis strains tested at concentrations up to 16 µg/ml, E5700 and ER-119884 showed antifungal activity against C. tropicalis ATCC 28707, a strain resistant to FLC, ITC, and AMB, with IC(50) and IC(90) values (i.e., the minimum inhibitory concentrations of the drugs determined as the lowest drug concentrations leading to a 50 and 90% of reduction in turbidity at 492 nm, respectively, after 48 h of incubation) of 1 and 4 µg/ml, respectively. Analysis of free sterols showed that non-treated C. tropicalis ATCC 28707 cells contained only 14-methylated sterols and that treatment with E5700 or ER-119884 led to a marked reduction of squalene content and the complete disappearance of the endogenous sterols. The fatty acid and phospholipid profiles in C. tropicalis ATCC 28707 cells grown in the presence of E5700 and ER-119884 were also markedly altered, with a large increase in the content of linolenic acid (C18:3), associated with a reduction in the content of linoleic (C18:2) and oleic (C18:1) acids. Treatment of C. tropicalis ATCC 28707 with E5700 or ER-119884 IC(50) values induced several ultrastructural alterations, including a marked increase in the thickness of the cell wall and the appearance of a large number of electron-dense vacuoles. In conclusion, our results indicated that E5700 and ER-119884 inhibited the growth and altered the lipid prolife and the ultrastructure of a multiple drug-resistant C. tropicalis strain. Therefore, such compounds could act as leads for the development of new treatment options against multidrug resistant Candida species.

[In vitro synergisms among hydrazones, ajoeno and posaconazole against Cryptococcus spp]. / Sinergismo in vitro entre hidrazonas, ajoeno y posaconazol sobre aislados de Cryptococcus spp.

The aim of this study was to assess the in vitro susceptibility to novel antifungal compounds, the steroidal hydrazones, and to compare their antifungal activity and synergistic effects with other compounds, such as ajoeno and posaconazole on Cryptocococus spp isolates. Three Cryptococcus strains were used for this study (42794, 4050 and 44192) and their antifungal sensitivity and synergistic effects with ajoeno and posaconazole were evaluated according to the CLSI protocol number M27-A2. Candida albicans (ATCC 90028) and Candida parapsilosis (ATCC 22019) were used as controls. A plateau effect with hydrazones (H1, H2, H3, H4) was observed after 10 microM (CMI). However, with H4 only a mild inhibition on the growth was obtained. Combining hydrazone and ajoeno, CMI values between 25 and 50 microM were obtained. The highest inhibitions values were obtained with posaconazole and a CMI value of 6 microM for the strains 42794 and 44192, and a CMI value of 20 microM for the strain 4050. Synergy was observed combining posaconazole with ajoeno, ajoeno with hydrazone 3 and posaconazole with hydrazone 3. Fractional inhibitory concentrations were 0.24, 0.16 and 0.09 respectively, which might indicate a synergistic effect. Important synergistic effects were obtained with posaconazole and ajoeno, ajoeno and hydrazone 3 and posaconazole with hydrazone 3, which would be very useful for clinical trials in the future.

Metallodrugs for the Treatment of Trypanosomatid Diseases: Recent Advances and New Insights.

Trypanosomatid parasites are responsible for many Neglected Tropical Diseases (NTDs). NTDs are a group of illnesses that prevail in low-income populations, such as in tropical and subtropical areas of Africa, Asia, and the Americas. The three major human diseases caused by trypanosomatids are African trypanosomiasis, Chagas disease and leishmaniasis. There are known drugs for the treatment of these diseases that are used extensively and are affordable; however, the use of these medicines is limited by several drawbacks such as the development of chemo-resistance, side effects such as cardiotoxicity, low selectivity, and others. Therefore, there is a need to develop new chemotherapeutic against these tropical parasitic diseases. Metal-based drugs against NTDs have been discussed over the years as alternative ways to overcome the difficulties presented by approved antiparasitic agents. The study of late transition metal-based drugs as chemotherapeutics is an exciting research field in chemistry, biology, and medicine due to the ability to develop multitarget antiparasitic agents. The evaluation of the late transition metal complexes for the treatment of trypanosomatid diseases is provided here, as well as some insights about their mechanism of action.

Amiodarone destabilizes intracellular Ca2+ homeostasis and biosynthesis of sterols in Leishmania mexicana.

Leishmaniasis represents a serious public health problem worldwide. The first line of treatment is based on glucantime and pentostan, which generate toxic effects in treated patients. We have recently shown that amiodarone, frequently used as an antiarrhythmic, possesses activity against Trypanosoma cruzi through the disruption of mitochondrial Ca(2+) homeostasis and the inhibition of parasite ergosterol biosynthesis, specifically at the level of oxidosqualene cyclase activity (G. Benaim, J. Sanders, Y. Garcia-Marchan, C. Colina, R. Lira, A. Caldera, G. Payares, C. Sanoja, J. Burgos, A. Leon-Rossell, J. Concepcion, A. Schijman, M. Levin, E. Oldfield, and J. Urbina, J. Med. Chem. 49:892-899, 2006). Here we show that at therapeutic concentrations, amiodarone has a profound effect on the viability of Leishmania mexicana promastigotes. Additionally, its effect on the viability of the parasite was greater against intracellular amastigotes than against promastigotes, and it did not affect the host cell. Using fluorimetric and confocal microscopy techniques, we also demonstrated that the mechanism of action of amiodarone was related to the disruption of intracellular Ca(2+) homeostasis through a direct action not only on the mitochondria but also on the acidocalcisomes. On the other hand, analysis of the free sterols in promastigotes incubated with amiodarone showed that this drug also affected the biosynthesis of 5-dehydroepisterol, which results in squalene accumulation, thus suggesting that amiodarone inhibits the squalene epoxidase activity of the parasite. Taken together, the results obtained in the present work point to a more general effect of amiodarone in trypanosomatids, opening potential therapeutic possibilities for this infectious disease.

Synthesis and characterization of platinum-sterol hydrazone complexes with biological activity against Leishmania (L.) mexicana.

Leishmaniasis is a parasitic zoonosis caused by protozoans of the genus Leishmania transmitted by insects known as phlebotomines, which are found in wild or urban environments. The disease occurs in tropical and sub-tropical areas, mainly in Asia, Europe, Africa and the Americas. At present, there is no effective treatment for this disease. In the search for new rational chemotherapeutic alternatives, two novel trans [Pt(Hpy1)(2)(Cl)(2)] (1) and trans [Pt(Hpy2)(2) (Cl)(2)] (2) complexes were synthesized by the reaction of K(2)PtCl(4) with sterol hydrazone ligands 20-hydrazone-pyridin-2-yl-5alpha-pregnan-3beta-ol (Hpy1) and 22-hydrazone-pyridin-2-yl-chol-5-ene-3beta-ol (Hpy2). These organic compounds are specific inhibitors of sterol methyl transferase (SMT). The new platinum complexes were characterized by a combination of ESI-MS (electrospray ionization-mass spectroscopy), UV-vis, infrared and NMR spectroscopies; elemental analysis and molar conductivity. Promastigotes of Leishmania (L.) mexicana were treated for 48 h with 10 microM of the sterol hydrazones Hpy1 or Hpy2 alone or coordinated to Pt. Hpy1 produced higher leishmanistatic activity than Hpy2 (39% growth inhibition vs. 16%), which significatively increased (71%, p<0.001) when the complex trans-[Pt(Hpy1)(2)(Cl)(2)] was used. This complex represents a new chemotherapeutic alternative to be evaluated in depth in experimental models of leishmaniasis.