New Insights into the Mechanism of Action of the Cyclopalladated Complex (CP2) in Leishmania: Calcium Dysregulation, Mitochondrial Dysfunction, and Cell Death.

The current treatment of leishmaniasis is based on a few drugs that present several drawbacks, such as high toxicity, difficult administration route, and low efficacy. These disadvantages raise the necessity to develop novel antileishmanial compounds allied with a comprehensive understanding of their mechanisms of action. Here, we elucidate the probable mechanism of action of the antileishmanial binuclear cyclopalladated complex [Pd(dmba)(µ-N3)]2 (CP2) in Leishmania amazonensis. CP2 causes oxidative stress in the parasite, resulting in disruption of mitochondrial Ca2+ homeostasis, cell cycle arrest at the S-phase, increasing the reactive oxygen species (ROS) production and overexpression of stress-related and cell detoxification proteins, and collapsing the Leishmania mitochondrial membrane potential, and promotes apoptotic-like features in promastigotes, leading to necrosis, or directs programmed cell death (PCD)-committed cells toward necrotic-like destruction. Moreover, CP2 reduces the parasite load in both liver and spleen in Leishmania infantum-infected hamsters when treated for 15 days with 1.5 mg/kg body weight/day CP2, expanding its potential application in addition to the already known effectiveness on cutaneous leishmaniasis for the treatment of visceral leishmaniasis, showing the broad spectrum of action of this cyclopalladated complex. The data presented here bring new insights into the CP2 molecular mechanisms of action, assisting the promotion of its rational modification to improve both safety and efficacy.

Insights into agar and secondary metabolite pathways from the genome of the red alga Gracilaria domingensis (Rhodophyta, Gracilariales).

Gracilariales is a clade of florideophycean red macroalgae known for being the main source of agar. We present a de novo genome assembly and annotation of Gracilaria domingensis, an agarophyte alga with flattened thallus widely distributed along Central and South American Atlantic intertidal zones. In addition to structural analysis, an organizational comparison was done with other Rhodophyta genomes. The nuclear genome has 78 Mbp, with 11,437 predicted coding genes, 4,075 of which did not have hits in sequence databases. We also predicted 1,567 noncoding RNAs, distributed in 14 classes. The plastid and mitochondrion genome structures were also obtained. Genes related to agar synthesis were identified. Genes for type II galactose sulfurylases could not be found. Genes related to ascorbate synthesis were found. These results suggest an intricate connection of cell wall polysaccharide synthesis and the redox systems through the use of L-galactose in Rhodophyta. The genome of G. domingensis should be valuable to phycological and aquacultural research, as it is the first tropical and Western Atlantic red macroalgal genome to be sequenced.

Chlorin, Phthalocyanine, and Porphyrin Types Derivatives in Phototreatment of Cutaneous Manifestations: A Review.

Recent scientific research has shown the use of chlorin, phthalocyanines, and porphyrins derivatives as photosensitizers in photodynamic therapy in the treatment of various pathologies, including some of the major skin diseases. Thus, the main goal of this critical review is to catalog the papers that used these photosensitizers in the treatment of acne vulgaris, psoriasis, papillomavirus infections, cutaneous leishmaniasis, and skin rejuvenation, and to explore the photodynamic therapy mechanisms against these conditions alongside their clinical benefits.

Efficacy of a Binuclear Cyclopalladated Compound Therapy for Cutaneous Leishmaniasis in the Murine Model of Infection with Leishmania amazonensis and Its Inhibitory Effect on Topoisomerase 1B.

Leishmaniasis is a disease found throughout the (sub)tropical parts of the world caused by protozoan parasites of the Leishmania genus. Despite the numerous problems associated with existing treatments, pharmaceutical companies continue to neglect the development of better ones. The high toxicity of current drugs combined with emerging resistance makes the discovery of new therapeutic alternatives urgent. We report here the evaluation of a binuclear cyclopalladated complex containing Pd(II) and N,N'-dimethylbenzylamine (Hdmba) against Leishmania amazonensis The compound [Pd(dmba)(µ-N3)]2 (CP2) inhibits promastigote growth (50% inhibitory concentration [IC50] = 13.2 ± 0.7 µM) and decreases the proliferation of intracellular amastigotes in in vitro incubated macrophages (IC50 = 10.2 ± 2.2 µM) without a cytotoxic effect when tested against peritoneal macrophages (50% cytotoxic concentration = 506.0 ± 10.7 µM). In addition, CP2 was also active against T. cruzi intracellular amastigotes (IC50 = 2.3 ± 0.5 µM, selective index = 225), an indication of its potential for use in Chagas disease therapy. In vivo assays using L. amazonensis-infected BALB/c showed an 80% reduction in parasite load compared to infected and nontreated animals. Also, compared to amphotericin B treatment, CP2 did not show any side effects, which was corroborated by the analysis of plasma levels of different hepatic and renal biomarkers. Furthermore, CP2 was able to inhibit Leishmania donovani topoisomerase 1B (Ldtopo1B), a potentially important target in this parasite. (This study has been registered at ClinicalTrials.gov under identifier NCT02169141.).

The 2',4'-dihydroxychalcone could be explored to develop new inhibitors against the glycerol-3-phosphate dehydrogenase from Leishmania species.

The enzyme glycerol-3-phosphate dehydrogenase (G3PDH) from Leishmania species is considered as an attractive target to design new antileishmanial drugs and a previous in silico study reported on the importance of chalcones to achieve its inhibition. Here, we report the identification of a synthetic chalcone in our in vitro assays with promastigote cells from Leishmania amazonensis, its biological activity in animal models, and docking followed by molecular dynamics simulation to investigate the molecular interactions and structural patterns that are crucial to achieve the inhibition complex between this compound and G3PDH. A molecular fragment of this natural product derivative can provide new inhibitors with increased potency and selectivity.

Synthesis and evaluation of novel prenylated chalcone derivatives as anti-leishmanial and anti-trypanosomal compounds.

Chalcones form a class of compounds that belong to the flavonoid family and are widely distributed in plants. Their simple structure and the ease of preparation make chalcones attractive scaffolds for the synthesis of a large number of derivatives enabling the evaluation of the effects of different functional groups on biological activities. In this Letter, we report the successful synthesis of a series of novel prenylated chalcones via Claisen-Schmidt condensation and the evaluation of their effect on the viability of the Trypanosomatidae parasites Leishmania amazonensis, Leishmania infantum and Trypanosoma cruzi.

Leishmanicidal activities of novel synthetic furoxan and benzofuroxan derivatives.

A novel series of furoxan (1,2,5-oxadiazole 2-oxide) (compounds 3, 4a and -b, 13a and -b, and 14a to -f) and benzofuroxan (benzo[c][1,2,5]oxadiazole 1-oxide) (compounds 7 and 8a to -c) derivatives were synthesized, characterized, and evaluated for in vitro activity against promastigote and intracellular amastigote forms of Leishmania amazonensis. The furoxan derivatives exhibited the ability to generate nitric oxide at different levels (7.8% to 27.4%). The benzofuroxan derivative 8a was able to increase nitrite production in medium supernatant from murine macrophages infected with L. amazonensis at 0.75 mM after 48 h. Furoxan and benzofuroxan derivatives showed remarkable leishmanicidal activity against both promastigote and intracellular amastigote forms. Compounds 8a, 14a and -b, and 14d exerted selective leishmanicidal activities superior to those of amphotericin B and pentamidine. In vitro studies at pH 5.4 reveal that compound 8a is stable until 8 h and that compound 14a behaves as a prodrug, releasing the active aldehyde 13a. These compounds have emerged as promising novel drug candidates for the treatment of leishmaniasis.

Peptide selection via phage display to inhibit Leishmania-macrophage interactions.

Introduction: Leishmaniasis comprises a complex group of diseases caused by protozoan parasites from the Leishmania genus, presenting a significant threat to human health. Infection starts by the release into the skin of metacyclic promastigote (MP) form of the parasite by an infected sand fly. Soon after their release, the MPs enter a phagocytic host cell. This study focuses on finding peptides that can inhibit MP-phagocytic host cell interaction. Methods: We used a phage display library to screen for peptides that bind to the surface of L. amazonensis (causative agent for cutaneous leishmaniasis) and L. infantum (causative agent for cutaneous and visceral leishmaniasis) MPs. Candidate peptide binding to the MP surface and inhibition of parasite-host cell interaction were tested in vitro. Peptide Inhibition of visceral leishmaniasis development was assessed in BALB/c mice. Results: The selected L. amazonensis binding peptide (La1) and the L. infantum binding peptide (Li1) inhibited 44% of parasite internalization into THP-1 macrophage-like cells in vitro. While inhibition of internalization by La1 was specific to L. amazonensis, Li1 was effective in inhibiting internalization of both parasite species. Importantly, Li1 inhibited L. infantum spleen and liver infection of BALB/c mice by 84%. Conclusion: We identified one peptide that specifically inhibits L. amazonensis MP infection of host cells and another that inhibits both, L. amazonensis and L. infantum, MP infection. Our findings suggest a promising path for the development of new treatments and prevention of leishmaniasis.

Antiprotozoal activity of quinonemethide triterpenes from Maytenus ilicifolia (Celastraceae).

The present study describes the leishmanicidal and trypanocidal activities of two quinonemethide triterpenes, maytenin (1) and pristimerin (2), isolated from Maytenus ilicifolia root barks (Celastraceae). The compounds were effective against the Trypanosomatidae Leishmania amazonensis and Leishmania chagasi and Trypanosoma cruzi, etiologic agents of leishmaniasis and Chagas' disease, respectively. The quinonemethide triterpenes 1 and 2 exhibited a marked in vitro leishmanicidal activity against promastigotes and amastigotes with 50% inhibitory concentration (IC(50)) values of less than 0.88 nM. Both compounds showed IC(50) lower than 0.3 nM against Trypanosoma cruzi epimastigotes. The selectivity indexes (SI) based on BALB/c macrophages for L. amazonensis and L. chagasi were 243.65 and 46.61 for (1) and 193.63 and 23.85 for (2) indicating that both compounds presented high selectivity for Leishmania sp. The data here presented suggests that these compounds should be considered in the development of new and more potent drugs for the treatment of leishmaniasis and Chagas' disease.

Antileishmanial activity of tetra-cationic porphyrins with peripheral Pt(II) and Pd(II) complexes mediated by photodynamic therapy.

Leishmaniasis is a neglected disease that impacts more than one billion people in endemic areas of the globe. Several drawbacks are associated with the currently existing drugs for treatment such as low effectiveness, toxicity, and the emergence of resistant strains that demonstrate the importance of looking for novel therapeutic alternatives. Photodynamic therapy (PDT) is a promising novel alternative for cutaneous leishmaniasis treatment because its topical application avoids potential side effects generally associated with oral/parenteral application. A light-sensitive compound known as photosensitizer (PS) interacts with light and molecular oxygen to generate reactive oxygen species (ROS), which promote cell death by oxidative stress through PDT approaches. Here, for the first time, we demonstrate the antileishmanial effect of tetra-cationic porphyrins with peripheral Pt(II)- and Pd(II)-polypyridyl complexes using PDT. The isomeric tetra-cationic porphyrins in the meta positions, 3-PtTPyP, and 3-PdTPyP, exhibited the highest antiparasitic activity against promastigote (IC50-pro = 41.8 nM and 46.1 nM, respectively) and intracellular amastigote forms (IC50-ama = 27.6 nM and 38.8 nM, respectively) of L. amazonensis under white light irradiation (72 J cm-2) with high selectivity (SI > 50) for both forms of parasites regarding mammalian cells. In addition, these PS induced the cell death of parasites principally by a necrotic process in the presence of white light by mitochondrial and acidic compartments accumulation. This study showed that porphyrins 3-PtTPyP and 3-PdTPyP displayed a promising antileishmanial-PDT activity with potential application for cutaneous leishmaniasis treatment.

Development of New Leishmanicidal Compounds via Bioconjugation of Antimicrobial Peptides and Antileishmanial Guanidines.

Leishmaniasis refers to a collection of diseases caused by protozoa from the Leishmania genus. These diseases, along with other parasitic afflictions, pose a significant public health issue, particularly given the escalating number of at-risk patients. This group includes immunocompromised individuals and those residing in impoverished conditions. The treatment of leishmaniasis is crucial, particularly in light of the mortality rate associated with nontreatment, which stands at 20-30,000 deaths per year globally. However, the therapeutic options currently available are limited, often ineffective, and potentially toxic. Consequently, the pursuit of new therapeutic alternatives is warranted. This study aims to design, synthesize, and evaluate the leishmanicidal activity of antimicrobial peptides functionalized with guanidine compounds and identify those with enhanced potency and selectivity against the parasite. Accordingly, three bioconjugates were obtained by using the solid-phase peptide synthesis protocol. Each proved to be more potent against intracellular amastigotes than their respective peptide or guanidine compounds alone and demonstrated higher selectivity to the parasites than to the host cells. Thus, the conjugation strategy employed with these compounds effectively contributes to the development of new molecules with leishmanicidal activity.

Antiprotozoal sesquiterpene pyridine alkaloids from Maytenus ilicifolia.

As part of a bioprospecting program aimed at the discovery of antiprotozoal agents from the Brazilian flora, two new sesquiterpene pyridine alkaloids, ilicifoliunines A (1) and B (2), along with the known alkaloids aquifoliunine E-I (3) and mayteine (4), were isolated from the root bark of Maytenus ilicifolia. The structures of 1 and 2 were established on the basis of spectroscopic data interpretation. Alkaloid 3 displayed potent in vitro antiprotozoal activity against Leishmania chagasi and Trypanosoma cruzi, with IC(50) values of 1.4 and 41.9 µM, respectively, as well as low cytotoxicity against murine peritoneal macrophages (IC(50) of 1.8 mM).

Baccharis trimera (Less.) DC leaf derivatives and eupatorin activities against Leishmania amazonensis.

Natural products have been largely explored as treatments for leishmaniasis, neglected diseases with few toxic therapeutic options, as scaffolds for the development of new drugs. Herein, derivatives from the aerial parts of Baccharis trimera (Less.) DC (extract and its fractions) were evaluated against Leishmania amazonensis and macrophage cells. The ethyl acetate extract was fractionated by solid-phase extraction, resulting in eight fractions (F1-F8). Fractions F3-4 were further separated into 149 subfractions; subfraction 148 (IC50-PRO = 1.56 ± 0.1 µg mL-1) was selected for purification and constituent(s) characterization by high-performance liquid chromatography, as well as 1H and 13C nuclear magnetic resonance spectroscopy. The flavonoid eupatorin (3',5-dihydroxy-4',6,7-trimethoxyflavone) was identified. This compound was 3.7 times more effective against intracellular amastigotes (IC50-AMA = 1.6 ± 0.1 µM) than amphotericin B and presented low cytotoxicity (CC50 > 100 µM), being almost 62 times more selective for the parasite, showing great potential in drug development for cutaneous leishmaniasis treatment.

Novel Selective and Low-Toxic Inhibitor of LmCPB2.8ΔCTE (CPB) One Important Cysteine Protease for Leishmania Virulence.

Leishmaniasis is a highly prevalent, yet neglected disease caused by protozoan parasites of the genus Leishmania. In the search for newer, safer, and more effective antileishmanial compounds, we herein present a study of the mode of action in addition to a detailed structural and biological characterization of LQOF-G6 [N-benzoyl-N'-benzyl-N″-(4-tertbutylphenyl)guanidine]. X-ray crystallography and extensive NMR experiments revealed that LQOF-G6 nearly exclusively adopts the Z conformation stabilized by an intramolecular hydrogen bond. The investigated guanidine showed selective inhibitory activity on Leishmania major cysteine protease LmCPB2.8ΔCTE (CPB) with ~73% inhibition and an IC50-CPB of 6.0 µM. This compound did not show any activity against the mammalian homologues cathepsin L and B. LQOF-G6 has been found to be nontoxic toward both organs and several cell lines, and no signs of hepatotoxicity or nephrotoxicity were observed from the analysis of biochemical clinical plasma markers in the treated mice. Docking simulations and experimental NMR measurements showed a clear contribution of the conformational parameters to the strength of the binding in the active site of the enzyme, and thus fit the differences in the inhibition values of LQOF-G6 compared to the other guanidines. Furthermore, the resulting data render LQOF-G6 suitable for further development as an antileishmanial drug.

Design, synthesis and biological evaluation of N-oxide derivatives with potent in vivo antileishmanial activity.

Leishmaniasis is a neglected disease that affects 12 million people living mainly in developing countries. Herein, 24 new N-oxide-containing compounds were synthesized followed by in vitro and in vivo evaluation of their antileishmanial activity. Compound 4f, a furoxan derivative, was particularly remarkable in this regard, with EC50 value of 3.6 µM against L. infantum amastigote forms and CC50 value superior to 500 µM against murine peritoneal macrophages. In vitro studies suggested that 4f may act by a dual effect, by releasing nitric oxide after biotransformation and by inhibiting cysteine protease CPB (IC50: 4.5 µM). In vivo studies using an acute model of infection showed that compound 4f at 7.7 mg/Kg reduced ~90% of parasite burden in the liver and spleen of L. infantum-infected BALB/c mice. Altogether, these outcomes highlight furoxan 4f as a promising compound for further evaluation as an antileishmanial agent.

The antileishmanial activity of the antarctic brown alga Ascoseira mirabilis Skottsberg.

Leishmaniasis is a group of diseases that have limited and high toxic therapeutic options. Herein, we evaluated the antileishmanial potential and cytotoxicity of hexanic extract obtained from the Antarctic brown alga Ascoseira mirabilis using bioguided fractionation against Leishmania amazonensis and murine macrophages, which was fractionated by SPE, yielding seven fractions (F1-F7). The fraction F6 showed good anti-amastigote activity (IC50 = 73.4 ± 0.4 µg mL-1) and low cytotoxicity (CC50 > 100 µg mL-1). Thus, in order to identify the bioactive constituent(s) of F6, the fraction was separated in a semipreparative HPLC, yielding four fractions (F6.1-F6.4). F6.2 was the most bioactive fraction (IC50 = 66.5 ± 4.5 µg mL-1) and GC-MS analyses revealed that the compounds octadecane, propanoic acid, 1-monomyristin and azelaic acid correspond to 61% of its composition. These data show for the first time the antileishmanial potential of the Antarctic alga A. mirabilis.

Antileishmanial activity of amphiphilic chlorin derivatives mediated by photodynamic therapy.

Leishmaniasis is a serious and neglected disease that affects 14 million people around the World. The currently available drugs for treatment present several drawbacks such as low efficacy and severe side effects, contributing to patients' low compliance. Photodynamic therapy (PDT) is rising as a promising treatment of cutaneous leishmaniasis, mainly considering its topical administration that circumvents any potential adverse effects commonly related to oral/parenteral administration. PDT depends on the interaction between a light-sensitive compound (photosensitizer - PS), light and molecular oxygen. The reaction generates reactive oxygen species (ROS) which induce cell death by oxidative stress. The main goal of this study is to demonstrate the antileishmanial effect of three chlorin derivatives (CHL-OH-A, CHL-OH-B, CHL-TRISMA) using PDT, as well as to investigate their cell death pathway on Leishmania amazonensis promastigote forms after chlorin-PDT application. The chlorin derivatives herein studied did not exhibit aggregates in aqueous medium and showed fast accumulation in Leishmania acidic compartments. CHL-OH-A exhibited the highest antiparasitic activity at 24 h (0.33 µmol L-1) and 48 h (0.14 µmol L-1) after irradiation at 660 nm (6.0 Jcm-2). CHL-OH-A, CHL-OH-B and CHL-TRISMA molecules induced the cell death of parasites mainly by an apoptotic-like process in the presence of light. These chlorin derivatives are 80-fold more active against Leishmania when compared to other PSs reported in the literature. In this study, we have shown that these amphiphilic chlorins, and in particular, CHL-OH-A, exert an interesting leishmanicidal activity suggesting that the use of these PSs associated with PDT could be a promising strategy for treatment of cutaneous leishmaniasis.

Antimicrobial activity of RP-1 peptide conjugate with ferrocene group.

Parasitic diseases are a neglected and serious problem, especially in underdeveloped countries. Among the major parasitic diseases, Leishmaniasis figures as an urgent challenge due to its high incidence and severity. At the same time, the indiscriminate use of antibiotics by the population is increasing together with resistance to medicines. To address this problem, new antibiotic-like molecules that directly kill or inhibit the growth of microorganisms are necessary, where antimicrobial peptides (AMPs) can be of great help. In this work, the ferrocene molecule, one active compound with low levels of in vivo toxicity, was coupled to the N-terminus of the RP1 peptide (derived from the human chemokine CXCL4), aiming to evaluate how this change modifies the structure, biological activity, and toxicity of the peptide. The peptide and the conjugate were synthesized using the solid phase peptide synthesis (SPPS). Circular dichroism assays in PBS showed that the RP1 peptide and its conjugate had a typical spectrum for disordered structures. The Fc-RP1 presented anti-amastigote activity against Leishmania amazonensis (IC50 = 0.25 µmol L-1). In comparison with amphotericin B, a second-line drug approved for leishmaniasis treatment, (IC50 = 0.63 µmol L-1), Fc-RP1 was more active and showed a 2.5-fold higher selectivity index. The RP1 peptide presented a MIC of 4.3 µmol L-1 against S. agalactiae, whilst Fc-RP1 was four times more active (MIC = 0.96 µmol L-1), indicating that ferrocene improved the antimicrobial activity against Gram-positive bacteria. The Fc-RP1 peptide also decreased the minimum inhibitory concentration (MIC) in the assays against E. faecalis (MIC = 7.9 µmol L-1), E. coli (MIC = 3.9 µmol L-1) and S. aureus (MIC = 3.9 µmol L-1). The cytotoxicity of the compounds was tested against HaCaT cells, and no significant activity at the highest concentration tested (500 µg. mL-1) was observed, showing the high potential of this new compound as a possible new drug. The coupling of ferrocene also increased the vesicle permeabilization of the peptide, showing a direct relation between high peptide concentration and high carboxyfluorescein release, which indicates the action mechanism by pore formation on the vesicles. Several studies have shown that ferrocene destabilizes cell membranes through lipid peroxidation, leading to cell lysis. It is noteworthy that the Fc-RP1 peptide synthesized here is a prototype of a bioconjugation strategy, but it still is a compound with great biological activity against neglected and fish diseases.

Nanotechnological Strategies for Treatment of Leishmaniasis--A Review.

The World Health Organization (WHO) estimates that more than one billion people suffer from neglected tropical diseases. Leishmaniasis is a widespread disease, affecting 12 million people around the world with about 1­2 million estimated new cases occurring every year. Although pentavalent antimonial drugs are the most frequently prescribed treatments for leishmaniasis, they produce severe side effects, including cardiotoxicity and hepatotoxicity. Other compounds, such as amphotericin B, pentamidine and miltefosine, are second choice drugs, but they also produce side effects that can endanger the patient's life. Nowadays, there are two approaches to develop new therapies: one is the search for new drugs and the other is the optimization of actual drug formulation. Traditional drug discovery takes 10 to 12 years in general and involves high costs; around one billion dollars on average to develop a drug. A possibility to improve leishmaniasis treatment would be the application of nanotechnology-drug delivery systems which can enhance the therapeutic potency of existing drugs by optimizing their adsorption, distribution, metabolism and excretion (ADME) and reducing toxicity. In this review we will discuss examples how nanotechnology-drug delivery systems have been used to improve the therapeutic aspects of existing antileishmanial drugs.