Solange L. de Castro

In vitro and in vivo experimental models for drug screening and development for Chagas disease

Chagas disease, a neglected illness, affects nearly 12-14 million people in endemic areas of Latin America. Although the occurrence of acute cases sharply has declined due to Southern Cone Initiative efforts to control vector transmission, there still remain serious challenges, including the maintenance of sustainable public policies for Chagas disease control and the urgent need for better drugs to treat chagasic patients. Since the introduction of benznidazole and nifurtimox approximately 40 years ago, many natural and synthetic compounds have been assayed against Trypanosoma cruzi, yet only a few compounds have advanced to clinical trials. This reflects, at least in part, the lack of consensus regarding appropriate in vitro and in vivo screening protocols as well as the lack of biomarkers for treating parasitaemia. The development of more effective drugs requires (i) the identification and validation of parasite targets, (ii) compounds to be screened against the targets or the whole parasite and (iii) a panel of minimum standardised procedures to advance leading compounds to clinical trials. This third aim was the topic of the workshop entitled Experimental Models in Drug Screening and Development for Chagas Disease, held in Rio de Janeiro, Brazil, on the 25th and 26th of November 2008 by the Fiocruz Program for Research and Technological Development on Chagas Disease and Drugs for Neglected Diseases Initiative. During the meeting, the minimum steps, requirements and decision gates for the determination of the efficacy of novel drugs for T. cruzi control were evaluated by interdisciplinary experts and an in vitro and in vivo flowchart was designed to serve as a general and standardised protocol for screening potential drugs for the treatment of Chagas disease.

The Trypanocidal Activity of Naphthoquinones: A Review

Naphthoquinones are compounds present in several families of higher plants. Their molecular structures confer redox properties, and they are involved in multiple biological oxidative processes. In folk medicine, especially among Indian populations, plants containing naphthoquinones have been employed for the treatment of various diseases. The biological redox cycle of quinones can be initiated by one electron reduction leading to the formation of semiquinones, unstable intermediates that react rapidly with molecular oxygen, generating free radicals. Alternatively, the reduction by two electrons, mediated by DT-diphorase, leads to the formation of hydroquinone. Lapachol, alpha-lapachone and beta-lapachone, which are isolated from the heartwood of trees of the Bignoniaceae family, are examples of bioactive naphthoquinones. In this review, we will discuss studies investigating the activity of these natural products and their derivatives in the context of the search for alternative drugs for Chagas disease, caused by Trypanosoma cruzi, a neglected illness that is endemic in Latin America.

Flavonoids and trypanocidal activity of Bulgarian propolis

Acetone and ethanol extracts of two Bulgarian propolis samples (Bur and Lov) were investigated by high temperature high resolution gas chromatography coupled to mass spectrometry (HT-HRGC-MS), and their activity against Trypanosoma cruzi was evaluated. The ethanol extracts--Et-Bur and Et-Lov--showed similar composition, with a high content of flavonoids, and strong inhibitory activity against T. cruzi proliferative epimastigotes, which were more susceptible than trypomastigotes. In the presence of blood, the activity of Et-Bur or Et-Lov against trypomastigotes was similar to that of the standard drug, crystal violet. Both extracts also showed similar and significant activity against Staphylococcus aureus and Candida albicans, while being inactive against Escherichia coli. The acetone extract, Ket-Bur, was more active than Et-Bur against both forms of T. cruzi.

Autophagy in protists.

Autophagy is the degradative process by which eukaryotic cells digest their own components using acid hydrolases within the lysosome. Originally thought to function almost exclusively in providing starving cells with nutrients taken from their own cellular constituents, autophagy is in fact involved in numerous cellular events including differentiation, turnover of macromolecules and organelles, and defense against parasitic invaders. During the last 10-20 years, molecular components of the autophagic machinery have been discovered, revealing a complex interactome of proteins and lipids, which, in a concerted way, induce membrane formation to engulf cellular material and target it for lysosomal degradation. Here, our emphasis is autophagy in protists. We discuss experimental and genomic data indicating that the canonical autophagy machinery characterized in animals and fungi appeared prior to the radiation of major eukaryotic lineages. Moreover, we describe how comparative bioinformatics revealed that this canonical machinery has been subject to moderation, outright loss or elaboration on multiple occasions in protist lineages, most probably as a consequence of diverse lifestyle adaptations. We also review experimental studies illustrating how several pathogenic protists either utilize autophagy mechanisms or manipulate host-cell autophagy in order to establish or maintain infection within a host. The essentiality of autophagy for the pathogenicity of many parasites, and the unique features of some of the autophagy-related proteins involved, suggest possible new targets for drug discovery. Further studies of the molecular details of autophagy in protists will undoubtedly enhance our understanding of the diversity and complexity of this cellular phenomenon and the opportunities it offers as a drug target.

Different cell death pathways induced by drugs in Trypanosoma cruzi: An ultrastructural study

Electron microscopy has proven to be a reliable and essential tool to determine morphological alterations and target organelles in the investigation of new drugs for Chagas disease. In this review, we focused on evaluating different agents that induce death of Trypanosoma cruzi, i.e. lysophospholipids analogues, naphthoquinones and derivatives, cytoskeletal inhibitors and natural products. Apoptosis-like presents as morphological characteristics DNA fragmentation, membrane blebbing and apoptotic body formation. Autophagy involves autophagosome formation, with the appearance of membranes surrounding organelles and cytosolic structures. Necrosis causes the loss of osmotic balance, an increase of cytoplasmic vacuolization and plasma membrane disruption. Mitochondrion appears as a central checkpoint in both apoptosis and necrosis. Our evidences of ultrastructural changes to T. cruzi treated with the different classes of compounds point to dramatic mitochondrial alterations and similar autophagic phenotypes. Lysophospholipid analogues interfere in the lipid biosynthesis in epimastigotes, altering the amount of both phospholipids and sterols, and consequently the physical properties of the membrane. Naphthoquinone derivatives led to a strong DNA fragmentation in trypomastigotes and to the release of cysteine proteases from reservosomes to cytosol in epimastigotes, starting a proteolytic process which results in parasite death. The susceptibility of reservosomes was also observed in parasites treated with propolis, suggesting impairment of lipid metabolism, compromising membrane fluidity and leading to lysis. The cytoskeletal agents blocked mitosis of epimastigotes, arresting cell cycle and impairing the parasite proliferation. The variety of drug stimuli converge to the same pathway of death suggests an intense cross-talking between the three types of PCD in the protozoa.

Trypanocidal activity of isolated naphthoquinones from Tabebuia and some heterocyclic derivatives: a review from an interdisciplinary study

Naphthoquinones isolated from the wood of trees of the families Bignoniaceae and Verbenaceae have been subjected to an interdisciplinary study since the seventies, when Dr. Benjamin Gilbert, at the Federal University of Rio de Janeiro, launched a program on the chemistry of natural products active against endemic diseases. In this paper we describe the synthesis of five naphthoimidazoles, derived from this program and their activity towards T. cruzi, the etiologic agent of Chagas disease. We also review the influence of chemical structure on trypanocidal action of naphthoquinones and of derived heterocycles with imidazole, oxazole, phenoxazine, indole, dipyrane and cyclopentene rings. The overall analysis corroborates the tendency of trypanocidal activity in compounds with an imidazole or oxazole ring linked to a naphthopyrane structure. Two naphthoimidazoles presented higher activities (14.5x and 34.8x) than the standard crystal violet. Emphasis is given to the biodiversity of the Brazilian flora as a starting point for the development of an autonomous and creative medicinal chemistry.

Trypanosoma cruzi targets for new chemotherapeutic approaches

Background: Strategies for development of anti-parasite chemotherapy involve identification of active principles of plants, investigation of drugs already licensed for other pathologies, or validation of specific targets identified within key metabolic pathways. Objective: To review the state of the art of drug targets against Trypanosoma cruzi with emphasis on sterol metabolism, kinetoplast DNA (kDNA) sites, trypanothione reductase, cysteine proteinase, hypoxanthine–guanine phosphoribosyltransferase, dihydrofolate reductase and glyceraldehyde-3-phosphate dehydrogenase. Methods: Current knowledge, accumulated over the last three decades, on targets for design and development of new trypanocidal compounds is described. Results/conclusion: There is an urgent need for better drugs to treat chagasic patients. Since the introduction of benznidazole and nifurtimox only allopurinol and a few sterol inhibitors have moved to clinical trials, despite the long list of natural and synthetic compounds assayed against T. cruzi. This reflects, at least in part, the absence of well-established universal protocols to screen and compare drug activity associated with a lack of definitive preclinical evidence of parasitological cure in animal models.

The effects on Trypanosoma cruzi of novel synthetic naphthoquinones are mediated by mitochondrial dysfunction.

Despite ongoing efforts, the current treatment for Chagas disease is still unsatisfactory, mainly because of the severe side effects and variable efficacy of the available nitroheterocycles. Our group has been assaying natural quinones isolated from Brazilian flora, and their derivatives, as alternative chemotherapeutic agents against Trypanosoma cruzi. From C-allyl lawsone three naphthofuranquinones were synthesized, which were active against trypomastigotes and epimastigotes. Here, we further investigated the activity and the mechanisms of action of these quinones. They exhibited powerful effects on intracellular amastigotes, presenting low toxicity to the host cells. Ultrastructural analyses of treated epimastigotes and trypomastigotes indicated a potent effect of the three naphthofuranquinones on the parasite mitochondrion, which appeared drastically swollen and with a washed-out matrix profile. Fluorescence-activated cell sorting analysis of rhodamine 123-stained T. cruzi showed that the three naphthofuranquinones caused a potent dose-dependent collapse of the mitochondrial membrane potential, especially in the epimastigote form. Naphthofuranquinones also decreased specifically mitochondrial complex I-III activity in both epimastigotes and trypomastigotes, parallel to a reduction in succinate-induced oxygen consumption. Mitochondrial hydrogen peroxide formation was also increased in epimastigotes after treatment with the naphthofuranquinones. Our results indicate that the trypanocidal action of the naphthofuranquinones is associated with mitochondrial dysfunction, leading to increased reactive oxygen species generation and parasite death.

Synthesis of quinoidal molecules: strategies towards bioactive compounds with an emphasis on lapachones.

Naphthoquinoidal compounds are of great interest in medicinal chemistry. In recent years, several synthetic routes have been developed to obtain bioactive molecules derived from lapachones. In this mini-review, we focus on the synthetic aspects and strategies used to design these compounds and on the biological activities of these substances for the development of drugs against the neglected diseases leishmaniasis and Chagas disease as well as malaria, tuberculosis and cancer. Three strategies used to develop bioactive naphthoquinoidal compounds are discussed: (i) C-ring modification, (ii) redox centre modification and (iii) A-ring modification. Among these strategies, reactions such as copper-catalysed azide-alkyne cycloaddition (click chemistry), palladium-catalysed cross couplings, and heterocyclisations will be discussed for the development of naphthoquinoidal compounds against Trypanosoma cruzi, Leishmania and cancer. The aim of derivatisation is the generation of novel molecules that inhibit cellular organelles/processes, generate reactive oxygen species (ROS) and increase lipophilicity to enhance penetration through the plasma membrane. Modified lapachones have emerged as promising prototypes for the development of drugs against neglected diseases and cancer.

Effect of the alkyl-lysophospholipids on the proliferation and differentiation of Trypanosoma cruzi.

Alkyl-lysophospholipids (ALPs), designed as potential immunomodulators, have been shown to be cytotoxic for a variety of tumour cells and are under clinical studies for cancer chemotherapy. ET-18-OCH(3), hexadecylphosphocholine and ilmofosine were assayed against the three forms of Trypanosoma cruzi. Incubation with bloodstream trypomastigotes resulted, under different experimental conditions, in higher activity of the compounds in comparison with crystal violet. The ED(50)/24 h values were 13.4+/-2.8 microM and 11. 7+/-0.6 microM for amastigotes and epimastigotes, respectively. ET-18-OCH(3) (0.3 and 0.6 microM) inhibited the differentiation of epimastigotes to trypomastigotes (Dm28C clone) in the range 40-57%. This drug (3.75-15 microM) also caused a time- and dose-dependent inhibition of the intracellular proliferation of amastigotes in heart muscle cells with ED(50) values of 14.3+/-4.2, 8.9+/-1.9 and 6. 8+/-0.4 microM, after 1, 2 and 3 days of treatment. Pre-treatment of the parasite with this drug inhibited its interiorization into the host cell. Interestingly, the intracellular differentiation of amastigotes to trypomastigotes was not hampered by the drug. The present results demonstrate the lytic effect of ALPs on the three forms of T. cruzi, as well as the inhibition of both the differentiation to the infective form and the proliferation of parasites interiorized in heart cells. Ultrastructural analysis of epimastigotes treated with the three ALPs showed extensive blebing of the flagellar membrane. As described in tumour cells, the membrane seems to be a primary target of the drugs.