Aline Araujo Zuma

Unveiling Benznidazole's mechanism of action through overexpression of DNA repair proteins in Trypanosoma cruzi

Benznidazole (BZ) is the most commonly used drug for the treatment of Chagas disease. Although BZ is known to induce the formation of free radicals and electrophilic metabolites within the parasite Trypanosoma cruzi, its precise mechanisms of action are still elusive. Here, we analyzed the survival of T. cruzi exposed to BZ using genetically modified parasites overexpressing different DNA repair proteins. Our results indicate that BZ induces oxidation mainly in the nucleotide pool, as heterologous expression of the nucleotide pyrophosphohydrolase MutT (but not overexpression of the glycosylase TcOgg1) increased drug resistance in the parasite. In addition, electron microscopy indicated that BZ catalyzes the formation of double‐stranded breaks in the parasite, as its genomic DNA undergoes extensive heterochromatin unpacking following exposure to the drug. Furthermore, the overexpression of proteins involved in the recombination‐mediated DNA repair increased resistance to BZ, reinforcing the idea that the drug causes double‐stranded breaks. Our results also show that the overexpression of mitochondrial DNA repair proteins increase parasite survival upon BZ exposure, indicating that the drug induces lesions in the mitochondrial DNA as well. These findings suggest that BZ preferentially oxidizes the nucleotide pool, and the extensive incorporation of oxidized nucleotides during DNA replication leads to potentially lethal double‐stranded DNA breaks in T. cruzi DNA. Environ. Mol. Mutagen. 55:309–321, 2014.

Effect of topoisomerase inhibitors and DNA-binding drugs on the cell proliferation and ultrastructure of Trypanosoma cruzi

Trypanosomatids present unusual organelles, such as the kinetoplast that contains the mitochondrial DNA arranged in catenated circles. The nucleus of these protozoa presents distinct domains during interphase as well as a closed mitosis. DNA topoisomerases modulate the topological state of DNA by regulating supercoiling of the double-stranded DNA during replication, transcription, recombination and repair. Because topoisomerases play essential roles in cellular processes, they constitute a potential target for antitumour and antimicrobial drugs. In this study, the effects of various topoisomerase inhibitors and DNA-binding drugs were tested on the cellular proliferation and ultrastructure of the Trypanosoma cruzi epimastigote form Blastocrithidia culicis was used as a comparative model, which has a more relaxed kinetoplast DNA (kDNA) organization. The results showed that the eukaryotic topoisomerase I inhibitors camptothecin and rebeccamycin were the most effective compounds in the arrest of T. cruzi proliferation. Of the eukaryotic topoisomerase II inhibitors, mitoxantrone, but not merbarone, was effective against cell proliferation. The prokaryotic topoisomerase II inhibitors norfloxacin and enoxacin targeted the kinetoplast specifically, thus promoting ultrastructural kDNA rearrangement in B. culicis. Of the DNA-binding drugs, berenil caused remarkable kDNA disorganization. With the exception of camptothecin, there have been no previous evaluations of the compounds tested here on trypanosomatid ultrastructure. In conclusion, inhibitors of the same class may have different effects on trypanosomatid proliferation and ultrastructure. The results obtained in this work may help to reveal the mechanism of action of different topoisomerase inhibitors in trypanosomatids.

Endosymbiosis in trypanosomatid protozoa: the bacterium division is controlled during the host cell cycle

Mutualism is defined as a beneficial relationship for the associated partners and usually assumes that the symbiont number is controlled. Some trypanosomatid protozoa co-evolve with a bacterial symbiont that divides in coordination with the host in a way that results in its equal distribution between daughter cells. The mechanism that controls this synchrony is largely unknown, and its comprehension might provide clues to understand how eukaryotic cells evolved when acquiring symbionts that later became organelles. Here, we approached this question by studying the effects of inhibitors that affect the host exclusively in two symbiont-bearing trypanosomatids, Strigomonas culicis and Angomonas deanei. We found that inhibiting host protein synthesis using cycloheximide or host DNA replication using aphidicolin did not affect the duplication of bacterial DNA. Although the bacteria had autonomy to duplicate their DNA when host protein synthesis was blocked by cycloheximide, they could not complete cytokinesis. Aphidicolin promoted the inhibition of the trypanosomatid cell cycle in the G1/S phase, leading to symbiont filamentation in S. culicis but not in A. deanei. Treatment with camptothecin blocked the host protozoa cell cycle in the G2 phase and induced the formation of filamentous symbionts in both species. Oryzalin, which affects host microtubule polymerization, blocked trypanosomatid mitosis and abrogated symbiont division. Our results indicate that host factors produced during the cell division cycle are essential for symbiont segregation and may control the bacterial cell number.

Unveiling the effects of berenil, a DNA-binding drug, on Trypanosoma cruzi: implications for kDNA ultrastructure and replication

Trypanosoma cruzi, the etiological agent of Chagas disease, exhibits a single mitochondrion with an enlarged portion termed kinetoplast. This unique structure harbors the mitochondrial DNA (kDNA), composed of interlocked molecules: minicircles and maxicircles. kDNA is a hallmark of kinetoplastids and for this reason constitutes a valuable target in chemotherapeutic and cell biology studies. In the present work, we analyzed the effects of berenil, a minor-groove-binding agent that acts preferentially at the kDNA, thereby affecting cell proliferation, ultrastructure, and mitochondrial activity of T. cruzi epimastigote form. Our results showed that berenil promoted a reduction on parasite growth when high concentrations were used; however, cell viability was not affected. This compound caused significant changes in kDNA arrangement, including the appearance of membrane profiles in the network and electron-lucent areas in the kinetoplast matrix, but nuclear ultrastructure was not modified. The use of the TdT technique, which specifically labels DNA, conjugated to atomic force microscopy analysis indicates that berenil prevents the minicircle decatenation of the network, thus impairing DNA replication and culminating in the appearance of dyskinetoplastic cells. Alterations in the kinetoplast network may be associated with kDNA lesions, as suggested by the quantitative PCR (qPCR) technique. Furthermore, parasites treated with berenil presented higher levels of reactive oxygen species and a slight decrease in the mitochondrial membrane potential and oxygen consumption. Taken together, our results reveal that this DNA-binding drug mainly affects kDNA topology and replication, reinforcing the idea that the kinetoplast represents a potential target for chemotherapy against trypanosomatids.

Interactions between 4-aminoquinoline and heme: Promising mechanism against Trypanosoma cruzi.

Chagas disease is a neglected tropical disease caused by the flagellated protozoan Trypanosoma cruzi. The current drugs used to treat this disease have limited efficacy and produce severe side effects. Quinolines, nitrogen heterocycle compounds that form complexes with heme, have a broad spectrum of antiprotozoal activity and are a promising class of new compounds for Chagas disease chemotherapy. In this study, we evaluated the activity of a series of 4-arylaminoquinoline-3-carbonitrile derivatives against all forms of Trypanosoma cruzi in vitro. Compound 1g showed promising activity against epimastigote forms when combined with hemin (IC50<1 μM), with better performance than benznidazole, the reference drug. This compound also inhibited the viability of trypomastigotes and intracellular amastigotes. The potency of 1g in combination with heme was enhanced against epimastigotes and trypomastigotes, suggesting a similar mechanism of action that occurs in Plasmodium spp. The addition of hemin to the culture medium increased trypanocidal activity of analog 1g without changing the cytotoxicity of the host cell, reaching an IC50 of 11.7 μM for trypomastigotes. The mechanism of action was demonstrated by the interaction of compound 1g with hemin in solution and prevention of heme peroxidation. Compound 1g and heme treatment induced alterations of the mitochondrion-kinetoplast complex in epimastigotes and trypomastigotes and also, accumulation of electron-dense deposits in amastigotes as visualized by transmission electron microscopy. The trypanocidal activity of 4-aminoquinolines and the elucidation of the mechanism involving interaction with heme is a neglected field of research, given the parasites lack of heme biosynthetic pathway and the importance of this cofactor for parasite survival and growth. The results of this study can improve and guide rational drug development and combination treatment strategies.

Voacamine alters Leishmania ultrastructure and kills parasite by poisoning unusual bi-subunit topoisomerase IB

ABSTRACT Indole alkaloids possess a large spectrum of biological activities including anti‐protozoal action. Here we report for the first time that voacamine, isolated from the plant Tabernaemontana coronaria, is an antiprotozoal agent effective against a large array of trypanosomatid parasites including Indian strain of Leishmania donovani and Brazilian strains of Leishmania amazonensis and Trypanosoma cruzi. It inhibits the relaxation activity of topoisomerase IB of L. donovani (LdTop1B) and stabilizes the cleavable complex. Voacamine is probably the first LdTop1B‐specific poison to act uncompetitively. It has no impact on human topoisomerase I and II up to 200 &mgr;M concentrations. The study also provides a thorough insight into ultrastructural alterations induced in three kinetoplastid parasites by a specific inhibitor of LdTop1B. Voacamine is also effective against intracellular amastigotes of different drug unresponsive field isolates of Leishmania donovani obtained from endemic zones of India severely affected with visceral leishmaniasis. Most importantly, this is the first report demonstrating the efficacy of a compound to reduce the burden of drug resistant parasites, unresponsive to SAG, amphotericin B and miltefosine, in experimental BALB/c mice model of visceral leishmaniasis. The findings cumulatively provide a strong evidence that voacamine can be a promising drug candidate against trypanosomatid infections.

Chaetocin—A histone methyltransferase inhibitor—Impairs proliferation, arrests cell cycle and induces nucleolar disassembly in Trypanosoma cruzi

The Trypanosomatidae family includes pathogenic species of medical and veterinary interest. Chagas disease is endemic in Latin America, and about 8 million people are infected worldwide. There is a need for more effective drugs for the acute, undetermined and chronic phases of the disease that, in addition, do not cause side effects, stimulating the search for identification of new drug targets, as well as new chemotherapeutic targets. Trypanosomatids contain characteristic structures, such as the nucleus that undergoes a closed mitosis without chromosome formation and variations of chromatin packing in the different protozoa developmental stages. The nuclear DNA is condensed by histones that suffer post-translational modifications, such as addition of methyl groups by histone methyltransferases (MHT) and addition of acetyl groups by acetyltransferases. These processes modulate gene expression and chromatin organization, which are crucial to transcription, replication, repair and recombination. In the present study, the effects of chaetocin, a HMT inhibitor, on T. cruzi epimastigote proliferation, viability, ultrastructure and cell cycle were investigated. Results indicate that chaetocin promoted irreversible inhibition of protozoa growth, evident unpacking of nuclear heterochromatin and intense nucleolus fragmentation, which is associated with parasite cell cycle arrest and RNA transcription blockage. Taken together, data obtained with chaetocin treatment stimulate the use of histone methyltransferase inhibitors against pathogenic trypanosomatids.

Effects of camptothecin derivatives and topoisomerase dual inhibitors on Trypanosoma cruzi growth and ultrastructure

BackgroundTrypanosoma cruzi is the etiological agent of Chagas’ disease that is an endemic disease in Latin America and affects about 8 million people. This parasite belongs to the Trypanosomatidae family which contains a single mitochondrion with an enlarged region, named kinetoplast that harbors the mitochondrial DNA (kDNA). The kinetoplast and the nucleus present a great variety of essential enzymes involved in DNA replication and topology, including DNA topoisomerases. Such enzymes are considered to be promising molecular targets for cancer treatment and for antiparasitic chemotherapy. In this work, the proliferation and ultrastructure of T. cruzi epimastigotes were evaluated after treatment with eukaryotic topoisomerase I inhibitors, such as topotecan and irinotecan, as well as with dual inhibitors (compounds that block eukaryotic topoisomerase I and topoisomerase II activities), such as baicalein, luteolin and evodiamine. Previous studies have shown that such inhibitors were able to block the growth of tumor cells, however most of them have never been tested on trypanosomatids.ResultsConsidering the effects of topoisomerase I inhibitors, our results showed that topotecan decreased cell proliferation and caused unpacking of nuclear heterochromatin, however none of these alterations were observed after treatment with irinotecan. The dual inhibitors baicalein and evodiamine decreased cell growth; however the nuclear and kinetoplast ultrastructures were not affected.ConclusionsTaken together, our data showed that camptothecin is more efficient than its derivatives in decreasing T. cruzi proliferation. Furthermore, we conclude that drugs pertaining to a certain class of topoisomerase inhibitors may present different efficiencies as chemotherapeutical agents.

Isobenzofuranone derivative JVPH3, an inhibitor of L. donovani topoisomerase II, disrupts mitochondrial architecture in trypanosomatid parasites

Kinetoplast DNA (kDNA) bearing unusual mitochondrion of trypanosomatid parasites offers a new paradigm in chemotherapy modality. Topoisomerase II of Leishmania donovani (LdTopII), a key enzyme associated with kDNA replication, is emerging as a potential drug target. However, mode of action of LdTopII targeted compounds in the parasites at sub-cellular level remains largely unknown. Previously, we reported that an isobenzofuranone derivative, namely 3,5-bis(4-chlorophenyl)-7-hydroxyisobenzofuran-1(3H)-one (JVPH3), targets LdTopII and induces apoptosis-like cell death in L. donovani. Here, we elucidate the phenotypic changes and the events occurring at sub-cellular level caused by JVPH3 in L. donovani. In addition, we have evaluated the cytotoxicity and ultrastructural alterations caused by JVPH3 in two brazilian trypanosomatid pathogens viz. L. amazonensis and Trypanosoma cruzi. Despite killing these parasites, JVPH3 caused significantly different phenotypes in L. donovani and L. amazonensis. More than 90% population of parasites showed altered morphology. Mitochondrion was a major target organelle subsequently causing kinetoplast network disorganization in Leishmania. Altered mitochondrial architecture was evident in 75–80% Leishmania population being investigated. Quantification of mitochondrial function using JC-1 fluorophore to measure a possible mitochondrial membrane depolarization further confirmed the mitochondrion as an essential target of the JVPH3 corroborating with the phenotype observed by electron microscopy. However, the impact of JVPH3 was lesser on T. cruzi than Leishmania. The molecule caused mitochondrial alteration in 40% population of the epimastigotes being investigated. To our knowledge, this is the first report to evaluate the proliferation pattern and ultrastructural alterations caused in Brazilian kinetoplastid pathogens by a synthetic LdTopII inhibitor previously established to have promising in vivo activity against Indian strain of L. donovani.

Histone deacetylases as targets for antitrypanosomal drugs

Parasitic protozoa comprise several species that are causative agents of important diseases. These diseases are distributed throughout the world and include leishmaniasis, Chagas disease and sleeping sickness, malaria and toxoplasmosis. Treatment is based on drugs that were developed many years ago, which have side effects and produce resistant parasites. One approach for the development of new drugs is the identification of new molecular targets. We summarize the data on histone deacetylases, a class of enzymes that act on histones, which are closely associated with DNA and its regulation. These enzymes may constitute new targets for the development of antiparasitic protozoa drugs. Although several protozoan species are mentioned, members of the Trypanosomatidae family are the main focus of this short review.