Mode of action of natural and synthetic drugs against Trypanosoma cruzi and their interaction with the mammalian host

Current knowledge of the biochemistry of Trypanosoma cruzi has led to the development of new drugs and the understanding of their mode of action. Some trypanocidal drugs such as nifurtimox and benznidazole act through free radical generation during their metabolism. T. cruzi is very susceptible to the cell damage induced by these metabolites because enzymes scavenging free radicals are absent or have very low activities in the parasite. Another potential target is the biosynthetic pathway of glutathione and trypanothione, the low molecular weight thiol found exclusively in trypanosomatids. These thiols scavenge free radicals and participate in the conjugation and detoxication of numerous drugs. Inhibition of this key pathway could render the parasite much more susceptible to the toxic action of drugs such as nifurtimox and benznidazole without affecting the host significantly. Other drugs such as allopurinol and purine analogs inhibit purine transport in T. cruzi, which cannot synthesize purines de novo. Nitroimidazole derivatives such as itraconazole inhibit sterol metabolism. The parasite's respiratory chain is another potential therapeutic target because of its many differences with the host enzyme complexes. The pharmacological modulation of the host's immune response against T. cruzi infection as a possible chemotherapeutic target is discussed. A large set of chemicals of plant origin and a few animal metabolites active against T. cruzi are enumerated and their likely modes of action are briefly discussed.     

Role of glutathione in the susceptibility of Trypanosoma cruzi to drugs

1. Glutathione (G-SH) concentration, gamma-glutamyltranspeptidase and glutathione S-transferase activities were studied in several strains of T. cruzi epimastigotes. GSH varied from 1.04 mM for the LQ strain to 0.61 mM for the Tulahuen strain. 2. Cultures of the LQ strain presented more resistance to drugs than those of the Tulahuen. It was necessary a concentration of nifurtimox 4 times higher and one of benznidazole 10 times higher in order to inhibit approximately to 50% the growth of LQ strain cultures when compared with the Tulahuen strain. 3. Buthionine sulfoximine decreased the concentration of glutathione to about 50% in the LQ and Tulahuen strains and potentiated the toxicity of nifurtimox and benznidazole in T. cruzi epimastigote cultures. These results suggest that glutathione is an important factor in the resistance of T. cruzi to nifurtimox and benznidazole.     

Trypanosoma cruzi: activities of lapachol and alpha- and beta-lapachone derivatives against epimastigote and trypomastigote forms

Derivatives of natural quinones with biological activities, such as lapachol, alpha- and beta-lapachones, have been synthesized and their trypanocidal activity evaluated in vitro in Trypanosoma cruzi cells. All tested compounds inhibited epimastigote growth and trypomastigote viability. Several compounds showed similar or higher activity as compared with current trypanocidal drugs, nifurtimox and benznidazole. The results presented here show that the anti-T. cruzi activity of the alpha-lapachone derivatives can be increased by the replacement of the benzene ring by a pyridine moiety. Free radical production and consequently oxidative stress through redox cycling or production of electrophilic metabolites are the potential biological mechanism of action for these synthetic quinones.     

In vitro and in vivo investigation of the efficacy of arylimidamide DB1831 and its mesylated salt form--DB1965--against Trypanosoma cruzi infection

Chagas disease is caused by infection with the intracellular protozoan parasite Trypanosoma cruzi. At present, nifurtimox and benznidazole, both compounds developed empirically over four decades ago, represent the chemotherapeutic arsenal for treating this highly neglected disease. However, both drugs present variable efficacy depending on the geographical area and the occurrence of natural resistance, and are poorly effective against the later chronic stage. As a part of a search for new therapeutic opportunities to treat chagasic patients, pre-clinical studies were performed to characterize the activity of a novel arylimidamide (AIA--DB1831 (hydrochloride salt) and DB1965 (mesylate salt)) against T. cruzi. These AIAs displayed a high trypanocidal effect in vitro against both relevant forms in mammalian hosts, exhibiting a high selectivity index and a very high efficacy (IC(50) value/48 h of 5-40 nM) against intracellular parasites. DB1965 shows high activity in vivo in acute experimental models (mouse) of T. cruzi, showing a similar effect to benznidazole (Bz) when compared under a scheme of 10 daily consecutive doses with 12.5 mg/kg. Although no parasitological cure was observed after treating with 20 daily consecutive doses, a combined dosage of DB1965 (5 mg/kg) with Bz (50 mg/kg) resulted in parasitaemia clearance and 100% animal survival. In summary, our present data confirmed that aryimidamides represent promising new chemical entities against T. cruzi in therapeutic schemes using the AIA alone or in combination with other drugs, like benznidazole.     

Mechanism of action of a nitroimidazole-thiadiazole derivate upon Trypanosoma cruzi tissue culture amastigotes

Megazol (CL 64,855) a very effective drug in experimental infections by Trypanosoma cruzi, and also in in vitro assays with vertebrate forms of the parasite, had its activity upon macromolecule biosynthesis tested using tissue culture-derived amastigote forms. Megazol presented a drastic inhibition of [3H]-leucine incorporation, and only a partial inhibition of [3H]-thymidine and [3H]-uridine incorporation, suggesting a selective activity upon protein synthesis. Comparing the three drugs, megazol was more potent than nifurtimox and benznidazole in inhibiting protein and DNA synthesis. Megazol showed a 91% of inhibition of [3H]-leucine incorporation whereas nifurtimox and benznidazole, 0% and 2%, respectively. These latter two drugs inhibited the incorporation of all the precursors tested at similar levels, but the concentration of benznidazole was always three times higher, suggesting different mechanisms of action or, more probably, a greater efficiency of the 5-nitrofuran derivate in relation to the 2-nitroimidazole. So, we conclude that the mode of action of megazol is different from the ones of nifurtimox and benznidazole and that its primary effect is associated with an impairment of protein synthesis.     

Effect of the host specific treatment in the phagocytosis of Trypanosoma cruzi blood forms by mouse peritoneal macrophages

Single doses of drugs active against Trypanosoma cruzi (megazol, nifurtimox and benznidazole) induce a rapid clearance of the blood parasites in experimentally infected mice. Furthermore, the in vitro phagocytosis and intracellular destruction by mouse peritoneal macrophage of blood forms collected from the treated animals is strongly enhanced as compared with parasites from untreated controls. The uptake of the blood forms by macrophages is significantly higher with megazol than with benznidazole and nifurtimox, a finding that concurs with data showing that megazol is also the most active compound in the living host. The possibility that macrophages participate in a synergic effect between the host immune response and chemotherapeutic effect is discussed.     

Chagas disease: Present status of pathogenic mechanisms and chemotherapy

There are approximately 7.8 million people in Latin America, including Chile, who suffer from Chagas disease and another 28 million who are at risk of contracting it. Chagas is caused by the flagellate protozoan Trypanosoma cruzi. It is a chronic disease, where 20%-30% of infected individuals develop severe cardiopathy, with heart failure and potentially fatal arrhythmias. Currently, Chagas disease treatment is more effective in the acute phase, but does not always produce complete parasite eradication during indeterminate and chronic phases. At present, only nifurtimox or benznidazole have been proven to be superior to new drugs being tested. Therefore, it is necessary to find alternative approaches to treatment of chronic Chagas. The current treatment may be rendered more effective by increasing the activity of anti-Chagasic drugs or by modifying the host's immune response. We have previously shown that glutathione synthesis inhibition increases nifurtimox and benznidazole activity. In addition, there is increasing evidence that cyclooxygenase inhibitors present an important effect on T. cruzi infection. Therefore, we found that aspirin reduced the intracellular infection in RAW 264.7 cells and, decreased myocarditis extension and mortality rates in mice. However, the long-term benefit of prostaglandin inhibition for Chagasic patients is still unknown.     

Free radical metabolism of antiparasitic agents

In recent years it has been apparent that many of the known antiparasitic drugs produce free radicals. Intracellular reduction followed by autooxidation yielding O.-2 and H2O2 has been suggested as the mode of action of nifurtimox on Trypanosoma cruzi and as the basis of its toxicity in mammals. On the other hand, free radical intermediates that do not generate oxygen-reduction products under physiological conditions have been found in the metabolic pathways of other antiparasitic nitro compounds (benznidazole, metronidazole, and other 5-nitroimidazoles) used in the treatment of diseases such as Chagas' disease, trichomoniasis, giardiasis, balantidiasis, amebiasis, and schistosomiasis. In these cases, as well as in the case of niridazole (used in the treatment of schistosomiasis), covalent binding or other interactions of the intermediates of nitroreduction with parasite macromolecules are possibly involved in their toxicity. Redox cycling of these compounds under aerobic conditions appears to be a detoxification reaction by inhibiting net reduction of the drugs.     

Biological characterization of Trypanosoma cruzi strains

Biological parameters of five Trypanosoma cruzi strains from different sources were determined in order to know the laboratory behaviour of natural populations. The parameters evaluated were growth kinetics of epimastigotes, differentiation into metacyclic forms, infectivity in mammalian cells grown in vitro and parasite susceptibility to nifurtimox, benznidazole and gentian violet. Differences in transformation to metacyclic, in the percentage of infected cells as well as in the number of amastigotes per cell were observed among the strains. Regarding to pharmacological assays, Y strain was the most sensitive to the three assayed compounds. These data demonstrate the heterogeneity of natural populations of T. cruzi, the only responsible of infection in humans.     

Trypanosoma cruzi: In vitro effect of aspirin with nifurtimox and benznidazole

Nifurtimox and benznidazole are the only active drugs against Trypanosoma cruzi; however, they have limited efficacy and severe side effects. During primoinfection, T. cruzi infected macrophages mount an antiparasitic response, which the parasite evades through an increase of tumor growth factor β and PGE₂ activation as well as decreased iNOS activity. Thus, prostaglandin synthesis inhibition with aspirin might increase macrophage antiparasitic activity and increase nifurtimox and benznidazole effect.Aspirin alone demonstrated a low effect upon macrophage antiparasitic activity. However, isobolographic analysis of the combined effects of aspirin, nifurtimox and benznidazole indicated a synergistic effect on T. cruzi infection of RAW cells, with combinatory indexes of 0.71 and 0.61, respectively.The observed effect of aspirin upon T. cruzi infection was not related with the PGE₂ synthesis inhibition. Nevertheless, NO levels were restored by aspirin in T. cruzi-infected RAW cells, contributing to macrophage antiparasitic activity improvement.Thus, the synergy of aspirin with nifurtimox and benznidazole is due to the capability of aspirin to increase antiparasitic activity of macrophages.     

Inhibition of Trypanosoma cruzi alpha-hydroxyacid dehydrogenase-isozyme II by N-isopropyl oxamate and its effect on intact epimastigotes

The effect of N-isopropyl oxamate on the activity of alpha-hydroxyacid dehydrogenase-isozyme II (HADH-isozyme II) from Trypanosoma cruzi was investigated. The kinetic studies showed that this substance was a competitive inhibitor of this isozyme. The attachment of the nonpolar isopropylic branched chain to the nitrogen of oxamate increased 12-fold the affinity of N-isopropyl oxamate for the active site of T. cruzi HADH-isozyme II. N-isopropyl oxamate was a selective inhibitor of HADH-isozyme II, since other T. cruzi dehydrogenases were not inhibited by this substance. Since HADH-isozyme II participates in the energy metabolism of T. cruzi, a trypanocidal effect can be expected with inhibitors of this isozyme. However, although it was not possible to detect any trypanocidal activity with N-isopropyl oxamate when the ethyl ester was tested as a possible trypanocidal prodrug, the expected trypanocidal effect was obtained, comparable to that obtained with nifurtimox and benznidazole.     

Synthesis, in vitro evaluation, and SAR studies of a potential antichagasic 1H-pyrazolo[3,4-b]pyridine series

The development of new drugs against Trypanosoma cruzi is still required since the only two drugs currently used cause severe side effects. In this work we described the synthesis, the in vitro biological evaluation, and the SAR results of 1H-pyrazolo[3,4-b]pyridine derivatives, a new antichagasic agent series. The presence of fluorine, hydroxyl or nitro group at Y position resulted in at least one or two promising compounds in each set of derivatives (6f, 6g, 6i, 6l, and 6m). The SAR study showed that trypanocidal activity observed depends on both geometric and stereoelectronic parameters (MEP and frontier molecular orbitals HOMO and LUMO). We also used the Osiris program for calculating and comparing the fragment based druglikeness of the most active derivative (6g) (IC(50)=1.9microg/mL), the inactive compound (6o), and the current toxic antichagasic drugs (nifurtimox and benznidazole). Interestingly 6g presented a potential druglikeness higher than nifurtimox and benznidazole while 6o presented the lowest value among them.     

Identification of benzoylisoquinolines as potential anti-Chagas agents

A set of three 3-benzoyl substituted isoquinolones was synthesized in good yields and assayed for in vitro trypanocidal activity against Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease. Depending on the concentration evaluated, a greater or equivalent reduction in the number of bloodborne trypomastigotes compared to that observed with benznidazole, a drug currently used to attack the parasite, was observed for two of the samples. In order to assess the potential of the 3-benzoylisoquinolone nucleus as a possible scaffold in the design of novel anti-trypanosomal lead structures, a computational analysis was performed using structural and inhibition information from both functional and target assays archived in the online database, ChEMBL. Chemical space projection of the synthesized compounds along with 3067 structures with known activities against T. cruzi shows that the isoquinolones occupy a sparsely-populated region of chemical space, indicating their potential for development as a novel class of trypanocidals. In addition, 2D and 3D structural similarity analyses revealed micromolar and submicromolar inhibitors of T. cruzi in ChEMBL with high similarity to the synthesized structures.     

Oxidative stress in malaria parasite-infected erythrocytes: host-parasite interactions

Experimenta naturae, like the glucose-6-phosphate dehydrogenase deficiency, indicate that malaria parasites are highly susceptible to alterations in the redox equilibrium. This offers a great potential for the development of urgently required novel chemotherapeutic strategies. However, the relationship between the redox status of malarial parasites and that of their host is complex. In this review article we summarise the presently available knowledge on sources and detoxification pathways of reactive oxygen species in malaria parasite-infected red cells, on clinical aspects of redox metabolism and redox-related mechanisms of drug action as well as future prospects for drug development. As delineated below, alterations in redox status contribute to disease manifestation including sequestration, cerebral pathology, anaemia, respiratory distress, and placental malaria. Studying haemoglobinopathies, like thalassemias and sickle cell disease, and other red cell defects that provide protection against malaria allows insights into this fine balance of redox interactions. The host immune response to malaria involves phagocytosis as well as the production of nitric oxide and oxygen radicals that form part of the host defence system and also contribute to the pathology of the disease. Haemoglobin degradation by the malarial parasite produces the redox active by-products, free haem and H(2)O(2), conferring oxidative insult on the host cell. However, the parasite also supplies antioxidant moieties to the host and possesses an efficient enzymatic antioxidant defence system including glutathione- and thioredoxin-dependent proteins. Mechanistic and structural work on these enzymes might provide a basis for targeting the parasite. Indeed, a number of currently used drugs, especially the endoperoxide antimalarials, appear to act by increasing oxidant stress, and novel drugs such as peroxidic compounds and anthroquinones are being developed.     

Biomarkers of therapeutic responses in chronic Chagas disease: state of the art and future perspectives

The definition of a biomarker provided by the World Health Organization is any substance, structure, or process that can be measured in the body, or its products and influence, or predict the incidence or outcome of disease. Currently, the lack of prognosis and progression markers for chronic Chagas disease has posed limitations for testing new drugs to treat this neglected disease. Several molecules and techniques to detect biomarkers in Trypanosoma cruzi-infected patients have been proposed to assess whether specific treatment with benznidazole or nifurtimox is effective. Isolated proteins or protein groups from different T. cruzi stages and parasite-derived glycoproteins and synthetic neoglycoconjugates have been demonstrated to be useful for this purpose, as have nucleic acid amplification techniques. The amplification of T. cruzi DNA using the real-time polymerase chain reaction method is the leading test for assessing responses to treatment in a short period of time. Biochemical biomarkers have been tested early after specific treatment. Cytokines and surface markers represent promising molecules for the characterisation of host cellular responses, but need to be further assessed.     

Cytotoxicity and trypanocidal activity of nifurtimox encapsulated in ethylcyanoacrylate nanoparticles

The aim of the present study was to study the trypanocidal activity of nanoparticles loaded with nifurtimox in comparison with the free drug against Trypanosoma cruzi, responsible for Chagas' disease. Ethylcyanoacrylate nanoparticles acted as the delivery system into cells. As the obligate replicative intracellular form is amastigote, in vitro studies were performed on this form of parasite as well as on cell culture derived trypomastigotes. The fluorescence method used here was very useful as it allowed for the simultaneous study of trypanocide activity and cytotoxicity by determining living or dead parasites within living or dead host cells. According to these results, the greatest trypanocide activity on cell culture-derived trypomastigotes was recorded for nifurtimox-loaded nanoparticles with a 50% inhibitory concentration (IC50) twenty times less than that of the free drug. The cytotoxicity of unloaded nanoparticles at low concentrations was similar to that obtained by free drug when evaluated on Vero cells. Furthermore, nifurtimox-loaded nanoparticles showed increased trypanocide activity on intracellular amastigotes with an IC50 thirteen times less than that of nifurtimox. We also observed that the unloaded nanoparticles possess the previously-described trypanocide activity, similar to the standard solution of nifurtimox, although the mechanism for this has not yet been elucidated. In conclusion, it was possible to establish in vitro conditions using nifurtimox encapsulated nanoparticles in order to decrease the doses of the drug and thus to obtain high trypanocidal activity on both free trypomastigotes and intracellular amastigotes with low cytotoxicity for the host cell.     

New potent 5-nitroindazole derivatives as inhibitors of Trypanosoma cruzi growth: Synthesis,biological evaluation,and mechanism of action studies

New 5-nitroindazole derivatives were developed and their antichagasic properties studied. Eight compounds (14–18, 20, 26 and 28) displayed remarkable in vitro activities against Trypanosoma cruzi (T. cruzi). Its unspecific cytotoxicity against macrophages was evaluated being not toxic at a concentration at least twice that of T. cruzi IC₅₀, for some derivatives. The electrochemical studies, parasite respiration studies and ESR experiment showed that 5-nitroindazole derivatives not be able to yield a redox cycling with molecular oxygen such as occurs with nifurtimox (Nfx). The study on the mechanism of action proves to be related to the production of reduced species of the nitro moiety similar to that observed with benznidazole.     

DNA repair BER pathway inhibition increases cell death caused by oxidative DNA damage in Trypanosoma cruzi

Trypanosoma cruzi, a parasitic protozoan, is the etiological agent of Chagas disease, an endemic and neglected pathology in Latin America. It presents a life cycle that involves a hematophagous insect and man as well as domestic and wild mammals. The parasitic infection is not eliminated by the immune system of mammals; thus, the vertebrate host serves as a parasite reservoir. Additionally, chronic processes leading to dysfunction of the cardiac and digestive systems are observed. To establish a chronic infection some parasites should resist the oxidative damage to its DNA exerted by oxygen and nitrogen free radicals (ROS/RNS) generated in host cells. Till date there are no reports directly showing oxidative DNA damage and repair in T. cruzi. We establish that ROS/RNS generate nuclear and kinetoplastid DNA damage in T. cruzi that may be partially repaired by the parasite. Furthermore, we determined that both oxidative agents diminish T. cruzi cell viability. This effect is significantly augmented in parasites subsequently incubated with methoxyamine, a DNA base excision repair (BER) pathway inhibitor, strongly suggesting that the maintenance of T. cruzi viability is a consequence of DNA repair mechanisms.     

Specific chemotherapy of Chagas disease: controversies and advances

Chagas disease, caused by Trypanosoma cruzi, is a major public health problem in Latin America, where it constitutes one of the largest parasitic disease burdens. Specific treatment of this condition has been controversial, but there is a growing consensus that elimination of T. cruzi could be a prerequisite to arrest the evolution of the disease. Currently available chemotherapy, based on a nitrofuran (nifurtimox) and a nitroimidazole (benznidazole), is unsatisfactory because of their limited efficacy in the prevalent chronic stage of the disease and their toxic side effects. New approaches to specific chemotherapy are being advanced. Biochemical routes such as the de novo sterol biosynthesis pathway, cruzipain-mediated proteolysis and pyrophosphate metabolism have been chemically validated, and the selective in vitro and in vivo anti-T. cruzi activities of inhibitors of these pathways have been demonstrated. Several of these compounds have now completed pre-clinical studies and are poised for clinical trials in the near future. Other promising approaches include interference with trypanothione synthesis and redox metabolism, in addition to inhibition of purine salvage, dihydrofolate reductase, phospholipid biosynthesis, and protein prenylation and acylation.