Synthesis and biological evaluation of 2-alkylaminoethyl-1,1-bisphosphonic acids against Trypanosoma cruzi and Toxoplasma gondii targeting farnesyl diphosphate synthase

The effect of a series of 2-alkylaminoethyl-1,1-bisphosphonic acids against proliferation of the clinically more relevant form of Trypanosoma cruzi, the etiologic agent of American trypanosomiasis (Chagas' disease), and against tachyzoites of Toxoplasma gondii has been studied. Most of these drugs exhibited an extremely potent inhibitory action against the intracellular form of T. cruzi, exhibiting IC(50) values at the low micromolar level. This cellular activity was associated with a strong inhibition of the enzymatic activity of T. cruzi farnesyl diphosphate synthase (TcFPPS), which constitutes a valid target for Chagas' disease chemotherapy. Compound 17 was an effective agent against amastigotes exhibiting an IC(50) value of 0.84 microM, while this compound showed an IC(50) value of 0.49 microM against the target enzyme TcFPPS. Interestingly, compound 19 was very effective against both T. cruzi and T. gondii exhibiting IC(50) values of 4.1 microM and 2.6 microM, respectively. In this case, 19 inhibited at least two different enzymes of T. cruzi (TcFPPS and solanesyl diphosphate synthase (TcSPPS); 1.01 microM and 0.25 microM, respectively), while it inhibited TgFPPS in T. gondii. In general, this family of drugs was less effective against the activity of T. cruzi SPPS and against T. gondii growth in vitro. As bisphosphonate-containing compounds are FDA-approved drugs for the treatment of bone resorption disorders, their potential low toxicity makes them good candidates to control tropical diseases.     

Synthesis and biological evaluation of new 2-alkylaminoethyl-1,1-bisphosphonic acids against Trypanosoma cruzi and Toxoplasma gondii targeting farnesyl diphosphate synthase

The effect of long-chain 2-alkylaminoethyl-1,1-bisphosphonates against proliferation of the clinically more relevant form of Trypanosoma cruzi, the etiologic agent of American trypanosomiasis (Chagas' disease), and against tachyzoites of Toxoplasma gondii was investigated. Particularly, compound 26 proved to be an extremely potent inhibitor against the intracellular form of T. cruzi, exhibiting IC(50) values at the nanomolar range. This cellular activity was associated with a strong inhibition of the enzymatic activity of T. cruzi farnesyl diphosphate synthase (TcFPPS), which constitutes a valid target for Chagas' disease chemotherapy. Compound 26 was an effective agent against T. cruzi (amastigotes) exhibiting an IC(50) value of 0.67 μM, while this compound showed an IC(50) value of 0.81 μM against the target enzyme TcFPPS. This drug was less effective against the enzymatic activity of T. cruzi solanesyl diphosphate synthase TcSPPS showing an IC(50) value of 3.2 μM. Interestingly, compound 26 was also very effective against T. gondii (tachyzoites) exhibiting IC(50) values of 6.23 μM. This cellular activity was also related to the inhibition of the enzymatic activity towards the target enzyme TgFPPS (IC(50)=0.093 μM) As bisphosphonate-containing compounds are FDA-approved drugs for the treatment of bone resorption disorders, their potential low toxicity makes them good candidates to control different tropical diseases.     

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.     

Bisphosphonates derived from fatty acids are potent growth inhibitors of Trypanosoma cruzi

We have investigated the effect of a series of bisphosphonates derived from fatty acids against Trypanosoma cruzi proliferation in in vitro assays. Some of these drugs proved to be potent inhibitors against the intracellular form of the parasite exhibiting IC50 values at the low micromolar level. As bisphosphonates are FDA clinically approved for treatment of bone resorption, their potential innocuousness makes them good candidates to control tropical diseases.     

Trypanosoma cruzi contains a single detectable uracil-DNA glycosylase and repairs uracil exclusively via short patch base excision repair

Enzymes involved in genomic maintenance of human parasites are attractive targets for parasite-specific drugs. The parasitic protozoan Trypanosoma cruzi contains at least two enzymes involved in the protection against potentially mutagenic uracil, a deoxyuridine triphosphate nucleotidohydrolase (dUTPase) and a uracil-DNA glycosylase belonging to the highly conserved UNG-family. Uracil-DNA glycosylase activities excise uracil from DNA and initiate a multistep base-excision repair (BER) pathway to restore the correct nucleotide sequence. Here we report the biochemical characterisation of T.cruzi UNG (TcUNG) and its contribution to the total uracil repair activity in T.cruzi. TcUNG is shown to be the major uracil-DNA glycosylase in T.cruzi. The purified recombinant TcUNG exhibits substrate preference for removal of uracil in the order ssU>U:G>U:A, and has no associated thymine-DNA glycosylase activity. T.cruzi apparently repairs U:G DNA substrate exclusively via short-patch BER, but the DNA polymerase involved surprisingly displays a vertebrate POLdelta-like pattern of inhibition. Back-up UDG activities such as SMUG, TDG and MBD4 were not found, underlying the importance of the TcUNG enzyme in protection against uracil in DNA and as a potential target for drug therapy.     

The activity of a metronidazole analogue and its β-cyclodextrin complex against Trypanosoma cruzi

In this study we prepared an inclusion complex between an iodide analogue of metronidazole (MTZ-I) and cyclodextrin (CD) to develop a safer and more effective method of treating Trypanosoma cruzi infections. According to our results, MTZ-I and MTZ-I:β-CD were 10 times more active than MTZ, demonstrating that the presence of an iodine atom on the side chain increased the trypanocidal activity while maintaining its cytotoxicity. The selective index shows that MTZ-I was 10 times more active against T. cruzi than it was against mammalian cells. The modification of MTZ side chains provides a promising avenue for the development of new drugs.     

Fluorine-containing aryloxyethyl thiocyanate derivatives are potent inhibitors of Trypanosoma cruzi and Toxoplasma gondii proliferation

As a part of our project aimed at developing new safe chemotherapeutic and chemoprophylactic agents against tropical diseases, fluorine-containing drugs structurally related to 4-phenoxyphenoxyethyl thiocyanate (1) were designed, synthesized, and evaluated as antiproliferative agents against Trypanosoma cruzi, the parasite responsible of American trypanosomiasis (Chagas' disease), and Toxoplasma gondii, the etiological agent of toxoplasmosis. This thiocyanate derivative had previously proven to be an effective agent against T. cruzi proliferation. Fluorine-containing thiocyanate derivatives 2 and 3 were threefold more potent than our lead drug 1 against intracellular T. cruzi. The biological evaluation against T. gondii was also very promising. The IC(50) values corresponding to 2 and 3 were at the very low micromolar level against tachyzoites of T. gondii. Both of these drugs are interesting examples of effective antiparasitic agents that have outstanding potential not only as lead drugs but also to be used for further in vivo studies.     

A rapid method for testing in vivo the susceptibility of different strains of Trypanosoma cruzi to active chemotherapeutic agents

A method is described which permits to determine in vivo and in a short period of time (4-6 hours) the sensitivity of T. cruzi strains to known active chemotherapeutic agents. By using resistant- and sensitive T. cruzi strains a fairly good correlation was observed between the results obtained with this rapid method (which detects activity against the circulating blood forms) and those obtained with long-term schedules which involve drug administration for at least 20 consecutive days and a prolonged period of assessment. This method may be used to characterize susceptibility to active drugs used clinically, provide information on the specific action against circulating trypomastigotes and screen active compounds.     

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.     

Antileishmanial and trypanocidal activity of Brazilian Cerrado plants

The side effects and the emerging resistance to the available drugs against leishmaniasis and trypanosomiasis led to the urgent need for new therapeutic agents against these diseases. Thirty one extracts of thirteen medicinal plants from the Brazilian Cerrado were therefore evaluated in vitro for their antiprotozoal activity against promastigotes of Leishmania donovani, and amastigotes of Trypanosoma cruzi. Among the selected plants, Casearia sylvestris var. lingua was the most active against both L. donovani and T. cruzi. Fifteen extracts were active against promastigotes of L. donovani with concentrations inhibiting 50% of parasite growth (IC50) between 0.1-10 microg/ml, particularly those of Annona crassiflora (Annonaceae), Himatanthus obovatus (Apocynaceae), Guarea kunthiana (Meliaceae), Cupania vernalis (Sapindaceae), and Serjania lethalis (Sapindaceae). With regard to amastigotes of T. cruzi, extracts of A. crassiflora, Duguetia furfuracea (Annonaceae), and C. sylvestris var. lingua were active with IC50 values between 0.3-10 microg/ml. Bioassay fractionations of the more active extracts are under progress to identify the active antiparasite compounds.     

Trypanocidal action of eupomatenoid-5 is related to mitochondrion dysfunction and oxidative damage in Trypanosoma cruzi

Because of its severe side effects and variable efficacy, the current treatment for Chagas disease is unsatisfactory. Natural compounds are good alternative chemotherapeutic agents for the treatment of this infection. Recently, our group reported the antiproliferative activity and morphological alterations in epimastigotes and intracellular amastigotes of Trypanosoma cruzi treated with eupomatenoid-5, a neolignan isolated from leaves of Piper regnellii var. pallescens. Here, we demonstrate that eupomatenoid-5 exhibited activity against trypomastigotes, the infective form of T. cruzi (EC?? 40.5 μM), leading to ultrastructural alteration and lipoperoxidation in the cell membrane. Additionally, eupomatenoid-5 induced depolarization of the mitochondrial membrane, lipoperoxidation and increased G6PD activity in epimastigotes of T. cruzi. These findings support the possibility that different mechanisms may be targeted, according to the form of the parasite, and that the plasma membrane and mitochondria are the structures that are most affected in trypomastigotes and epimastigotes, respectively. Thus, the trypanocidal action of eupomatenoid-5 may be associated with mitochondrial dysfunction and oxidative damage, which can trigger destructive effects on biological molecules of T. cruzi, leading to parasite death.     

Bisphosphonates derived from fatty acids are potent inhibitors of Trypanosoma cruzi farnesyl pyrophosphate synthase

Studies on the mode of action of a series of bisphosphonates derived from fatty acids, which had previously proved to be potent inhibitors against Trypanosoma cruzi proliferation in in vitro assays, have been performed. Some of these drugs proved to be potent inhibitors against the intracellular form of the parasite, exhibiting IC(50) values at the low micromolar level. As bisphosphonates are FDA clinically approved for treatment of bone resorption disorders, their potential innocuousness makes them good candidates to control tropical diseases.     

Toxic effects of natural piperine and its derivatives on epimastigotes and amastigotes of Trypanosoma cruzi

We describe herein an evaluation of trypanocidal effects of the natural alkaloid piperine and twelve synthetic derivatives against epimastigote and amastigote forms of the protozoan parasite Trypanosoma cruzi, the causative agent of the incurable human disease, Chagas' disease. The results obtained point to piperine as a suitable template for the development of new drugs with trypanocidal activity.     

In vitro activity of N-benzenesulfonylbenzotriazole on Trypanosoma cruzi epimastigote and trypomastigote forms

Chagas disease is still an important health problem in Central and South America. However, the only drugs currently available for specific treatment of this disease may induce toxic side effects in the host. The aim of this work was to determine the activity of N-benzenesulfonylbenzotriazole (BSBZT) against the protozoan parasite Trypanosoma cruzi. The effects of BSBZT and benzotriazole (BZT) were compared to those of benznidazole (BZL) on epimastigote and trypomastigote forms. BSBZT was found to have an in vitro growth inhibitory dose-dependent activity against epimastigotes, with flow cytometry analysis confirming that the treated parasites presented size reduction. BSBZT showed an IC(50) of 21.56 μg/mL (81.07 μM) against epimastigotes at 72 h of incubation, whereas BZT did not affect the growth of this parasite form. Furthermore, the toxic effect of BSBZT, was stronger and appeared earlier (at 24h) in trypomastigotes than in epimastigotes, with the LC(50) of this compound being 28.40 μg/mL (106.79 μM) against trypomastigotes. The concentrations of BSBZT used in this study presented low hemolytic activity and cytotoxicity. Consequently, at concentrations near IC(50) and LC(50) (25μg/mL), BSBZT caused only 2.4% hemolysis and 15% of RAW 264.7 cell cytotoxicity. These results reveal the potential of BSBZT as a prototype in drug design for developing new anti-T. cruzi compounds.     

Trypanocidal activity of extracts from Brazilian Atlantic Rain Forest plant species.

The trypanocidal activity of crude hydro alcoholic extracts and several fractions of 13 plants from Brazilian Atlantic Rain Forest were tested in vitro against epimastigote and trypomastigote forms of Trypanosoma cruzi, the etiological agent of Chagas disease. Crude ethanol extracts with promising in vitro activity (DL50 between 5-10 microg/ml) against epimastigotes were fractionated by solvent partition and further tested against bloodstream form of the parasite. Activity against bloodstream parasites was observed in both dichloromethane and hexane fractions of Polygala sabulosa and P. paniculata.     

In vitro antileishmanial and antitrypanosomal activities of flavanones from Baccharis retusa DC. (Asteraceae)

Leishmaniasis and Chagas' are parasitic protozoan diseases that affect the poorest population in the world, causing a high mortality and morbidity. As a result of highly toxic and long-term treatments, novel, safe and more efficacious drugs are essential. In this work, the CH(2)Cl(2) phase from MeOH extract from the leaves of Baccharis retusa DC. (Asteraceae) was fractioned to afford two flavonoids: naringenin (1) and sakuranetin (2). These compounds were in vitro tested against Leishmania spp. promastigotes and amastigotes and Trypanosoma cruzi trypomastigotes and amastigotes. Compound 2 presented activity against Leishmania (L.) amazonensis, Leishmania (V.) braziliensis, Leishmania (L.) major, and Leishmania (L.) chagasi with IC(50) values in the range between 43 and 52 μg/mL and against T. cruzi trypomastigotes (IC(50)=20.17 μg/mL). Despite of the chemical similarity, compound 1 did not show antiparasitic activity. Additionally, compound 2 was subjected to a methylation procedure to give sakuranetin-4'-methyl ether (3), which resulted in an inactive compound against both Leishmania spp. and T. cruzi. The obtained results indicated that the presence of one hydroxyl group at C-4' associated to one methoxyl group at C-7 is important to the antiparasitic activity. Further drug design studies aiming derivatives could be a promising tool for the development of new therapeutic agents for Leishmaniasis and Chagas' disease.     

Dinitroaniline herbicides against protozoan parasites: the case of Trypanosoma cruzi

The drugs presently in use against Chagas disease are very toxic, inducing a great number of side effects. Alternative treatments are necessary, not only for Chagas disease but also for other diseases caused by protozoan parasites where current drugs pose toxicity problems. The plant microtubule inhibitor trifluralin has previously been tested with success against Leishmania, Trypanosoma brucei and several other protozoan parasites. Trypanosoma cruzi, the causative agent of Chagas disease, is also sensitive to the drug. This sensitivity has been correlated with the deduced amino acid sequences of alpha- and beta-tubulin of T. cruzi as compared with plant, mammal and other parasite sequences.     

Crystal structure of the tryparedoxin peroxidase from the human parasite Trypanosoma cruzi

Tryparedoxin peroxidase from Trypanosoma cruzi (TcTXNPx) belongs to the family of typical 2-Cys peroxiredoxins. These enzymes function as antioxidants through their peroxidase and peroxynitrite reductase activities. In T. cruzi, as in all trypanosomatids, this enzyme is the final electron acceptor of a unique system for detoxifying hydroperoxides, constituting a relevant target for drug design. We have determined the crystal structure of TcTXPNx in the reduced active state. The structure comprises 10 subunits in the asymmetric unit, associated to form a decamer of toroidal shape obeying 52 (D5) point group symmetry. We have analyzed the structure of TcTXNPx by comparing it with other structures of typical 2-Cys peroxiredoxins in both redox states, and have identified key residues in the structural rearrangement taking place in the enzymatic cycle. This is the first report of the structure of an active peroxiredoxin that has peroxidase and peroxynitrite reductase activity, and it is noteworthy that it is from a human parasite. This knowledge is of interest for further understanding peroxide metabolism in these parasites, and in the design of new trypanosomatidal drugs against Chagas disease.     

2,3-diphenyl-1,4-naphthoquinone: a potential chemotherapeutic agent against Trypanosoma cruzi

Chagas disease, caused by Trypanosoma cruzi, is a widespread infection in Latin America. Currently, only 2 partially effective and highly toxic drugs, i.e., benznidazole and nifurtimox, are available for the treatment of this disease, and several efforts are underway in the search for better chemotherapeutic agents. Here, we have determined the trypanocidal activity of 2,3-diphenyl-1 ,4-naphthoquinone (DPNQ), a novel quinone derivative. In vitro, DPNQ was highly cytotoxic at a low, micromolar concentration (LD50 = 2.5 microM) against epimastigote, cell-derived trypomastigote, and intracellular amastigote forms of T. cruzi, but not against mammalian cells (LD50 = 130 microM). In vivo studies on the murine model of Chagas disease revealed that DPNQ-treated animals (3 doses of 10 mg/kg/day) showed a significant delay in parasitemia peak and higher (up to 60%) survival rate 70 days post-infection, when compared with the control group (infected, untreated). We also observed a 2-fold decrease in parasitemia between the control group (infected, untreated) and the treated group (infected, treated). No apparent drug toxicity effects were noticed in the control group (uninfected, treated). In addition, we determined that DPNQ is the first competitive inhibitor of T. cruzi lipoamide dehydrogenase (TcLipDH) thus far described. Our results indicate that DPNQ is a promising chemotherapeutic agent against T. cruzi.