Drug screening and development cascade for Chagas disease: an update of in vitro and in vivo experimental models.

Chagas disease is a tropical neglected disease that affects millions of people worldwide, still demanding a more effective and safer therapy, especially in its chronic phase which lacks a treatment that promotes substantial parasitological cure. The technical note of Romanha and collaborators published in 2010 aimed establish a guideline with the set of minimum criteria and decision gates for the development of new agents against Trypanosoma cruzi with the focus on developing new antichagasic drugs. In this sense, the present review aims to update this technical note, bringing the state of the art and new advances on this topic in recent years.

Anti-Trypanosoma cruzi Activity, Mutagenicity, Hepatocytotoxicity and Nitroreductase Enzyme Evaluation of 3-Nitrotriazole, 2-Nitroimidazole and Triazole Derivatives.

Chagas disease (CD), which is caused by Trypanosoma cruzi and was discovered more than 100 years ago, remains the leading cause of death from parasitic diseases in the Americas. As a curative treatment is only available for the acute phase of CD, the search for new therapeutic options is urgent. In this study, nitroazole and azole compounds were synthesized and underwent molecular modeling, anti-T. cruzi evaluations and nitroreductase enzymatic assays. The compounds were designed as possible inhibitors of ergosterol biosynthesis and/or as substrates of nitroreductase enzymes. The in vitro evaluation against T. cruzi clearly showed that nitrotriazole compounds are significantly more potent than nitroimidazoles and triazoles. When their carbonyls were reduced to hydroxyl groups, the compounds showed a significant increase in activity. In addition, these substances showed potential for action via nitroreductase activation, as the substances were metabolized at higher rates than benznidazole (BZN), a reference drug against CD. Among the compounds, 1-(2,4-difluorophenyl)-2-(3-nitro-1H-1,2,4-triazol-1-yl)ethanol (8) is the most potent and selective of the series, with an IC50 of 0.39 µM and selectivity index of 3077; compared to BZN, 8 is 4-fold more potent and 2-fold more selective. Moreover, this compound was not mutagenic at any of the concentrations evaluated, exhibited a favorable in silico ADMET profile and showed a low potential for hepatotoxicity, as evidenced by the high values of CC50 in HepG2 cells. Furthermore, compared to BZN, derivative 8 showed a higher rate of conversion by nitroreductase and was metabolized three times more quickly when both compounds were tested at a concentration of 50 µM. The results obtained by the enzymatic evaluation and molecular docking studies suggest that, as planned, nitroazole derivatives may utilize the nitroreductase metabolism pathway as their main mechanism of action against Trypanosoma cruzi. In summary, we have successfully identified and characterized new nitrotriazole analogs, demonstrating their potential as promising candidates for the development of Chagas disease drug candidates that function via nitroreductase activation, are considerably selective and show no mutagenic potential.

Trypanocidal Activity of Flavanone Derivatives.

Chagas disease, also known as American trypanosomiasis, is classified as a neglected disease by the World Health Organization. For clinical treatment, only two drugs have been on the market, Benznidazole and Nifurtimox, both of which are recommended for use in the acute phase but present low cure rates in the chronic phase. Furthermore, strong side effects may result in discontinuation of this treatment. Faced with this situation, we report the synthesis and trypanocidal activity of 3-benzoyl-flavanones. Novel 3-benzoyl-flavanone derivatives were prepared in satisfactory yields in the 3-step synthetic procedure. According to recommended guidelines, the whole cell-based screening methodology was utilized that allowed for the simultaneous use of both parasite forms responsible for human infection. The majority of the tested compounds displayed promising anti-Trypanosoma cruzi activity and the most potent flavanone bearing a nitrofuran moiety was more potent than the reference drug, Benznidazole.

In Vitro Trypanocidal and Antibacterial Activities of Essential Oils from Four Species of the Family Annonaceae.

The objective of this study was to evaluate the chemical composition, and the trypanocidal and antibacterial activities of the essential oils from four species of Annonaceae: Bocageopsis multiflora (Mart.) R.E.Fr., Duguetia quitarensis Benth., Fusaea longifolia (Aubl.) Saff., and Guatteria punctata (Aubl.) R.A.Howard. The chemical composition of the essential oils from the aerial parts yielded 23, 20, 21 and 23 constituents, respectively, which were identified by GC/MS. The trypanocidal activity was evaluated against the amastigote and trypomastigote forms of T. cruzi. The antibacterial activity was evaluated by the microdilution method against enterohemorrhagic Escherichia coli, Pseudomonas aeruginosa, Streptococcus mutans, Streptococcus pyogenes, and methicillin-resistant Staphylococcus aureus. The results of trypanocidal activity showed that the essential oils of the four species were active at the tested concentrations, with G. punctata essential oil being the most active, with IC50 =0.029 µg/mL, and selectivity index (SI)=32, being 34 times more active than the reference drug benznidazole. All EOs showed strong antibacterial activity (minimum inhibitory concentrations of 4.68-37.5 µg/mL) against strains of S. mutans.

Synthesis and biological evaluation of potential inhibitors of the cysteine proteases cruzain and rhodesain designed by molecular simplification.

Analogues of 8-chloro-N-(3-morpholinopropyl)-5H-pyrimido[5,4-b]indol-4-amine 1, a known cruzain inhibitor, were synthesized using a molecular simplification strategy. Five series of analogues were obtained: indole, pyrimidine, quinoline, aniline and pyrrole derivatives. The activity of the compounds was evaluated against the enzymes cruzain and rhodesain as well as against Trypanosoma cruzi amastigote and trypomastigote forms. The 4-aminoquinoline derivatives showed promising activity against both enzymes, with IC50 values ranging from 15 to 125µM. These derivatives were selective inhibitors for the parasitic proteases, being unable to inhibit mammalian cathepsins B and S. The most active compound against cruzain (compound 5a; IC50=15µM) is considerably more synthetically accessible than 1, while retaining its ligand efficiency. As observed for the original lead, compound 5a was shown to be a competitive enzyme inhibitor. In addition, it was also active against T. cruzi (IC50=67.7µM). Interestingly, the pyrimidine derivative 4b, although inactive in enzymatic assays, was highly active against T. cruzi (IC50=3.1µM) with remarkable selectivity index (SI=128) compared to uninfected fibroblasts. Both 5a and 4b exhibit drug-like physicochemical properties and are predicted to have a favorable ADME profile, therefore having great potential as candidates for lead optimization in the search for new drugs to treat Chagas disease.

18-Des-hydroxy Cytochalasin: an antiparasitic compound of Diaporthe phaseolorum-92C, an endophytic fungus isolated from Combretum lanceolatum Pohl ex Eichler.

Chemical investigation of the ethyl acetate extract from the endophytic fungus Diaporthe phaseolorum-92C (92C) isolated from the roots of Combretum lanceolatum led to the isolation of 18-des-hydroxy Cytochalasin H (compound 1). The trypanocidal and schistosomicidal activity and cytotoxicity of the extract from 92C were evaluated. The schistosomicidal, leishmanicidal, antimicrobial, and antioxidant actions, as well as the antitumor activity against the breast cancer cells MDA-MB-231 and MCF-7, and the cytotoxicity towards normal human lung fibroblasts GM07492A of compound 1 was tested. The extract from 92C (20 µg/mL) exerted potent trypanocidal activity, reducing 82% of the number of amastigotes and trypomastigotes of Trypanosoma cruzi. Compound 1 at 50 µg/mL killed 50% of Schistosoma mansoni adult worms. Compound 1 reduced the viability of Leishmania amazonenses promastigotes (IC50 = 9.2 µg/mL) and of the cancer cells MDA-MB-231 and MCF-7 (IC50 = 17.5 and 8.88 µg/mL, respectively), presented moderate antioxidant activity, and gave IC50 of 2049.7 ± 39.9 µg/mL for the cytotoxicity towards normal cells GM07492A. This knowledge is highly relevant to the search for new promising compounds for therapeutic purposes.

Synthesis of Xylitan Derivatives and Preliminary Evaluation of in Vitro Trypanocidal Activity.

A series of novel xylitan derivatives derived from xylitol were synthesized using operationally simple procedures. A xylitan acetonide was the key intermediate used to prepare benzoate, arylsulfonate esters and 1,2,3-triazole derivatives of xylitan. These compounds were evaluated for their in vitro anti-Trypanosoma cruzi activity against trypomastigote and amastigote forms of the parasite in T. cruzi-infected cell lineages. Benznidazole was used as positive control against T. cruzi and cytotoxicity was determined in mammalian L929 cells. The arylsulfonate xylitan derivative bearing a nitro group displayed the best activity of all the compounds tested, and was slightly more potent than the reference drug benznidazole. The importance of the isopropylidene ketal moiety was established and the greater lipophilicity of these compounds suggests enhancement in cell penetration.

Synthesis of a sugar-based thiosemicarbazone series and structure-activity relationship versus the parasite cysteine proteases rhodesain, cruzain, and Schistosoma mansoni cathepsin B1.

The pressing need for better drugs against Chagas disease, African sleeping sickness, and schistosomiasis motivates the search for inhibitors of cruzain, rhodesain, and Schistosoma mansoni CB1 (SmCB1), the major cysteine proteases from Trypanosoma cruzi, Trypanosoma brucei, and S. mansoni, respectively. Thiosemicarbazones and heterocyclic analogues have been shown to be both antitrypanocidal and inhibitory against parasite cysteine proteases. A series of compounds was synthesized and evaluated against cruzain, rhodesain, and SmCB1 through biochemical assays to determine their potency and structure-activity relationships (SAR). This approach led to the discovery of 6 rhodesain, 4 cruzain, and 5 SmCB1 inhibitors with 50% inhibitory concentrations (IC50s) of ≤ 10 µM. Among the compounds tested, the thiosemicarbazone derivative of peracetylated galactoside (compound 4i) was discovered to be a potent rhodesain inhibitor (IC50 = 1.2 ± 1.0 µM). The impact of a range of modifications was determined; removal of thiosemicarbazone or its replacement by semicarbazone resulted in virtually inactive compounds, and modifications in the sugar also diminished potency. Compounds were also evaluated in vitro against the parasites T. cruzi, T. brucei, and S. mansoni, revealing active compounds among this series.

In vitro activity of 1,3-bisaryloxypropanamines against Trypanosoma cruzi-infected L929 cultures.

We describe herein the antitrypanosomal activity of 20 novel 1,3-bis(aryloxy)propan-2-amine derivatives. Compounds 2, 4, 6, 12, 15, 16 and 19 were significantly active against amastigote and trypomastigote forms, with half maximal inhibitory concentrationvalues in the range of 6-18 µM. In the cytotoxicity tests against L929 cells, only compound 4 presented selectivity index value above 10, indicating low toxicity.

The in vitro activity of fatty diamines and amino alcohols against mixed amastigote and trypomastigote Trypanosoma cruzi forms.

Four diamines and three amino alcohols derived from 1-decanol, 1-dodecanol and 1,2-dodecanediol were evaluated in an in vitro assay against a mixture of trypomastigote and intracellular amastigote forms of Trypanosoma cruzi. Two of these compounds (6 and 7) showed better activity against both proliferative stages of T. cruzi than the positive control benznidazole, three were of similar potency (1, 2 and 5) and two were less active (3 and 4).

New insights into Trypanosoma cruzi genetic diversity, and its influence on parasite biology and clinical outcomes.

Chagas disease, caused by Trypanosoma cruzi, remains a serious public health problem worldwide. The parasite was subdivided into six distinct genetic groups, called "discrete typing units" (DTUs), from TcI to TcVI. Several studies have indicated that the heterogeneity of T. cruzi species directly affects the diversity of clinical manifestations of Chagas disease, control, diagnosis performance, and susceptibility to treatment. Thus, this review aims to describe how T. cruzi genetic diversity influences the biology of the parasite and/or clinical parameters in humans. Regarding the geographic dispersion of T. cruzi, evident differences were observed in the distribution of DTUs in distinct areas. For example, TcII is the main DTU detected in Brazilian patients from the central and southeastern regions, where there are also registers of TcVI as a secondary T. cruzi DTU. An important aspect observed in previous studies is that the genetic variability of T. cruzi can impact parasite infectivity, reproduction, and differentiation in the vectors. It has been proposed that T. cruzi DTU influences the host immune response and affects disease progression. Genetic aspects of the parasite play an important role in determining which host tissues will be infected, thus heavily influencing Chagas disease's pathogenesis. Several teams have investigated the correlation between T. cruzi DTU and the reactivation of Chagas disease. In agreement with these data, it is reasonable to suppose that the immunological condition of the patient, whether or not associated with the reactivation of the T. cruzi infection and the parasite strain, may have an important role in the pathogenesis of Chagas disease. In this context, understanding the genetics of T. cruzi and its biological and clinical implications will provide new knowledge that may contribute to additional strategies in the diagnosis and clinical outcome follow-up of patients with Chagas disease, in addition to the reactivation of immunocompromised patients infected with T. cruzi.

Novel diamides inspired by protein kinase inhibitors as anti-Trypanosoma cruzi agents: in vitro and in vivo evaluations.

Background: Chagas disease is a life-threatening illness caused by Trypanosoma cruzi. The involvement of serine-/arginine-rich protein kinase in the T. cruzi life cycle is significant. Aims: To synthesize, characterize and evaluate the trypanocidal activity of diamides inspired by kinase inhibitor, SRPIN340. Material & Methods: Synthesis using a three-step process and characterization by infrared, nuclear magnetic resonance and high-resolution mass spectrometry were conducted. The selectivity index was obtained by the ratio of CC50/IC50 in two in vitro models. The most active compound, 3j, was evaluated using in vitro cytokine assays and assessing in vivo trypanocidal activity. Results: 3j activity in the macrophage J774 lineage showed an anti-inflammatory profile, and mice showed significantly reduced parasitemia and morbidity at low compound dosages. Conclusion: Novel diamide is active against T. cruzi in vitro and in vivo.

Structural design, synthesis, and anti-Trypanosomatidae profile of new Pyridyl-thiazolidinones.

The present work reports the synthesis of a novel series of pyridine-thiazolidinones with anti-Trypanosoma cruzi and leishmanicidal activities (compounds 10-27), derived from 2 or 4-pyridine thiosemicarbazones (1-9). The in vitro assays were performed with Trypanosoma cruzi trypomastigotes and amastigotes, as well as with Leishmania amazonensis promastigotes and amastigotes. The cytotoxicity profile was evaluated using the cell line RAW 264.7. From the 18 pyridine-thiazolidinones, 5 were able to inhibit trypomastigotes. Overall, all compounds inhibited amastigotes, highlighting compounds 15 (0.60 µM), 18 (0.64 µM), 17 (0.81 µM), and 27 (0.89 µM). Compounds 15 and 18 were able to induce parasite cell death through necrosis induction. Analysis by scanning electron microscopy showed that T. cruzi trypomastigotes treated with compounds 15 and 18 induced morphological changes such as shortening, retraction and curvature of the parasite body and leakage of internal content. Regarding the antiparasitic evaluation against Leishmania amazonensis, only compound 27 had a higher selectivity compared to Miltefosine against the amastigote form (IC50 = 5.70 µM). Our results showed that compound 27 presented an antiparasitic activity for both Trypanosoma cruzi and Leishmania amazonensis. After in silico evaluation, it was suggested that the new pyridine-thiazolidinones had an appropriate drug-likeness profile. Our results pointed out a new chemical frame with an anti-Trypanosomatidae profile. The pyridine-thiazolidinones presented here for the first time could be used as a starting point for the development of new antiparasitic agents.

In vitro trypanocidal activity of DB745B and other novel arylimidamides against Trypanosoma cruzi.

OBJECTIVES: As part of a search for new therapeutic opportunities to treat chagasic patients, in vitro efficacy studies were performed to characterize the activity of five novel arylimidamides (AIAs) against Trypanosoma cruzi. METHODS: The trypanocidal effect against T. cruzi was evaluated by light microscopy through the determination of IC50 values. Cytotoxicity was determined by MTT assays against mouse cardiomyocytes. RESULTS: Our data demonstrated the trypanocidal efficacy of these new compounds against bloodstream trypomastigotes and intracellular amastigotes, exhibiting IC50 values ranging from 0.015 to 2.5 and 0.02 to0.2 µM, respectively. One of the compounds, DB745B, was also highly active against a broad panel of isolates, including those naturally resistant to benznidazole. DB745B showed higher in vitro efficacy than the reference drugs used to treat patients (benznidazole IC50= 12.94 µM) and to prevent blood bank infection (gentian violet IC50= 30.6 µM). CONCLUSIONS: AIAs represent promising new chemical entities against T. cruzi and are also potential trypanocidal agents to prevent transfusion-associated Chagas' disease.

The activity of a metronidazole analogue and its ß-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:ß-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.

Trypanocidal Trixikingolides From Trixis Vauthieri.

Chagas disease is an illness caused by the protozoan parasite Trypanosoma cruzi. Only two drugs are available, with the drawback of low rate of cure in the chronic phase of the disease and undesirable side effects. These facts highlight the need to find new compounds for Chagas disease chemotherapy. We describe the isolation and identification of an inseparable mixture of two new trixikingolides from Trixis vauthieri, a plant from family Asteraceae, which present outstanding in vitro trypanocidal activity, with IC50 value of 0.053 µM against the intracellular trypomastigotes and amastigotes forms of T. cruzi infecting L929 cells. The IC50 of the mixture against the host cells is 68 times higher and about 70 times more potent than benznidazole, the reference drug used as control at the experiments. The next step, which depends on obtaining larger quantities of the mixture, is to test it on mice infected with T. cruzi.

Arylimidamide DB766, a potential chemotherapeutic candidate for Chagas' disease treatment.

Chagas' disease, a neglected tropical illness for which current therapy is unsatisfactory, is caused by the intracellular parasite Trypanosoma cruzi. The goal of this work is to investigate the in vitro and in vivo effects of the arylimidamide (AIA) DB766 against T. cruzi. This arylimidamide exhibits strong trypanocidal activity and excellent selectivity for bloodstream trypomastigotes and intracellular amastigotes (Y strain), giving IC(50)s (drug concentrations that reduce 50% of the number of the treated parasites) of 60 and 25 nM, respectively. DB766 also exerts striking effects upon different parasite stocks, including those naturally resistant to benznidazole, and displays higher activity in vitro than the reference drugs. By fluorescent and transmission electron microscopy analyses, we found that this AIA localizes in DNA-enriched compartments and induces considerable damage to the mitochondria. DB766 effectively reduces the parasite load in the blood and cardiac tissue and presents efficacy similar to that of benznidazole in mouse models of T. cruzi infection employing the Y and Colombian strains, using oral and intraperitoneal doses of up to 100 mg/kg/day that were given after the establishment of parasite infection. This AIA ameliorates electrocardiographic alterations, reduces hepatic and heart lesions induced by the infection, and provides 90 to 100% protection against mortality, which is similar to that provided by benznidazole. Our data clearly show the trypanocidal efficacy of DB766, suggesting that this AIA may represent a new lead compound candidate to Chagas' disease treatment.

Design, synthesis, molecular modelling, and in vitro evaluation of tricyclic coumarins against Trypanosoma cruzi.

Chagas disease is caused by infection with the parasite protozoan Trypanosoma cruzi and affects about 8 million people in 21 countries in Latin America. The main form of treatment of this disease is still based on the use of two drugs, benznidazole and nifurtimox, which both present low cure rates in the chronic phase and often have serious side-effects. Herein, we describe the synthesis of tricyclic coumarins that were obtained via NHC organocatalysis and evaluation of their trypanocidal activity. Molecular docking studies against trypanosomal enzyme triosephosphate isomerase (TIM) were carried out, as well as a theoretical study of the physicochemical parameters. The tricyclic coumarins were tested in vitro against the intracellular forms of Trypanosoma cruzi. Among the 18 compounds tested, 10 were more active than the reference drug benznidazole. The trypanocidal activity of the lead compound was rationalized by molecular docking study which suggested the strong interaction with the enzyme TIM by T. cruzi and therefore indicating a possible mode of action. Furthermore, the selectivity index of eight tricyclic coumarins with high anti-T. cruzi activity was above 50 and thus showing that these lead compounds are viable candidates for further in vivo assays.

Accomplishing the genotype-specific serodiagnosis of single and dual Trypanosoma cruzi infections by flow cytometry Chagas-Flow ATE-IgG2a.

The methods currently available for genotype-specific diagnosis of T. cruzi infection still present relevant limitations, especially to identify mixed infection. In the present investigation, we have evaluated the performance of Chagas-Flow ATE-IgG2a test for early and late differential diagnosis of single and dual genotype-specific T. cruzi infections. Serum samples from Swiss mice at early and late stages of T. cruzi infection were assayed in parallel batches for genotype-specific diagnosis of single (TcI, TcVI or TcII) and dual (TcI+TcVI, TcVI+TcII or TcII+TcI) infections. The intrinsic reactivity to TcI, TcVI and TcII target antigens, including amastigote (AI/AVI/AII), trypomastigote-(TI/TVI/TII) and epimastigote (EI/EVI/EII), at specific reverse of serum dilutions (500 to 64,000), was employed to provide reliable decision-trees for "early" vs "late", "single vs "dual" and "genotype-specific" serology. The results demonstrated that selective set of attributes "EII 500/EI 2,000/AII 500" were able to provide high-quality accuracy (81%) to segregate early and late stages of T. cruzi infection. The sets "TI 2,000/AI 1,000/EII 1,000" and "TI 8,000/AII 32,000" presented expressive scores to discriminate single from dual T. cruzi infections at early (85%) and late stages (84%), respectively. Moreover, the attributes "TI 4,000/TVI 500/TII 1,000", "TI 16,000/EI 2,000/EII 2,000/AI 500/TVI 500" showed good performance for genotype-specific diagnosis at early stage of single (72%) and dual (80%) T. cruzi infections, respectively. In addition, the attributes "TI 4,000/AII 1,000/EVI 1,000", "TI 64,000/AVI 500/AI 2,000/AII 1,000/EII 4,000" showed moderate performance for genotype-specific diagnosis at late stage of single (69%) and dual (76%) T. cruzi infections, respectively. The sets of decision-trees were assembled to construct a sequential algorithm with expressive accuracy (81%) for serological diagnosis of T. cruzi infection. These findings engender new perspectives for the application of Chagas-Flow ATE-IgG2a method for genotype-specific diagnosis in humans, with relevant contributions for epidemiological surveys as well as clinical and post-therapeutic monitoring of Chagas disease.

The in vivo and in vitro roles of Trypanosoma cruzi Rad51 in the repair of DNA double strand breaks and oxidative lesions.

In Trypanosoma cruzi, the etiologic agent of Chagas disease, Rad51 (TcRad51) is a central enzyme for homologous recombination. Here we describe the different roles of TcRad51 in DNA repair. Epimastigotes of T. cruzi overexpressing TcRAD51 presented abundant TcRad51-labeled foci before gamma irradiation treatment, and a faster growth recovery when compared to single-knockout epimastigotes for RAD51. Overexpression of RAD51 also promoted increased resistance against hydrogen peroxide treatment, while the single-knockout epimastigotes for RAD51 exhibited increased sensitivity to this oxidant agent, which indicates a role for this gene in the repair of DNA oxidative lesions. In contrast, TcRad51 was not involved in the repair of crosslink lesions promoted by UV light and cisplatin treatment. Also, RAD51 single-knockout epimastigotes showed a similar growth rate to that exhibited by wild-type ones after treatment with hydroxyurea, but an increased sensitivity to methyl methane sulfonate. Besides its role in epimastigotes, TcRad51 is also important during mammalian infection, as shown by increased detection of T. cruzi cells overexpressing RAD51, and decreased detection of single-knockout cells for RAD51, in both fibroblasts and macrophages infected with amastigotes. Besides that, RAD51-overexpressing parasites infecting mice also presented increased infectivity and higher resistance against benznidazole. We thus show that TcRad51 is involved in the repair of DNA double strands breaks and oxidative lesions in two different T. cruzi developmental stages, possibly playing an important role in the infectivity of this parasite.