Open access peer-reviewed chapter

Parasite, Compartments, and Molecules: Trick versus Treatment on Chagas Disease

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Marcos André Vannier-Santos, Giselle V. Brunoro, Maria de Nazaré C. Soeiro, Solange L. DeCastro and Rubem F.S. Menna-Barreto

Submitted: September 6th, 2018 Reviewed: January 16th, 2019 Published: March 15th, 2019

DOI: 10.5772/intechopen.84472

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Chagas disease, caused by the protozoan Trypanosoma cruzi, is endemic to Latin America, standing out as a socio-economic problem for low-income tropical populations. Such disease affects millions of people worldwide and emerges in nonendemic areas due to migration and climate changes. The current chemotherapy is restricted to two nitroderivatives (benznidazole and nifurtimox), which is unsatisfactory due to limited efficacy (particularly in chronic phase) and adverse side effects. T. cruzi life cycle is complex, including invertebrate and vertebrate hosts and three developmental forms (epimastigotes, trypomastigotes, and amastigotes). In this chapter, we will discuss promising cellular and molecular targets present in the vertebrate-dwelling forms of the parasite (trypomastigotes and amastigotes). Among the cellular targets, the mitochondrion is the most frequently studied; while among the molecular ones, we highlight squalene synthase, C14α-sterol demethylase, and cysteine proteases. In this scenario, proteomics becomes a valuable tool for the identification of other molecular targets, and some previously identified candidates will be also discussed. Multidisciplinary studies are needed to identify novel key molecules in T. cruzi in order to increase trypanocidal activity and reduce mammalian toxicity, ensuring the development of novel drugs for Chagas disease.


  • Trypanosoma cruzi
  • Chagas disease
  • chemotherapy
  • drug targets
  • organelles
  • proteomics
  • oxidative metabolism

1. Introduction

Chagas disease, or American trypanosomiasis, was described in 1909 by the Brazilian physician Carlos Chagas, who identified the causative agent—Trypanosoma cruzi—the transmission vector, major reservoirs, mechanism of human infection, as well as some clinical manifestations [1]. This disease is primarily transmitted to humans by the feces of hematophagous insects, of the family Reduviidae, subfamily Triatominae. The impressive decrease in Chagas disease prevalence from 16–18 million people by 1990 to 6–8 million people by 2010 was essentially the consequence of the launching of transnational programs in Latin America focused on the elimination of domestic vectors and blood donors screening supported by Pan American Health Organization/World Health Organization (PAHO/WHO) [2]. Despite these successful control interventions, the Chagas disease prevalence was estimated to reach 1.0–2.4% of the Brazilian population [1]. This disease, classically associated with poor and rural populations, underwent an urbanization process in the 1970s and 1980s to Latin American cities and later on beyond endemic countries, creating new epidemiological, social, and political challenges [3, 4, 5].

With the success of vector and blood bank control programs, congenital [6, 7], and oral [8, 9] transmissions have become important sources of new cases of Chagas disease. Congenital infections represent an estimated 22% of new cases in Latin America [2], occurring also in nonendemic countries [10, 11]. The oral route, which is probably the most frequent mechanism among vectors and wild mammals, has recently become relevant, due to environmental changes caused by deforestation [12]. T. cruziDNA was recently shown in 10% among 140 samples of açai-based products marketed in Rio de Janeiro and Pará States in Brazil [13].

Chagas disease results from the establishment of T. cruziin host tissues, involving an initial acute phase followed by a chronic phase, classified as indeterminate, cardiac, and/or digestive syndromes. The acute phase is characterized by detectable parasitemia and is commonly asymptomatic [14]. Without treatment, approximately 5–10% of symptomatic patients die during this phase due to encephalomyelitis or severe cardiac failure and rarely due to cardiac arrest [15]. After 2–3 months, the infection enters the chronic phase, and without successful treatment, it is lifelong. Approximately, two-thirds of infected individuals have the indeterminate form of the chronic phase, which is asymptomatic and defined by the presence of T. cruziantibodies and normal electrocardiographic and radiologic exams. The remaining infected individuals, due to an unbalanced inflammatory response and persistent low parasitism, will develop years or even decades later symptomatic chronic disease with cardiac (20–30%) and/or digestive (15–20%) disorders.

The current etiological treatment for Chagas disease is restricted to two nitroheterocyclic drugs: benznidazole (Bz/LAFEPE, Abarax®, ELEA and Bz/Chemo Research, Exeltis) and nifurtimox (Nif, LAMPIT®, Bayer) (Figure 1). Bz has been recently FDA-approved for use in children aged 2–12 years, being the first treatment approved in the United States for Chagas disease [16]. The results obtained with these two nitroderivatives vary according to the phase of Chagas disease, the period, and dose of treatment, as well as the age and geographical origin of the patients [17]. Both drugs have often shown successful results with high parasitological cure rates during the acute phase, but the effectiveness decreases with advance of the infection; therefore, early detection and intervention are crucial for reaching high cure rates [18]. The high incidence of collateral effects, especially for adults, leads to treatment abandonment rates reaching over 30% of the patients [19, 20, 21]. In contrast, children have a markedly higher tolerance for treatment [1, 14].

Figure 1.

Clinical drugs for Chagas disease treatment: (A) benznidazole and (B) nifurtimox.

There are significant drawbacks on the use of these drugs, mostly related to the limited efficacy in the chronic phase [22], and so, new alternative therapies are urgently required. In the last decades, many chemical diversity libraries from several pharmaceutical companies have been screened in the search of novel anti-T. cruzicandidates. In these programs, different approaches have been used including target-oriented studies, combination therapies, new formulations for drugs in use, and drug repurposing, and thus, in the present review, some of these points will be addressed.


2. Trypanosoma cruziand drug targets

One important point to be addressed in the search of alternative molecular targets in T. cruziis their presence in parasite forms dwelling in vertebrates. Once the parasite stages present different metabolic profiles [23, 24], the most promising targets are involved in crucial metabolic pathways, such as key enzymes related to antioxidant metabolism or sterol biosynthesis. In this section, we revised some of the most studied targets for drug intervention.

2.1 Mitochondrion, glycosomes, and oxidative metabolism

Mitochondrion plays a pivotal role in the oxidative stress, since the electron leakage from the electron transport chain (ETC particularly from complexes I, III and coenzyme Q) leads to the partial reduction of oxygen, being the main source of reactive oxygen species (ROS) in the cells [25]. During electron leakage, ROS were produced that interfere with different biological processes [26]. Such production leads to the increase in the expression of antioxidant enzymes such as superoxide dismutase (SOD), trypanothione reductase (TR), and peroxidases in response to the oxidative burst, and TR is considered one of the most promising chemotherapy targets in Chagas disease [27]. In trypanosomatids, mitochondrial metabolism is quite similar to that of other eukaryotes. Complex I (NADH: ubiquinone oxidoreductase) is expressed (almost 19 subunits were detected) but its functionality is still controversial [26]. In this way, glucose metabolism results mostly in succinate (complex II substrate) in trypanosomatids, derived from glycosomal and mitochondrial NADH-dependent fumarate reductase activities [28, 29]. Since complex I is not functional, oxidative phosphorylation is exclusively dependent of complex II in these protozoa. On the other hand, complexes III (ubiquinol:cytochrome c oxidoreductase) and IV (cytochrome c oxidase) of high eukaryotes and trypanosomatids display no differences, being complex III considered the major mitochondrial source of ROS production [30]. Many studies pointed to the susceptibility of T. cruzimitochondrion to a great variety of compounds, and such mitochondrial damage (ultrastructural swelling, decreased mitochondrial membrane potential, etc.) may comprise early or late events in trypanocidal agent activity (Figure 2) [31].

Figure 2.

Most recurrent cellular drug targets inT. cruzimammalian stages. M: mitochondrion; K: kinetoplast; N: nucleus; F: flagellum; Gl: glycosomes; G: golgi; Ac: acidocalcisomes; ER: endoplasmic reticulum; and Ap: autophagosomes.

Glycosomes are organelles crucial for the energetic and antioxidant metabolisms of the parasite, and the compartmentalization of their enzymes (including the majority of the enzymes of the glycolytic pathway) has also been reported to be directly involved in the maintenance of T. cruziviability, indicating this organelle as a potential drug target [32]. Among the glycosomal oxidative, scavengers are SOD isoforms, tryparedoxin, and peroxidases [26]. Up to now, no specific inhibitors of glycosomal enzymes showed promising trypanocidal activity [33].

2.1.1 Mitochondrial ETC

T. cruzimitochondrion is the most recurrent cellular drug target described in mechanistic studies; however, the exact molecular machinery involved in the susceptibility of this organelle to different classes of compounds is still unclear [31]. No hypothesis about the molecular mechanism involved in the mitochondrial effect of the great majority of trypanocidal drugs was postulated hitherto, and the damage specificity in this organelle is very debatable. Some specific inhibitors of ETC complexes have already been tested on T. cruzi. Rotenone, a well-known complex I inhibitor, has a controversial activity in the parasite. However, rotenone at high doses inhibited the activity of T. cruziNADH-dependent enzymes [34]. The existence of complex I activity was not strongly supported by such inhibition and could be caused by nonspecific binding to other electron carriers. On the other hand, depolarization of mitochondrial membrane, ROS production, and apoptotic-like phenotype was detected in parasites after the treatment with inhibitors of complexes III and IV, antimycin A, and potassium cyanide, respectively [24]. Structural and functional similarities between mammalian and trypanosomatidae complexes are suggestive of high toxicity.

2.1.2 Trypanothione reductase

The presence of some antioxidant components that are absent in mammalian cells makes this pathway a promising target of drug intervention in trypanosomatids. The unusual spermidine-glutathione adduct named trypanothione or N1,N8-bis(glutathionyl)spermidine found solely in these parasites functions as an electron donor in many pathways by neutralizing diverse reactive species through redox reactions, also providing reducing equivalents to intermediate molecules in other antioxidant pathways and in biosynthetic pathways such as DNA synthesis [35, 36]. The catalysis of NADPH-dependent reduction of trypanothione disulfide to T(SH)2 is performed by trypanothione reductase (TR), enzyme that has been proposed as a molecular target, based on the specific inhibition of antioxidant defenses of the parasite [37, 38]. The central role of trypanothione makes other enzymes that influence its production also interesting drug targets such as trypanothione synthetase, ornithine decarboxylase (ODC), S-adenosylmethionine (AdoMet) decarboxylase, γ-glutamylcysteine synthetase as well as polyamine transporters [39, 40].

In the last decades, many TR inhibitors were developed, but only a few had a positive correlation between trypanocidal activity and binding to the enzyme demonstrated [41, 42, 43]. Recently, a high-throughput screening of 1.8 million compounds was performed, and specific inhibitors of LeishmaniaTR were identified. Since this enzyme is considered well-conserved among trypanosomatids, this study could represent a critical step for the identification of inhibitors also for T. cruziTR [44]. Up to the moment, the inhibition of trypanothione metabolism of this parasite was poorly assessed in animal models, and no clinical trial has been reported involving this target (Figure 3).

Figure 3.

Landmarks in the investigation of TR as a drug target. Despite many efforts up to now, specific inhibitors of this enzyme presented neither important trypanocidal activityin vitronorin vivo. TR DPB ID: 1BZL.

2.1.3 ROS inducers

Varying the dose or the time of drug treatment, injury to the mitochondrion usually leads to ROS production [45]. Despite many compounds having induced mitochondrial alterations, generating ROS, the molecular mechanistic action was not elucidated in most studies. In this section, we will discuss only quinones and nitrocompounds, compounds with oxidative mechanisms of action well-characterized.

Quinones: Chemical properties of quinoidal carbonyls lead to the direct ROS generation [46], and trypanocidal effect of natural quinones and derivatives have been assessed [47, 48, 49, 50]. In epimastigotes, the oxidative activity of β-lapachone was first reported almost 40 years ago [51, 52], and increase in ROS levels has also been related to the treatment of T. cruziwith other naphthoquinones [53, 54]. In 2009, we proposed the trypanocidal mechanism of action of naphthofuranquinones. Such quinones strongly impaired the parasite mitochondrion by the deviation of the electrons from ubiquinone, culminating in this organelle depolarization, loss of respiratory rates, inhibition of complexes I–III activities, and ROS production [54]. Increased levels of ROS were also detected in parasites after the treatment with other classes of compounds such as pyrazyl/pyridylhydrazones and thiosemicarbazones [55, 56, 57].

Nitrocompounds: These compounds are usually avoided in medicinal chemistry approaches because the presence of a nitro group creates concerns regarding toxicity issues associated with DNA damage [58]. Regarding Chagas disease, fexinidazole evaluated in vivo, led to high cure rates and reduced myocarditis [59]. Subsequently, a phase II clinical trial was performed in chronic chagasic patients in Bolivia using fexinidazole treatment (NCT02498782), and it was observed that parasitemia was cleared; however, after recruiting 47 participants, some safety and tolerability issues arose, and it was decided to conclude the trial without the inclusion of new participants. After a 12 month follow-up, a high efficacy rate was evidenced, without relapses [60]. Therefore, a new proof of concept study was initiated in Spain in 2017, with the results expected in 2019 (EudraCT Number 2016-004905-15).

Surprisingly, trypanocidal action of Nif and Bz is still controversial. Nif has the oxidative activity demonstrated in the early 1980s, being hydrogen peroxide and superoxide anion production detected, while no reactive species was found after the treatment with Bz [61]. Bz and Nif are considered prodrugs that require activation by nitroreductases—NTR-I, an oxygen insensitive class catalyzing the two-electron reduction of the nitro group and NTR-II, an oxygen-sensitive class catalyzing one-electron reduction [62]. The mechanistic proposal of Nif involves nitroanion radical metabolization by NTR-II, followed by reoxidation by molecular oxygen to form superoxide anion (∙O2), which is converted to hydrogen peroxide (H2O2) under catalysis by SOD (Figure 4) [63]. On the other hand, low molecular weight thiol reduction together with no redox cycling in trypanocidal doses supported the hypothesis that oxidative effect was not involved in the parasite killing by Nif [64]. Additionally, NTR-I activity has been related to the trypanocidal effect of Nif and Bz through a two-electron reduction in the nitro group. In an oxygen-independent way, the production of nitroso and hydroxylamine intermediates led to amine generation, using NADH as a cofactor. The cleavage of the Nif furane ring produces a highly reactive unsaturated open chain nitrile [65].

Figure 4.

Metobolization of nitrocompounds by NTR-1 and NTR-II. ROS is generated by reoxidation (one electron route). Two-electron reduction produces hydroxylamine intermediates and reactive nitroso [63].

Recently, some highly potent 3-nitro-1H-1,2,4-triazole derivatives emerged as excellent substrates for NTR-I, but the enzymatic activity was not required for the trypanocidal activity [66]. Alternative enzymes have been associated with the reduction of nitro compounds in T. cruzi, indicative of a secondary action for these drugs, and further studies about the molecular mechanisms involved must be performed. The high trypanocidal activity together with the identification of exclusive nitroreductases in trypanosomatids supports the hypothesis of selectivity [63].

2.1.4 Polyamines

Polyamines (PA) are ubiquitous organic polycations that play a plethora of ubiquitous biological roles in most cell types, including bacteria, protozoa, and higher organisms [67], with significant metabolic differences, therefore comprising promising drug targets for protozoal diseases [68]. PA metabolism among parasitic protozoa is defective in a number of pathways as compared to mammalian cells. T. cruziparasites lack ODC and so are auxotrophic for the diamine putrescine [69]. Therefore, the protozoan relies on the diamine uptake from the extracellular milieu via surface transporters or permeases [70], and so, these mechanisms comprise targets for chemotherapy agent development [71], and pentamidine was shown to inhibit polyamine transport by T. cruzi[72]. Putrescine uptake is required for the massive infection [73] and scape from stress conditions [74]. Spermidine is synthesized by the transfer of an aminopropyl group from decarboxylated S-adenosyl-l-methionine to putrescine and takes part in the biogenesis of T[SH]2 a pivotal adduct in oxidative stress endurance and involved in anti-T. cruzidrug resistance [75]. In this regard, the putrescine analog DAB (1,4-diamino-2-butanone) promotes oxidative stress in T. cruzi[76] and leads to T. cruzimitochondrial destruction [77]. It is noteworthy that DAB not only is involved on reactive species production but also inhibit putrescine synthesis [78] and incorporation [79].

Polyamines may play multiple functions in parasite endurance under oxidative stress conditions, not only for TSH is a spermidine adduct but also because these polycations per semay be antioxidant, protecting T. cruzifrom oxidative stress [80]. Polyamines are also relevant for controlling differentiation, including T. cruzimetacyclogenesis [81]. Thus, the enzymes involved in polyamine and TSH metabolism provide important drug targets for potential anti-T. cruzitherapy [40].

2.2 Biosynthesis of sterols

Sterols are essential lipid molecules, performing numerous cellular roles associated with membrane and signal functions [82]. Cholesterol is biosynthesized in humans, whereas ergosterol or other 24-alkylated sterols are biosynthesized in opportunistic fungi and parasitic protozoa and such difference is exploited in the drug development [83]. T. cruziand related trypanosomatids have a strict requirement for endogenous sterols (ergosterol and analogs) for survival that cannot be replaced by cholesterol found in the host. Thus, the biosynthesis of sterols is a major target in the drug development for Chagas disease [84]. Among enzymes of the sterol metabolism, squalene synthase (SQS) and C14α-sterol demethylase (CYP51) have been intensively investigated as drug targets (Figure 5).

Figure 5.

Ergosterol biosynthesis is an important drug target inT. cruzi. In red, the enzymes described as molecular targets and in blue, the classes of the speciffic inhibitors. Inside gray boxes, the intermediary steps of the conversion of lanosterol into 4,4-dimethyl-5a-cholesta-8,1,4,24-3b-ol by sterol 14-α-demethylase (CYP51).

2.2.1 Squalene synthase (SQS)

This enzyme catalyzes the dimerization of two molecules of farnesyl pyrophosphate (FPP) to produce squalene. This enzyme is under study as a possible target for cholesterol-lowering agents in humans [85]. SQS is a membrane-bound enzyme in T. cruziepimastigotes, being distributed between glycosome and mitochondrial/microsomal vesicles [86]. FPP is a branching point in isoprenoid biosynthesis: conversion to squalene and sterols by SQS or synthesis of other essential isoprenoids. The quinuclidine-based inhibitors of mammalian SQS, 3-(biphenyl-4-yl)-3-hydroxyquinuclidine (BPQ-OH) ER27856, E5700, and ER-119884 were assayed against T. cruzi, leading to the in vitroinhibition of epimastigote and intracellular amastigote proliferation, depletion of endogenous squalene and sterols, and marked ultrastructural alterations [86, 87] and in vivoE5700 led to 100% survival and parasitemia negativation [88]. However, E5700 and ER-119884 have no selectivity toward the parasite enzyme in comparative assays with the recombinant human enzyme [89]. In 2014, the X-ray crystallographic structure of SQS from T. cruziwas reported, confirming the binding of the enzyme to distinct classes of inhibitors such as the quinuclidines E5700 and ER119884 and the thiocyanate WC-9 opening possibilities to the development of alternative inhibitors [90].

In a screening of compounds containing the 4-phenoxyphenoxy skeleton, 4-phenoxyphenoxyethyl thiocyanate (WC-9) was highlighted due to the high activity against the proliferative form epimastigotes (low micromolar) and intracellular amastigotes (nanomolar) and a potent inhibitor of the enzymatic activity of both glycosomal and mitochondrial isoforms of SQS [91, 92]. Since then, different series of WC-9 analogs have been developed [91, 93], including seleno-containing analogs resulting in compounds, such as 4-phenoxyphenoxyethyl selenocyanate, with EC50 values at low nanomolar level and selectivity index (SI) higher than 900 [94].

2.2.2 C14α-sterol demethylase (CYP51)

This enzyme catalyzes the oxidative removal of the 14α-methyl group from of catalyzing the oxidative removal of the 14α-methyl group from sterol precursors such as lanosterol or eburicol, via a repetitive three-step process that uses NADPH and oxygen to produce 4,4-dimethyl-5α-cholesta-8,14,24-trien-3β-ol [83]. CYP51s are the most conserved cytochrome P450 enzymes [84]. Series of azoles originally developed for the treatment of fungal infections targets this enzyme leading to accumulation of lanosterol and other sterol intermediates and displaying activity in vitroand in vivoagainst T. cruzi[95, 96, 97]. This line of investigation led to the selection of the triazole posaconazole [98, 99] and E1224 (fosravuconazole) [100, 101] for Phase II clinical trials with chronic patients, which, however, led to therapeutic failure as compared to benznidazole, with parasitemia relapses: NCT01162967 (Chagazol) [102], NCT01377480 (Stopchagas) [103], and NCT01489228 (E1224 trial) [104].

VNI, a carboxamide-containing β-phenyl-imidazole, identified from a Novartis collection of azoles, was active in acute and chronic mouse models using Tulahuen strain [105]; whereas in experiments with other parasite strains, no complete parasitological clearance was achieved [106]. In subsequent work, VFV, a fluoro analog of VNI, designed to fill the deepest portion of the CYP51 substrate-binding cavity demonstrated 100% efficacy in experimental infection, displaying favorable oral bioavailability and pharmacokinetics [107]. Comparison between VNI and VFV, in murine models of infection, revealed that regardless of the treatment scheme or delivery vehicle, VFV was more potent in both genders [108]. VT-1161, a 1-tetrazole-based drug undergoing phase II antifungal clinical trials, is active in vitroand in vivoagainst T. cruzi. It was structurally characterized in a complex with TcCYP51, allowing for the optimization of new tetrazole-based analogs and presents good pharmacokinetic properties and an excellent safety profile [109]. Friggeri et al. [110] synthesized imidazolyl-2-phenylethanol derivatives, and several of them were active against intracellular amastigotes and inhibited TcCYP51. In sequence, eight new derivatives were prepared and assayed against the parasite, and the most active was a piperazinyl-carbamate derivative at nanomolar range, low cytotoxicity, and good chemical and metabolic stability [111]. Recently, a series of pyrazolo[3,4-e][1,4]thiazepin analogs, novel CYP51 inhibitors, were investigated revealing in vitroand in vivoactivity against T. cruzi, with several analogs displaying effect at low micromolar dosis and low host toxicity [112].

2.3 Cysteine proteases

Cysteine proteases are intensively used as molecular targets in trypanosomatid disease drug discovery efforts. Target-based screening, structure-based drug design, and medicinal chemistry approaches targeting cysteine proteases are strategies intensively used in the development of drugs for diseases caused by pathogenic trypanosomatids. T. cruzicysteine protease named cruzipain (or cruzain) is a cathepsin-L-like protease of the papain family and is essential for the intracellular replication, differentiation, and immune evasion of the parasite [113, 114]. Three-dimensional structures of cruzain with different ligands have been reported, allowing the design and synthesis of new hit compounds [115]. Based on the interaction with its active site, enzyme inhibitors have been classified as irreversible, forming covalent bonds with cysteine sulfur, or as reversible, forming 1,2-adducts with cysteine that are generally unstable [116]. Among irreversible peptidyl inhibitors of cruzipain, we highlight diazomethyl ketones, allyl sulfones, vinyl sulfonamides, and vinyl sulfones, including K777 and its arginine variant WRR-483 [117, 118, 119]. Among nonpeptidyl inhibitors of cruzipain, we found thiosemicarbazones, thiazolylhydrazones, thiazoles, and oxadiazoles (Figure 6) [120].

Figure 6.

Cruzipain as a molecular target inT. cruzi.Irreversible and reversible inhibition was demonstrated by different classes of compounds. Cruzipain PDB ID: 3Io6.

Another group of compounds that has been studied as cruzipain reversible inhibitors are those containing a nitrile head: purine nitriles [121], nitrile analogs of odanacatib [122, 123], and nonpeptidic nitriles [124]. Salas-Sarduy et al. [125] identified two new cruzipain inhibitory scaffolds from GlaxoSmithKline HAT and Chagas chemical boxes, both containing a nitrile moiety, with major structural differences between them. Benzimidazoles and oxidiazoles have also been explored as noncovalent cruzain inhibitors, using an approach combining high throughput and virtual screenings [126, 127].

Development of cruzipain inhibitors by structure activity relationship (SAR) studies, combinatorial chemistry, HTS, and virtual screening are also employed in repositioning strategies [128]. Bromocriptine (antiparkinson and antidiabetic drug), amiodarone (antiarrhythmic drug), and levothyroxine (hypothyroidism drug) were selected in a screening campaign for cruzain inhibitors of the DrugBank database [129], clofazimine (antileprosy drug) and benidipine (antihypertensive) from the Merck Index 12th database [130, 131], and etofyllin clofibrate (antilipemic drug) and piperacillin, cefoperazone, and flucloxacillin (β-lactam antibiotics) from a collection of 3180 FDA drugs [132].

Calpains are calcium-dependent nonlysosomal cysteine peptidases highly conserved among eukaryotes, but their precise biological function is not completely clear. In mammalian cells, calpains participate in many different calcium processes including proliferation, differentiation, cytoskeletal assembly, cellular signaling, among many others; however, T. cruzicalpains do not present a mapped active catalytic site up to now [133]. In man, uncontrolled activity of calpains has been associated with muscular and neurological disorders such as Alzheimer, Parkinson, multiple sclerosis, and arthritis, and the terapeutic effect of specific calpain inhibitors was suggested [134, 135]. Recently, the repurposing of calpain inhibitors was also postulated for neglected tropical diseases, including Chagas disease [133]. In T. cruzi, only the inhibitor MDL28170 was tested, and the trypanocidal activity at low micromolar range was shown on all three parasite forms, impairing the ultrastructural architecture of Golgi and reservosomes [136, 137]. Despite the study’s scarcity, calpains inhibition has been suggested as an attractive antitrypanosomatid approach even without the confirmation of their proteolytic activity in these parasites.

2.4 Nuclear and kinetoplast DNA

Both T. cruzikinetoplast and nucleus may be targeted by different classes of compounds with antiparasitic activity [138, 139]. Early studies [140] revealed that hydroxystilbamidine led to the disorganization of kinetoplast DNA (kDNA). Later other compounds such as vinblastine, geranylgeraniol, diaminobenzidine, and aromatic diamidines were reported to affect kDNA arrangement causing its fragmentation [138, 141, 142]. Trypanosomatid nucleus and kinetoplast display topoisomerases that show significant structural differences from host orthologs advocating their potential as drug targets [143]. Interestingly, T. cruzitopoisomerase I is inactivated by ROS [144], so the oxidative stress induced by both immune response and trypanocidal agents may also affect parasite chromatin organization.

Classic aromatic diamidines have been shown to bind noncovalently and through a nonintercalative manner to the minor groove of DNA; several hypotheses regarding their mode of action were proposed. They could act by complexation with DNA and subsequently lead to a selective inhibition of DNA-dependent enzymes and/or through the direct inhibition of transcription [145]. Thus, evidences suggest that diamidines interfere in the kinetoplast function of trypanosomatids through a selective association to the unique AT-rich regions of kDNA minicircles, perhaps involving DNA-processing enzymes [146]. Medicinal chemistry studies pointed to arylimidamides (AIAs) as the most promising antimicrobial diamidines [106]. DB702, DB786, DB811, and DB889 presented anti-T. cruziactivity in the low-micromolar range and led to ultrastructural alterations mainly associated with the nucleus and mitochondrion [147, 148]. On the other hand, recent study with novel bis-AIAs revealed their higher potency and in silicoanalysis showed DNA as the main target, but no DNA ultrastructural alterations were found [149].

On the other hand, enzymes involved in nucleic acid metabolism could be also promising targets. Topoisomerases play a crucial role for the DNA dynamics during the transcription, replication, or even in the repair. Due to their participation in essential cellular processes, interfering with DNA topology, and consequently leading to physiological implications, topoisomerases have been described as molecular targets for cancer and also parasitic illnesses such as Chagas disease. Up to now, innumerous topoisomerase inhibitors presented antitrypanosomatidae activities such as camptothecin, doxorubicin, etoposide, suramin, among many others [150]. Recently, voacamine and an isobenzofuranone derivative induced important morphological alterations in different trypanosomatids, including T. cruzi. In Leishmania parasites, the most affected organelle was mitochondrion (severely swelled presenting membrane depolarization), where the derivative led to kinetoplast network disorganization [151, 152].


3. Proteomic insights for the target identification in the parasite

The evaluation of the proteomic profile in trypanosomatids is particularly interesting because these protozoa exhibit open reading frames in long polycistronic regions, and the regulation of gene expression occurs only post-transcriptionally, justifying the importance on monitoring the protein expression by proteomic approach [153]. This section will focus on proteomic analysis of parasite forms dwelling in mammalian hosts. The first large-scale analysis was performed by Atwood and colleagues in 2005 [23], identifying 1486 proteins of culture-derived trypomastigotes, and 30 trans-sialidases, enzymes that play an important role in parasite host cell invasion, were among the top-scoring proteins exclusively detected in this developmental form. T. cruzisurface subproteome and basic proteins analysis confirmed the high distribution of trans-sialidases in this life form [154, 155]. Specific trypomastigote surface analysis also revealed membrane-associated enzymes that are involved in biosynthetic pathway of phospholipid and glycolysis [155]. The evaluation of chromatin fraction of this stage revealed RNA-binding proteins and histones, representing 29% of chromatin protein content [156], providing new insights into gene expression and histone modifications involved in the parasite cycle regulation. Likewise, T. cruziglycoproteome was assessed, and trypomastigote-specific glycoproteins were identified, including mucin family members [157, 158].

In metacyclic trypomastigotes, Atwood and co-workers [23] identified 2339 proteins, and different antioxidant enzymes were among the main proteins detected. The presence of these enzymes in this stage could be related to the parasite adaptation to the oxidative environment inside the vertebrate host circulation and particularly inside the phagocytes. The analysis of metacyclogenesis revealed increased expression of cytoskeletal proteins as well as proteins related to energetic and oxidative metabolisms, suggestive of the morphological and metabolic reorganization [159]. Plasma membrane subproteome pointed to a large repertoire of surface proteins in this parasite stage, including trans-sialidases, mucins, and GP63 protease [160]. Such glycoprotein diversity confers adaptation of the parasite to distinct environmental conditions. Morever, secretome of metacyclic trypomastigotes also demonstrated trans-sialidases and other surface molecules, playing a role in parasite invasion during acute and chronic infections [161, 162]. The blockage of this process could be an interesting strategy in novel drug development.

The first proteomic analysis of bloodstream trypomastigotes was performed by our group in 2015, identifying a total 5901 proteins [163]. In this work, a comparison among the proteomic maps of trypomastigotes (bloodstream, cultured-derived, and metacyclic forms) was also assessed, and 2202 proteins related to the parasite surface, cytoskeleton, redox metabolism, cell signaling, and energetic metabolism were exclusively detected in bloodstream forms. Overall, the proteomic profile of bloodstream form comprises an important tool to discover potential new drug targets and novel antigens for vaccines or diagnostics. The differences in the trypomastigote proteomic profiles were expected due to their environment, and huge number of stage-specific proteins in bloodstream forms, probably triggered by the exposure to the host immune system reinforces the necessity for drug validation on this developmental form. In relation to proteomic evaluation of trypanocidal action of drugs, β-lapachone-derived naphthoimidazoles induced the increase in the abundance of 27 proteins, involved in stress response, cell structure, energetic metabolism, nucleic and amino acid metabolisms, oxidative metabolism, among other pathways [164]. This large-scale study revealed an important set of proteins belonging to metabolic pathways that play pivotal functions for this parasite form, providing new insights for the understanding of the parasite biology and of potential drugable molecules for the treatment of Chagas disease.

In 2005, 1871 proteins of culture-derived amastigotes were identified, preferably involved in endoplasmic reticulum to Golgi trafficking, suggesting an intense traffic at this stage [23]. The analysis of amastigogenesis evidenced high abundance of glycolytic enzymes in amastigotes as well as the lower abundance of flagellar components, compatible with the morphology of this stage [165]. Later, the surface subproteome of vertebrate-dwelling parasite forms was characterized, displaying molecules involved in cell division, signal transduction, and lipid metabolism, crucial for the parasite intracellular self-maintenance [155].

Another interesting target is the posttranslational modification of parasite proteins. Acetylation at lysine residues exerts important role in both vertebrates and microbial cells. The NAD+-dependent lysine deacetylases are termed sirtuins. Humans present seven different sirtuins, whereas T. cruzi, solely two. TcSIR2RP1 andTcSIR2RP3 are found in the cytosolic and mitochondrial compartments, respectively. Parasites overexpressing TcSIR2RP1 display enhanced metacyclogenesis and host cell infection [166]. The sirtuin antagonist salermide diminishes intracellular parasite proliferation and parasitemia in murine infection [167]. Thus, acetylation of T. cruziproteins may provide useful targets for the development of antiparasitic agents [167, 168]. In addition, mammalian sirtuin targeting may be beneficial in chronic chagasic cardiomyopathy [169].


4. Conclusions

The clinical chemotherapy for Chagas disease (Nif and Bz) led to a parasitological cure in the great number of congenital, adult acute, or early chronic cases [170]. However, undesirable side effects and the resistance of some parasite strains [171], together with the limited efficacy in symptomatic chronic cases, drive the continuous search for novel chemotherapeutic agents [33]. Drug repurposing or even combinations with the current drugs could be options to minimize this problem [59, 120]. In this direction, phenotypic strategy has been considered the most valuable approach for the screening of antiparasitic compounds [172].

High throughput screening complemented by whole-cell phenotypic assays represents the more feasible option in the search for novel anti-T. cruzicompounds, also leading to an increment in sensitivity [173]. Preclinical in silicocombined to in vitroassays represents an essential step for the recognition of T. cruzidrug targets, generating knowledge about the metabolism, biochemical pathways, or biological processes allowing the target validation. In this way, the identification of selective targets comprises a logical startpoint to the reduction of the side effects in the hosts [153, 163]. Ultrastructural observations can predict the potential primary cellular targets due to their earlier alterations triggered by pharmacologic stimuli, helping the prospection of molecular modes of action of antiparasitic agents [141, 174].

Large-scale proteomics represents an alternative approach for the assessment of molecular mechanisms of trypanocidal drugs. Indeed, despite its potential, such technique is poorly employed in this context. The importance of the use of clinical relevant T. cruzistages in drug screening was evidenced by the remarkable differences found among the proteomic profile of parasite forms [23, 163]. The proteomic analysis of bloodstream trypomastigotes identified a huge variety of proteins from distinct biological processes, pointed to more than 2000 proteins present only in bloodstream forms (not in trypomastigotes from other sources), reinforcing the importance of the chemotherapeutic tests using recent isolated parasites from animals’ blood [163].

In the current scenario, the CYP51 still represents one the most promising alternatives. Large-scale screening pointed to the high activity of CYP51 inhibitors in vitro(even higher than Bz), but not producing T. cruzielimination [175, 176]. Unfortunately, the clinical trial with posaconazole and E1224 was also unsatisfactory [102]. In Argentina, a drug–drug interaction analysis of the combination Bz and E1224 in healthy volunteers demonstrated no improvement in tolerability or safety parameters [177], and a clinical study using this combination is planned [178]. Also, the use of new scaffolds with the design of inhibitors much more selective toward CYP51 has demonstrated its high trypanocidal activity in association or not with other licensed drugs [108, 179].

Among the parasite antioxidant defenses, the most promising drug target is TR, and the specific inhibitor development has been proposed in the last three decades [36]. However, no active inhibitors of this enzyme were described up to now [180]. The hypothesis that T. cruziis more susceptible to oxidative species than the vertebrate hosts is an old misleading concept, due to the highly efficient scavengers described in the parasite [51, 181]. Another aspect to be considered is the central role of ROS production and the mitochondrion for the trypanocial action of a great variety of preclinical compounds [31]. The mitochondrial swelling is frequently observed in T. cruziafter the treatment with different drugs, but this phenotype is rarely associated with a specific molecular mechanism, except for DNA ligands such as aromatic amidines [106]. The molecular mechanistic proposal opens the possibility of the mitochondrial dysfunction to be as a random consequences of indirect effect triggered by the impaired homeostasis, resulting in redox imbalance [31].

Bioinformatic, proteomic, and ultrastructural analyses are pivotal tools in the identification of drug targets; however, the use of specific inhibitors must be validated before the following studies. The absence of the predicted biological activity or even the specific binding to the respective molecular target is not uncommon [178, 182]. To improve the safety, mechanisms of action characterization should be performed in parallel to the high-throughput screening of the trypanocidal activity [183]. In case of obligatory intracellular parasites as T. cruzi, the direct analysis in a phenotypic assay is also essential, considering permeability of the host cells besides the therapeutic window related to the mammalian host toxicity aspects [182]. The adequate choice of the experimental design is also crucial. Animal models, parasite strains, and treatment protocols for preclinical assays (in vitroand in vivo) must be standardized, in order to reach better translation to humans [184].



This research was funded by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Rio de Janeiro (Faperj) and by Fundação Oswaldo Cruz (Fiocruz).


Conflict of interest

None to declare.


  1. 1. Dias JCP, Ramos AN Jr, Gontijo ED, Luquetti A, Shikanai-Yasuda MA, Coura JR, et al. 2 nd Brazilian consensus on Chagas disease, 2015. Revista da Sociedade Brasileira de Medicina Tropical. 2016;49:3-60. DOI: 10.1590/0037-8682-0505-2016
  2. 2. WHOChagas disease in Latin America: An epidemiological update based on 2010 estimates. Relevé Épidémiologique Hebdomadaire. 2015;90:33-43
  3. 3. Schmunis GA. Epidemiology of Chagas disease in non-endemic countries: The role of international migration. Memórias do Instituto Oswaldo Cruz. 2007;102(Suppl 1):75-85. DOI: 10.1590/S0074-02762007005000093
  4. 4. Antinori S, Galimberti L, Bianco R, Grande R, Galli M, Corbellino M. Chagas disease in Europe: A review for the internist in the globalized world. European Journal of Internal Medicine. 2017;43:6-15. DOI: 10.1016/j.ejim.2017.05.001
  5. 5. Monge-Maillo B, López-Vélez R. Challenges in the management of Chagas disease in Latin-American migrants in Europe. Clinical Microbiology and Infection. 2017;23:290-295. DOI: 10.1016/j.cmi.2017.04.013
  6. 6. Messenger LA, Bern C. Congenital Chagas disease: Current diagnostics, limitations and future perspectives. Current Opinion in Infectious Diseases. 2018;31:415, 421. DOI: 10.1097/QCO.0000000000000478
  7. 7. Carlier Y, Torrico F, Sosa-Estani S, Russomando G, Luquetti A, Freilij H, et al. Congenital Chagas disease: Recommendations for diagnosis, treatment and control of newborns, siblings and pregnant women. PLoS Neglected Tropical Diseases. 2011;5:e1250. DOI: 10.1371/journal.pntd.0001250
  8. 8. Shikanai-Yasuda MA, Carvalho NB. Oral transmission of Chagas disease. Clinical Infectious Diseases. 2012;54:845-852. DOI: 10.1093/cid/cir956
  9. 9. Silva-dos-Santos D, Barreto-de-Albuquerque J, Guerra B, Moreira OC, Berbert LR, Ramos MT, et al. Unraveling Chagas disease transmission through the oral route: Gateways toTrypanosoma cruziinfection and target tissues. PLoS Neglected Tropical Diseases. 2017;11:e0005507. DOI: 10.1371/journal.pntd.0005507
  10. 10. Centers for Disease Control and Prevention. Congenital transmission of Chagas disease—Virginia, 2010. Morbidity and Mortality Weekly Report. 2012;61:477-479
  11. 11. Rodari P, Angheben A, Gennati G, Trezzi L, Bargiggia G, Maino M, et al. Congenital Chagas disease in a non-endemic area: Results from a control programme in Bergamo province, Northern Italy. Travel Medicine and Infectious Disease. 2018;25:31-34. DOI: 10.1016/j.tmaid.2018.04.011
  12. 12. Coura JR, Junqueira AC. Surveillance, health promotion and control of Chagas disease in the Amazon region—Medical attention in the Brazilian Amazon region: A proposal. Memórias do Instituto Oswaldo Cruz. 2015;110:825-830. DOI: 10.1590/0074-02760150153
  13. 13. Ferreira RTB, Cabral ML, Martins RS, Araujo PF, da Silva SA, Britto C, et al. Detection and genotyping ofTrypanosoma cruzifrom açai products commercialized in Rio de Janeiro and Pará, Brazil. Parasites & Vectors. 2018;11. DOI: 10.1186/s13071-018-2699-6
  14. 14. WHO, editor. Control of Chagas Disease: Second Report of the WHO Expert Committee. Geneva: WHO; 2002
  15. 15. Prata A. Clinical and epidemiological aspects of Chagas disease. The Lancet Infectious Diseases. 2001;1:92-100. DOI: 10.1016/S1473-3099(01)00065-2
  16. 16. FDA. FDA approves first U.S. treatment for Chagas disease. US Food Drug Adm; 2017. Available from:[Accessed: September 20, 2018]
  17. 17. Coura J, de Castro SL. A critical review on Chagas disease chemotherapy. Memórias do Instituto Oswaldo Cruz. 2002;97:3-24. DOI: 10.1590/S0074-02762002000100001
  18. 18. Coura JR, Borges-Pereira J. Chronic phase of Chagas disease: Why should it be treated? A comprehensive review. Memórias do Instituto Oswaldo Cruz. 2011;106:641-645
  19. 19. Jackson Y, Alirol E, Getaz L, Wolff H, Combescure C, Chappuis F. Tolerance and safety of Nifurtimox in patients with chronic Chagas disease. Clinical Infectious Diseases. 2010;51:e69-e75. DOI: 10.1086/656917
  20. 20. Sperandio da Silva GM, Mediano MFF, Alvarenga Americano do Brasil PE, da Costa Chambela M, da Silva JA, de Sousa AS, et al. A clinical adverse drug reaction prediction model for patients with Chagas disease treated with benznidazole. Antimicrobial Agents and Chemotherapy. 2014;58:6371-6377. DOI: 10.1128/AAC.02842-14
  21. 21. Pérez-Molina JA, Molina I. Chagas disease. The Lancet. 2018;391:82-94. DOI: 10.1016/S0140-6736(17)31612-4
  22. 22. Rassi A Jr, Marin Neto JA, Rassi A. Chronic Chagas cardiomyopathy: A review of the main pathogenic mechanisms and the efficacy of aetiological treatment following the BENznidazole evaluation for interrupting Trypanosomiasis (BENEFIT) trial. Memórias do Instituto Oswaldo Cruz. 2017;112:224-235. DOI: 10.1590/0074-02760160334
  23. 23. Atwood JA, Weatherly DB, Minning TA, Bundy B, Cavola C, Opperdoes FR, et al. TheTrypanosoma cruziproteome. Science. 2005;309:473-476. DOI: 10.1126/science.1110289
  24. 24. Gonçalves RLS, Barreto RFSM, Polycarpo CR, Gadelha FR, Castro SL, Oliveira MF. A comparative assessment of mitochondrial function in epimastigotes and bloodstream trypomastigotes ofTrypanosoma cruzi. Journal of Bioenergetics and Biomembranes. 2011;43:651-661. DOI: 10.1007/s10863-011-9398-8
  25. 25. Venditti P, Di Stefano L, Di Meo S. Mitochondrial metabolism of reactive oxygen species. Mitochondrion. 2013;13:71-82. DOI: 10.1016/j.mito.2013.01.008
  26. 26. Bombaça ACS, Menna-Barreto RFS. The oxidative metabolism in trypanosomatids: Implications for these protozoa biology and perspectives for drugs development. In: Leon L, Torres-Santos EC, editors. Differ. Asp. Chemother. Trypanos. First. New York: Nova Science Publishers; 2017. pp. 93-129
  27. 27. Beltran-Hortelano I, Perez-Silanes S, Galiano S. Trypanothione reductase and superoxide dismutase as current drug targets forTrypanosoma cruzi: An overview of compounds with activity against Chagas disease. Current Medicinal Chemistry. 2017;24:1066-1138. DOI: 10.2174/0929867323666161227094049
  28. 28. Boveris A, Hertig CM, Turrens JF. Fumarate reductase and other mitochondrial activities inTrypanosoma cruzi. Molecular and Biochemical Parasitology. 1986;19:163-169
  29. 29. Coustou V, Besteiro S, Rivière L, Biran M, Biteau N, Franconi J-M, et al. A mitochondrial NADH-dependent fumarate reductase involved in the production of succinate excreted by procyclicTrypanosoma brucei. The Journal of Biological Chemistry. 2005;280:16559-16570. DOI: 10.1074/jbc.M500343200
  30. 30. Mehta A, Shaha C. Apoptotic death inLeishmania donovanipromastigotes in response to respiratory chain inhibition: Complex II inhibition results in increased pentamidine cytotoxicity. The Journal of Biological Chemistry. 2004;279:11798-11813. DOI: 10.1074/jbc.M309341200
  31. 31. Menna-Barreto RFS, de Castro SL. The double-edged sword in pathogenic trypanosomatids: The pivotal role of mitochondria in oxidative stress and bioenergetics. BioMed Research International. 2014;2014:1-14. DOI: 10.1155/2014/614014
  32. 32. Barros-Alvarez X, Gualdrón-López M, Acosta H, Cáceres AJ, Graminha MAS, Michels PAM, et al. Glycosomal targets for anti-trypanosomatid drug discovery. Current Medicinal Chemistry. 2014;21:1679-1706. DOI: 10.2174/09298673113209990139
  33. 33. Menna-Barreto RFS, d CSL. Clear shot at primary aim: Susceptibility ofTrypanosoma cruziorganelles, structures and molecular targets to drug treatment. Current Topics in Medicinal Chemistry. 2016;17:1212-1234. DOI: 10.2174/1568026616666161025161858
  34. 34. Hernandez FR, Turrens JF. Rotenone at high concentrations inhibits NADH-fumarate reductase and the mitochondrial respiratory chain of Trypanosoma brucei and T. cruzi. Mol Biochem Parasitol. 1998;93:135-137. DOI: 10.1016/S0166-6851(98)00015-2
  35. 35. Fairlamb AH, Blackburn P, Ulrich P, Chait BT, Cerami A. Trypanothione: A novel bis(glutathionyl)spermidine cofactor for glutathione reductase in trypanosomatids. Science. 1985;227:1485-1487
  36. 36. Leroux AE, Krauth-Siegel RL. Thiol redox biology of trypanosomatids and potential targets for chemotherapy. Molecular and Biochemical Parasitology. 2016;206:67-74. DOI: 10.1016/j.molbiopara.2015.11.003
  37. 37. Krieger S, Schwarz W, Ariyanayagam MR, Fairlamb AH, Krauth-Siegel RL, Clayton C. Trypanosomes lacking trypanothione reductase are avirulent and show increased sensitivity to oxidative stress. Molecular Microbiology. 2000;35:542-552
  38. 38. Krauth-Siegel RL, Bauer H, Schirmer RH. Dithiol proteins as guardians of the intracellular redox milieu in parasites: Old and new drug targets in trypanosomes and malaria-causing plasmodia. Angewandte Chemie, International Edition. 2005;44:690-715. DOI: 10.1002/anie.200300639
  39. 39. Flohé L. The trypanothione system and its implications in the therapy of trypanosomatid diseases. International Journal of Medical Microbiology. 2012;302:216-220. DOI: 10.1016/j.ijmm.2012.07.008
  40. 40. Maya JD, Salas CO, Aguilera-Venegas B, Diaz MV, López-Muñoz R. Key proteins in the polyamine-trypanothione pathway as drug targets againstTrypanosoma cruzi. Current Medicinal Chemistry. 2014;21:1757-1771
  41. 41. Beig M, Oellien F, Garoff L, Noack S, Krauth-Siegel RL, Selzer PM. Trypanothione reductase: A target protein for a combined in vitro and in silico screening approach. PLoS Neglected Tropical Diseases. 2015;9:e0003773. DOI: 10.1371/journal.pntd.0003773
  42. 42. Vázquez K, Paulino M, Salas CO, Zarate-Ramos JJ, Vera B, Rivera G. Trypanothione reductase: A target for the development of anti-Trypanosoma cruzidrugs. Mini Reviews in Medicinal Chemistry. 2017;17:939-946. DOI: 10.2174/1389557517666170315145410
  43. 43. Talevi A, Carrillo C, Comini MA. The thiol-polyamine metabolism ofTrypanosoma cruzi: Molecular targets and drug repurposing strategies. Current Medicinal Chemistry. 2018;25. DOI: 10.2174/0929867325666180926151059. [Epub ahead of print]
  44. 44. Ilari A, Genovese I, Fiorillo F, Battista T, DII, Fiorillo A, et al. Toward a drug against all kinetoplastids: From LeishBox to specific and potent trypanothione reductase inhibitors. Molecular Pharmaceutics. 2018;15:3069-3078. DOI: 10.1021/acs.molpharmaceut.8b00185
  45. 45. Docampo R, Gadelha FR, Moreno SN, Benaim G, Hoffmann ME, Vercesi AE. Disruption of Ca2+ homeostasis inTrypanosoma cruziby crystal violet. The Journal of Eukaryotic Microbiology. 1993;40:311-316
  46. 46. Monks TJ, Jones DC. The metabolism and toxicity of quinones, quinonimines, quinone methides, and quinone-thioethers. Current Drug Metabolism. 2002;3:425-438
  47. 47. Salas CO, Faúndez M, Morello A, Maya JD, Tapia RA. Natural and synthetic naphthoquinones active againstTrypanosoma cruzi: An initial step towards new drugs for Chagas disease. Current Medicinal Chemistry. 2011;18:144-161
  48. 48. Schmidt TJ, Khalid SA, Romanha AJ, Alves TM, Biavatti MW, Brun R, et al. The potential of secondary metabolites from plants as drugs or leads against protozoan neglected diseases—Part II. Current Medicinal Chemistry. 2012;19:2176-2228
  49. 49. da Silva Júnior EN, Jardim GAM, Menna-Barreto RFS, de Castro SL. Anti-Trypanosoma cruzicompounds: Our contribution for the evaluation and insights on the mode of action of naphthoquinones and derivatives. Journal of the Brazilian Chemical Society. 2014;25:1780-1798. DOI: 10.5935/0103-5053.20140180
  50. 50. Pieretti S, Haanstra JR, Mazet M, Perozzo R, Bergamini C, Prati F, et al. Naphthoquinone derivatives exert their Antitrypanosomal activity via a multi-target MECHANISM. PLoS Neglected Tropical Diseases. 2013;7:e2012. DOI: 10.1371/journal.pntd.0002012
  51. 51. Boveris A, Stoppani AOM. Hydrogen peroxide generation inTrypanosoma cruzi. Experientia. 1977;33:1306-1308. DOI: 10.1007/BF01920148
  52. 52. Cruz FS, Docampo R, de Souza W. Effect of beta-lapachone on hydrogen peroxide production inTrypanosoma cruzi. Acta Tropica. 1978;35:35-40
  53. 53. Lara LS, Moreira CS, Calvet CM, Lechuga GC, Souza RS, Bourguignon SC, et al. Efficacy of 2-hydroxy-3-phenylsulfanylmethyl-[1,4]-naphthoquinone derivatives against differentTrypanosoma cruzidiscrete type units: Identification of a promising hit compound. European Journal of Medicinal Chemistry. 2018;144:572-581. DOI: 10.1016/j.ejmech.2017.12.052
  54. 54. Menna-Barreto RFS, Goncalves RLS, Costa EM, Silva RSF, Pinto AV, Oliveira MF, et al. The effects onTrypanosoma cruziof novel synthetic naphthoquinones are mediated by mitochondrial dysfunction. Free Radical Biology & Medicine. 2009;47:644-653. DOI: 10.1016/j.freeradbiomed.2009.06.004
  55. 55. Soares ROA, Echevarria A, Bellieny MSS, Pinho RT, de Leo RMM, Seguins WS, et al. Evaluation of thiosemicarbazones and semicarbazones as potential agents anti-Trypanosoma cruzi. Experimental Parasitology. 2011;129:381-387. DOI: 10.1016/j.exppara.2011.08.019
  56. 56. Martins SC, Lazarin-Bidóia D, Desoti VC, Falzirolli H, da Silva CC, Ueda-Nakamura T, et al. 1,3,4-Thiadiazole derivatives of R-(+)-limonene benzaldehyde-thiosemicarbazones cause death inTrypanosoma cruzithrough oxidative stress. Microbes and Infection. 2016;18:787-797. DOI: 10.1016/j.micinf.2016.07.007
  57. 57. Lapier M, Zuniga-Lopez MC, Aguilera-Venegas B, Adam R, Abarca B, Ballesteros R, et al. Evaluation of the novel antichagasic activity of [1,2,3]Triazolo[1,5-a]pyridine derivatives. Current Topics in Medicinal Chemistry. 2017;17:399-411
  58. 58. Patterson S, Wyllie S. Nitro drugs for the treatment of trypanosomatid diseases: Past, present, and future prospects. Trends in Parasitology. 2014;30:289-298. DOI: 10.1016/
  59. 59. Bahia MT, Diniz L d F, Mosqueira VCF. Therapeutical approaches under investigation for treatment of Chagas disease. Expert Opinion on Investigational Drugs. 2014;23:1225-1237. DOI: 10.1517/13543784.2014.922952
  60. 60. DNDi. Fexinidazole (Chagas). Drugs Neglected Dis Initiat DNDi. 2018. Available from:[Accessed: November 2, 2018]
  61. 61. Docampo R, Moreno SN. Free radical metabolites in the mode of action of chemotherapeutic agents and phagocytic cells onTrypanosoma cruzi. Reviews of Infectious Diseases. 1984;6:223-238
  62. 62. Peterson FJ, Mason RP, Hovsepian J, Holtzman JL. Oxygen-sensitive and -insensitive nitroreduction byEscherichia coliand rat hepatic microsomes. The Journal of Biological Chemistry. 1979;254:4009-4014
  63. 63. Fairlamb AH, Patterson S. Current and future prospects of nitro-compounds as drugs for Trypanosomiasis and Leishmaniasis. Current Medicinal Chemistry. 2018;25. DOI: 10.2174/0929867325666180426164352. [Epub ahead of print]
  64. 64. Boiani M, Piacenza L, Hernández P, Boiani L, Cerecetto H, González M, et al. Mode of action of nifurtimox and N-oxide-containing heterocycles againstTrypanosoma cruzi: Is oxidative stress involved? Biochemical Pharmacology. 2010;79:1736-1745. DOI: 10.1016/j.bcp.2010.02.009
  65. 65. Hall BS, Wilkinson SR. Activation of benznidazole by trypanosomal type I nitroreductases results in glyoxal formation. Antimicrobial Agents and Chemotherapy. 2012;56:115-123. DOI: 10.1128/AAC.05135-11
  66. 66. Papadopoulou MV, Bloomer WD, Rosenzweig HS, O’Shea IP, Wilkinson SR, Kaiser M, et al. Discovery of potent nitrotriazole-based antitrypanosomal agents: In vitro and in vivo evaluation. Bioorganic & Medicinal Chemistry. 2015;23:6467-6476. DOI: 10.1016/j.bmc.2015.08.014
  67. 67. Vannier-Santos MA, Suarez-Fontes AM. Role of polyamines in parasite cell architecture and function. Current Pharmaceutical Design. 2017;23:3342-3358. DOI: 10.2174/1381612823666170703163458
  68. 68. Roberts S, Ullman B. Parasite polyamines as pharmaceutical targets. Current Pharmaceutical Design. 2017;23:3325-3341. DOI: 10.2174/1381612823666170601101644
  69. 69. Algranati ID. Polyamine metabolism inTrypanosoma cruzi: Studies on the expression and regulation of heterologous genes involved in polyamine biosynthesis. Amino Acids. 2010;38:645-651. DOI: 10.1007/s00726-009-0425-6
  70. 70. Hasne M-P, Coppens I, Soysa R, Ullman B. A high-affinity putrescine-cadaverine transporter fromTrypanosoma cruzi. Molecular Microbiology. 2010;76:78-91. DOI: 10.1111/j.1365-2958.2010.07081.x
  71. 71. Reigada C, Phanstiel O, Miranda MR, Pereira CA. Targeting polyamine transport inTrypanosoma cruzi. European Journal of Medicinal Chemistry. 2018;147:1-6. DOI: 10.1016/j.ejmech.2018.01.083
  72. 72. Díaz MV, Miranda MR, Campos-Estrada C, Reigada C, Maya JD, Pereira CA, et al. Pentamidine exerts in vitro and in vivo antiTrypanosoma cruziactivity and inhibits the polyamine transport inTrypanosoma cruzi. Acta Tropica. 2014;134:1-9. DOI: 10.1016/j.actatropica.2014.02.012
  73. 73. Hasne M-P, Soysa R, Ullman B. TheTrypanosoma cruzidiamine transporter is essential for robust infection of mammalian cells. PLoS One. 2016;11:e0152715. DOI: 10.1371/journal.pone.0152715
  74. 74. Reigada C, Sayé M, Vera EV, Balcazar D, Fraccaroli L, Carrillo C, et al.Trypanosoma cruzipolyamine transporter: Its role on parasite growth and survival under stress conditions. The Journal of Membrane Biology. 2016;249:475-481. DOI: 10.1007/s00232-016-9888-z
  75. 75. Mesías AC, Sasoni N, Arias DG, Pérez Brandán C, Orban OCF, Kunick C, et al. Trypanothione synthetase confers growth, survival advantage and resistance to anti-protozoal drugs inTrypanosoma cruzi. Free Radical Biology & Medicine. 2019;130:23-34. DOI: 10.1016/j.freeradbiomed.2018.10.436
  76. 76. Soares CO, Colli W, Bechara EJH, Alves MJM. 1,4-Diamino-2-butanone, a putrescine analogue, promotes redox imbalance inTrypanosoma cruziand mammalian cells. Archives of Biochemistry and Biophysics. 2012;528:103-110. DOI: 10.1016/
  77. 77. Menezes D, Valentim C, Oliveira MF, Vannier-Santos MA. Putrescine analogue cytotoxicity againstTrypanosoma cruzi. Parasitology Research. 2006;98:99-105. DOI: 10.1007/s00436-005-0010-1
  78. 78. Reis IA, Martinez MP, Yarlett N, Johnson PJ, Silva-Filho FC, Vannier-Santos MA. Inhibition of polyamine synthesis arrests trichomonad growth and induces destruction of hydrogenosomes. Antimicrobial Agents and Chemotherapy. 1999;43:1919-1923
  79. 79. Vannier-Santos MA, Menezes D, Oliveira MF, de Mello FG. The putrescine analogue 1,4-diamino-2-butanone affects polyamine synthesis, transport, ultrastructure and intracellular survival in Leishmania amazonensis. Microbiology. 2008;154:3104-3111. DOI: 10.1099/mic.0.2007/013896-0
  80. 80. Hernández SM, Sánchez MS, de Tarlovsky MNS. Polyamines as a defense mechanism against lipoperoxidation inTrypanosoma cruzi. Acta Tropica. 2006;98:94-102. DOI: 10.1016/j.actatropica.2006.02.005
  81. 81. Vanrell MC, Losinno AD, Cueto JA, Balcazar D, Fraccaroli LV, Carrillo C, et al. The regulation of autophagy differentially affectsTrypanosoma cruzimetacyclogenesis. PLoS Neglected Tropical Diseases. 2017;11:e0006049. DOI: 10.1371/journal.pntd.0006049
  82. 82. Nes WD. Biosynthesis of cholesterol and other sterols. Chemical Reviews. 2011;111:6423-6451. DOI: 10.1021/cr200021m
  83. 83. Leaver D. Synthesis and biological activity of sterol 14α-demethylase and sterol C24-methyltransferase inhibitors. Molecules. 2018;23:1753. DOI: 10.3390/molecules23071753
  84. 84. Lepesheva GI, Friggeri L, Waterman MR. CYP51 as drug targets for fungi and protozoan parasites: Past, present and future. Parasitology. 2018;145:1820-1836. DOI: 10.1017/S0031182018000562
  85. 85. Do R, Kiss R, Gaudet D, Engert J. Squalene synthase: A critical enzyme in the cholesterol biosynthesis pathway. Clinical Genetics. 2009;75:19-29. DOI: 10.1111/j.1399-0004.2008.01099.x
  86. 86. Urbina JA, Concepcion JL, Rangel S, Visbal G, Lira R. Squalene synthase as a chemotherapeutic target inTrypanosoma cruziand Leishmania mexicana. Molecular and Biochemical Parasitology. 2002;125:35-45
  87. 87. Braga MV, Urbina JA, de Souza W. Effects of squalene synthase inhibitors on the growth and ultrastructure ofTrypanosoma cruzi. International Journal of Antimicrobial Agents. 2004;24:72-78. DOI: 10.1016/j.ijantimicag.2003.12.009
  88. 88. Urbina JA, Concepcion JL, Caldera A, Payares G, Sanoja C, Otomo T, et al. In vitro and in vivo activities of E5700 and ER-119884, two novel orally active squalene synthase inhibitors, againstTrypanosoma cruzi. Antimicrobial Agents and Chemotherapy. 2004;48:2379-2387. DOI: 10.1128/AAC.48.7.2379-2387.2004
  89. 89. Sealey-Cardona M, Cammerer S, Jones S, Ruiz-Perez LM, Brun R, Gilbert IH, et al. Kinetic characterization of squalene synthase fromTrypanosoma cruzi: Selective inhibition by quinuclidine derivatives. Antimicrobial Agents and Chemotherapy. 2007;51:2123-2129. DOI: 10.1128/AAC.01454-06
  90. 90. Shang N, Li Q, Ko T-P, Chan H-C, Li J, Zheng Y, et al. Squalene synthase As a target for Chagas disease therapeutics. PLoS Pathogens. 2014;10:e1004114. DOI: 10.1371/journal.ppat.1004114
  91. 91. Rodriguez JB. WC-9 a Lead drug with great prospects for American Trypanosomiasis and Toxoplasmosis. Mini Reviews in Medicinal Chemistry. 2016;16:1195-1200
  92. 92. Urbina JA, Concepcion JL, Montalvetti A, Rodriguez JB, Docampo R. Mechanism of action of 4-phenoxyphenoxyethyl thiocyanate (WC-9) against Trypanosoma cruzi, the causative agent of Chagas’ disease. Antimicrobial Agents and Chemotherapy. 2003;47:2047-2050
  93. 93. Liñares GEG, Ravaschino EL, Rodriguez JB. Progresses in the field of drug design to combat tropical protozoan parasitic diseases. Current Medicinal Chemistry. 2006;13:335-360
  94. 94. Chao MN, Storey M, Li C, Rodríguez MG, Di Salvo F, Szajnman SH, et al. Selenium-containing analogues of WC-9 are extremely potent inhibitors ofTrypanosoma cruziproliferation. Bioorganic & Medicinal Chemistry. 2017;25:6435-6449. DOI: 10.1016/j.bmc.2017.10.016
  95. 95. Urbina JA. Ergosterol biosynthesis and drug development for Chagas disease. Memórias do Instituto Oswaldo Cruz. 2009;104(Suppl 1):311-318
  96. 96. Lepesheva GI, Villalta F, Waterman MR. TargetingTrypanosoma cruzisterol 14α-demethylase (CYP51). Advances in Parasitology. 2011;75:65-87. DOI: 10.1016/B978-0-12-385863-4.00004-6
  97. 97. Urbina JA, McKerrow JH. Drug susceptibility of genetically engineeredTrypanosoma cruzistrains and sterile cure in animal models as a criterion for potential clinical efficacy of anti-T. cruzidrugs. Antimicrobial Agents and Chemotherapy. 2015;59:7923-7924. DOI: 10.1128/AAC.01714-15
  98. 98. Urbina JA, Payares G, Contreras LM, Liendo A, Sanoja C, Molina J, et al. Antiproliferative effects and mechanism of action of SCH 56592 against Trypanosoma (Schizotrypanum) cruzi: In vitro and in vivo studies. Antimicrobial Agents and Chemotherapy. 1998;42:1771-1777
  99. 99. Molina J, Martins-Filho O, Brener Z, Romanha AJ, Loebenberg D, Urbina JA. Activities of the triazole derivative SCH 56592 (posaconazole) against drug-resistant strains of the protozoan parasite Trypanosoma (Schizotrypanum) cruzi in immunocompetent and immunosuppressed murine hosts. Antimicrobial Agents and Chemotherapy. 2000;44:150-155
  100. 100. Urbina JA, Payares G, Sanoja C, Lira R, Romanha AJ. In vitro and in vivo activities of ravuconazole onTrypanosoma cruzi, the causative agent of Chagas disease. International Journal of Antimicrobial Agents. 2003;21:27-38
  101. 101. Diniz L de F, Caldas IS, Guedes PM da M, Crepalde G, de Lana M, Carneiro CM, et al. Effects of ravuconazole treatment on parasite load and immune response in dogs experimentally infected withTrypanosoma cruzi. Antimicrobial Agents and Chemotherapy. 2010;54:2979-2986. DOI: 10.1128/AAC.01742-09
  102. 102. Molina I, Gómez i, Prat J, Salvador F, Treviño B, Sulleiro E, et al. Randomized trial of posaconazole and benznidazole for chronic Chagas’ disease. The New England Journal of Medicine. 2014;370:1899-1908. DOI: 10.1056/NEJMoa1313122
  103. 103. Morillo CA, Waskin H, Sosa-Estani S, del Carmen Bangher M, Cuneo C, Milesi R, et al. Benznidazole and Posaconazole in eliminating parasites in asymptomaticT. cruzicarriers. Journal of the American College of Cardiology. 2017;69:939-947. DOI: 10.1016/j.jacc.2016.12.023
  104. 104. Torrico F, Gascon J, Ortiz L, Alonso-Vega C, Pinazo M-J, Schijman A, et al. Treatment of adult chronic indeterminate Chagas disease with benznidazole and three E1224 dosing regimens: A proof-of-concept, randomised, placebo-controlled trial. The Lancet Infectious Diseases. 2018;18:419-430. DOI: 10.1016/S1473-3099(17)30538-8
  105. 105. Villalta F, Dobish MC, Nde PN, Kleshchenko YY, Hargrove TY, Johnson CA, et al. VNI cures acute and chronic experimental Chagas disease. The Journal of Infectious Diseases. 2013;208:504-511. DOI: 10.1093/infdis/jit042
  106. 106. Soeiro M de NC, de Souza EM, da Silva CF, Batista D da GJ, Batista MM, Pavão BP, et al.In vitroandin vivostudies of the Antiparasitic activity of sterol 14α-demethylase (CYP51) inhibitor VNI against drug-resistant strains ofTrypanosoma cruzi. Antimicrobial Agents and Chemotherapy. 2013;57:4151-4163. DOI: 10.1128/AAC.00070-13
  107. 107. Lepesheva GI, Hargrove TY, Rachakonda G, Wawrzak Z, Pomel S, Cojean S, et al. VFV as a new effective CYP51 structure-derived drug candidate for Chagas disease and visceral Leishmaniasis. The Journal of Infectious Diseases. 2015;212:1439-1448. DOI: 10.1093/infdis/jiv228
  108. 108. Guedes-da-Silva FH, Batista DGJ, Da Silva CF, De Araújo JS, Pavão BP, Simões-Silva MR, et al. Antitrypanosomal activity of sterol 14α-demethylase (CYP51) inhibitors VNI and VFV in the Swiss mouse models of Chagas disease induced by theTrypanosoma cruziY strain. Antimicrobial Agents and Chemotherapy. 2017;61:e02098-16. DOI: 10.1128/AAC.02098-16
  109. 109. Hoekstra WJ, Hargrove TY, Wawrzak Z, da Gama Jaen Batista D, da Silva CF, Nefertiti ASG, et al. Clinical candidate VT-1161’s antiparasitic effectin vitro, activity in a murine model of Chagas disease, and structural characterization in complex with the target enzyme CYP51 fromTrypanosoma cruzi. Antimicrobial Agents and Chemotherapy. 2016;60:1058-1066. DOI: 10.1128/AAC.02287-15
  110. 110. Friggeri L, Hargrove TY, Rachakonda G, Williams AD, Wawrzak Z, DSR, et al. Structural basis for rational design of inhibitors targetingTrypanosoma cruzisterol 14α-demethylase: Two regions of the enzyme molecule potentiate its inhibition. Journal of Medicinal Chemistry. 2014;57:6704-6717. DOI: 10.1021/jm500739f
  111. 111. De Vita D, Moraca F, Zamperini C, Pandolfi F, Di Santo R, Matheeussen A, et al. In vitro screening of 2-(1H-imidazol-1-yl)-1-phenylethanol derivatives as antiprotozoal agents and docking studies onTrypanosoma cruziCYP51. European Journal of Medicinal Chemistry. 2016;113:28-33. DOI: 10.1016/j.ejmech.2016.02.028
  112. 112. Ferreira de Almeida Fiuza L, Peres RB, Simões-Silva MR, da Silva PB, Batista D d GJ, da Silva CF, et al. Identification of Pyrazolo[3,4-e][1,4]thiazepin based CYP51 inhibitors as potential Chagas disease therapeutic alternative: In vitro and in vivo evaluation, binding mode prediction and SAR exploration. European Journal of Medicinal Chemistry. 2018;149:257-268. DOI: 10.1016/j.ejmech.2018.02.020
  113. 113. Ferreira LG, Andricopulo AD. Targeting cysteine proteases in trypanosomatid disease drug discovery. Pharmacology & Therapeutics. 2017;180:49-61. DOI: 10.1016/j.pharmthera.2017.06.004
  114. 114. Siqueira-Neto JL, Debnath A, McCall L-I, Bernatchez JA, Ndao M, Reed SL, et al. Cysteine proteases in protozoan parasites. PLoS Neglected Tropical Diseases. 2018;12:e0006512. DOI: 10.1371/journal.pntd.0006512
  115. 115. Martinez-Mayorga K, Byler KG, Ramirez-Hernandez AI, Terrazas-Alvares DE. Cruzain inhibitors: Efforts made, current leads and a structural outlook of new hits. Drug Discovery Today. 2015;20:890-898. DOI: 10.1016/j.drudis.2015.02.004
  116. 116. Nicoll-Griffith DA. Use of cysteine-reactive small molecules in drug discovery for trypanosomal disease. Expert Opinion on Drug Discovery. 2012;7:353-366. DOI: 10.1517/17460441.2012.668520
  117. 117. Engel JC, Doyle PS, Hsieh I, McKerrow JH. Cysteine protease inhibitors cure an experimentalTrypanosoma cruziinfection. The Journal of Experimental Medicine. 1998;188:725-734
  118. 118. Doyle PS, Zhou YM, Engel JC, McKerrow JH. A cysteine protease inhibitor cures Chagas’ disease in an Immunodeficient-mouse model of infection. Antimicrobial Agents and Chemotherapy. 2007;51:3932-3939. DOI: 10.1128/AAC.00436-07
  119. 119. Chen YT, Brinen LS, Kerr ID, Hansell E, Doyle PS, McKerrow JH, et al. In vitro and in vivo studies of the trypanocidal properties of WRR-483 againstTrypanosoma cruzi. PLoS Neglected Tropical Diseases. 2010;4:e825. DOI: 10.1371/journal.pntd.0000825
  120. 120. Salomão K, De Castro SL, editors. Recent Advances in Drug Development for Chagas Disease: Two Magic Words, Combination and Repositioning. Differ. Asp. Chemother. Trypanos. New York: Leon L & Torres-Santos EC; 2017. pp. 181-226
  121. 121. Mott BT, Ferreira RS, Simeonov A, Jadhav A, Ang KK-H, Leister W, et al. Identification and optimization of inhibitors of trypanosomal cysteine proteases: Cruzain, Rhodesain, and TbCatB. Journal of Medicinal Chemistry. 2010;53:52-60. DOI: 10.1021/jm901069a
  122. 122. Beaulieu C, Isabel E, Fortier A, Massé F, Mellon C, Méthot N, et al. Identification of potent and reversible cruzipain inhibitors for the treatment of Chagas disease. Bioorganic & Medicinal Chemistry Letters. 2010;20:7444-7449. DOI: 10.1016/j.bmcl.2010.10.015
  123. 123. Ndao M, Beaulieu C, Black WC, Isabel E, Vasquez-Camargo F, Nath-Chowdhury M, et al. Reversible cysteine protease inhibitors show promise for a Chagas disease cure. Antimicrobial Agents and Chemotherapy. 2014;58:1167-1178. DOI: 10.1128/AAC.01855-13
  124. 124. Burtoloso ACB, de Albuquerque S, Furber M, Gomes JC, Gonçalez C, Kenny PW, et al. Anti-trypanosomal activity of non-peptidic nitrile-based cysteine protease inhibitors. PLoS Neglected Tropical Diseases. 2017;11:e0005343. DOI: 10.1371/journal.pntd.0005343
  125. 125. Salas-Sarduy E, Landaburu LU, Karpiak J, Madauss KP, Cazzulo JJ, Agüero F, et al. Novel scaffolds for inhibition of Cruzipain identified from high-throughput screening of anti-kinetoplastid chemical boxes. Scientific Reports. 2017;7:12073. DOI: 10.1038/s41598-017-12170-4
  126. 126. Pauli I, Ferreira LG, de Souza ML, Oliva G, Ferreira RS, Dessoy MA, et al. Molecular modeling and structure–activity relationships for a series of benzimidazole derivatives as cruzain inhibitors. Future Medicinal Chemistry. 2017;9:641-657. DOI: 10.4155/fmc-2016-0236
  127. 127. de Souza AS, de Oliveira MT, Andricopulo AD. Development of a pharmacophore for cruzain using oxadiazoles as virtual molecular probes: Quantitative structure-activity relationship studies. Journal of Computer-Aided Molecular Design. 2017;31:801-816. DOI: 10.1007/s10822-017-0039-0
  128. 128. Kaiser M, Mäser P, Tadoori LP, Ioset J-R, Brun R. Antiprotozoal activity profiling of approved drugs: A starting point toward drug repositioning. PLoS One. 2015;10:e0135556. DOI: 10.1371/journal.pone.0135556
  129. 129. Bellera CL, Balcazar DE, Alberca L, Labriola CA, Talevi A, Carrillo C. Identification of levothyroxine antichagasic activity through computer-aided drug repurposing. Scientific World Journal. 2014;2014:1-9. DOI: 10.1155/2014/279618
  130. 130. Bellera CL, Balcazar DE, Vanrell MC, Casassa AF, Palestro PH, Gavernet L, et al. Computer-guided drug repurposing: Identification of trypanocidal activity of clofazimine, benidipine and saquinavir. European Journal of Medicinal Chemistry. 2015;93:338-348. DOI: 10.1016/j.ejmech.2015.01.065
  131. 131. Sbaraglini ML, Bellera CL, Fraccaroli L, Larocca L, Carrillo C, Talevi A, et al. Novel cruzipain inhibitors for the chemotherapy of chronic Chagas disease. International Journal of Antimicrobial Agents. 2016;48:91-95. DOI: 10.1016/j.ijantimicag.2016.02.018
  132. 132. Palos I, Lara-Ramirez EE, Lopez-Cedillo JC, Garcia-Perez C, Kashif M, Bocanegra-Garcia V, et al. Repositioning FDA drugs as potential Cruzain inhibitors fromTrypanosoma cruzi: Virtual screening, in vitro and in vivo studies. Molecules (Basel, Switzerland). 2017;22:E1015. DOI: 10.3390/molecules22061015
  133. 133. Branquinha MH, Marinho FA, Sangenito LS, Oliveira SSC, Goncalves KC, Ennes-Vidal V, et al. Calpains: Potential targets for alternative chemotherapeutic intervention against human pathogenic trypanosomatids. Current Medicinal Chemistry. 2013;20:3174-3185
  134. 134. Saez ME, Ramirez-Lorca R, Moron FJ, Ruiz A. The therapeutic potential of the calpain family: New aspects. Drug Discovery Today. 2006;11:917-923. DOI: 10.1016/j.drudis.2006.08.009
  135. 135. Donkor IO. An updated patent review of calpain inhibitors (2012-2014). Expert Opinion on Therapeutic Patents. 2015;25:17-31. DOI: 10.1517/13543776.2014.982534
  136. 136. Ennes-Vidal V, Menna-Barreto RFS, Santos ALS, Branquinha MH, d’Avila-Levy CM. MDL28170, a calpain inhibitor, affectsTrypanosoma cruzimetacyclogenesis, ultrastructure and attachment to Rhodnius prolixus midgut. PLoS One. 2011;6:e18371. DOI: 10.1371/journal.pone.0018371
  137. 137. Ennes-Vidal V, Menna-Barreto RFS, Santos ALS, Branquinha MH, d’Avila-Levy CM. Effects of the calpain inhibitor MDL28170 on the clinically relevant forms ofTrypanosoma cruziin vitro. The Journal of Antimicrobial Chemotherapy. 2010;65:1395-1398. DOI: 10.1093/jac/dkq154
  138. 138. Motta MCM. Kinetoplast as a potential chemotherapeutic target of trypanosomatids. Current Pharmaceutical Design. 2008;14:847-854
  139. 139. Soeiro M de NC, de Castro SL. Screening of potential anti-Trypanosoma cruzicandidates: In vitro and in vivo studies. Open Medicinal Chemistry Journal. 2011;5:21-30. DOI: 10.2174/1874104501105010021
  140. 140. Delain E, Brack C, Riou G, Festy B. Ultrastructural alterations ofTrypanosoma cruzikinetoplast induced by the interaction of a trypanocidal drug (hydroxystilbamidine) with the kinetoplast DNA. Journal of Ultrastructure Research. 1971;37:200-218
  141. 141. Vannier-Santos MA, De Castro SL. Electron microscopy in antiparasitic chemotherapy: A (close) view to a kill. Current Drug Targets. 2009;10:246-260. DOI: 10.2174/138945009787581168
  142. 142. Girard RMBM, Crispim M, Stolić I, Damasceno FS, Santos da Silva M, Pral EMF, et al. An aromatic diamidine that targets kinetoplast DNA, impairs the cell cycle inTrypanosoma cruzi, and diminishes trypomastigote release from infected mammalian host cells. Antimicrobial Agents and Chemotherapy. 2016;60:5867-5877. DOI: 10.1128/AAC.01595-15
  143. 143. Balaña-Fouce R, Álvarez-Velilla R, Fernández-Prada C, García-Estrada C, Reguera RM. Trypanosomatids topoisomerase re-visited. New structural findings and role in drug discovery. International Journal for Parasitology: Drugs and Drug Resistance. 2014;4:326-337. DOI: 10.1016/j.ijpddr.2014.07.006
  144. 144. Podestá D, Stoppani A, Villamil SF. Inactivation ofTrypanosoma cruziandCrithidia fasciculatatopoisomerase I by Fenton systems. Redox Report. 2003;8:357-363. DOI: 10.1179/135100003225003366
  145. 145. Wilson WD, Nguyen B, Tanious FA, Mathis A, Hall JE, Stephens CE, et al. Dications that target the DNA minor groove: Compound design and preparation, DNA interactions, cellular distribution and biological activity. Current Medicinal Chemistry—Anti-Cancer Agents. 2005;5:389-408
  146. 146. Werbovetz K. Diamidines as antitrypanosomal, antileishmanial and antimalarial agents. Current Opinion in Investigational Drugs (London, England: 2000). 2006;7:147-157
  147. 147. Silva CF, Meuser MB, De Souza EM, Meirelles MNL, Stephens CE, Som P, et al. Cellular effects of reversed amidines onTrypanosoma cruzi. Antimicrobial Agents and Chemotherapy. 2007;51:3803-3809. DOI: 10.1128/AAC.00047-07
  148. 148. Silva CF, Batista MM, Mota RA, de Souza EM, Stephens CE, Som P, et al. Activity of “reversed” diamidines againstTrypanosoma cruzi“in vitro”. Biochemical Pharmacology. 2007;73:1939-1946. DOI: 10.1016/j.bcp.2007.03.020
  149. 149. Santos CC, Lionel JR, Peres RB, Batista MM, da Silva PB, de Oliveira GM, et al.In vitro,in silico, andin vivoanalyses of novel aromatic amidines againstTrypanosoma cruzi. Antimicrobial Agents and Chemotherapy. 2017;62:e02205-17. DOI: 10.1128/AAC.02205-17
  150. 150. Das A, Dasgupta A, Sengupta T, Majumder HK. Topoisomerases of kinetoplastid parasites as potential chemotherapeutic targets. Trends in Parasitology. 2004;20:381-387. DOI: 10.1016/
  151. 151. Chowdhury SR, Godinho JLP, Vinayagam J, Zuma AA, Silva STDM, Jaisankar P, et al. Isobenzofuranone derivative JVPH3, an inhibitor ofL. donovanitopoisomerase II, disrupts mitochondrial architecture in trypanosomatid parasites. Scientific Reports. 2018;8:11940. DOI: 10.1038/s41598-018-30405-w
  152. 152. Chowdhury SR, Kumar A, Godinho JLP, De Macedo Silva ST, Zuma AA, Saha S, et al. Voacamine alters Leishmania ultrastructure and kills parasite by poisoning unusual bi-subunit topoisomerase IB. Biochemical Pharmacology. 2017;138:19-30. DOI: 10.1016/j.bcp.2017.05.002
  153. 153. Brunoro GV-F, Caminha MA, Menna-Barreto RF. From proteins to molecular targets:Trypanosoma cruziproteomic insights in drug development. In: Protozoan Parasitism: From Omics to Prevention and Control. Caister Academic Press; 2018;1:1-30. DOI: 10.21775/9781910190838.01
  154. 154. Magalhães AD, Charneau S, Paba J, Guércio RAP, Teixeira ARL, Santana JM, et al. Trypanosoma cruzi alkaline 2-DE: Optimization and application to comparative proteome analysis of flagellate life stages. Proteome Science. 2008;6:24. DOI: 10.1186/1477-5956-6-24
  155. 155. Queiroz RML, Charneau S, Bastos IMD, Santana JM, Sousa MV, Roepstorff P, et al. Cell surface proteome analysis of human-hostedTrypanosoma cruzilife stages. Journal of Proteome Research. 2014;13:3530-3541. DOI: 10.1021/pr401120y
  156. 156. de Jesus LTC, Calderano SG, Vitorino FN d L, Llanos RP, Lopes M d C, de Araújo CB, et al. Quantitative proteomic analysis of replicative and nonreplicative forms reveals important insights into chromatin biology ofTrypanosoma cruzi. Molecular & Cellular Proteomics. 2017;16:23-38. DOI: 10.1074/mcp.M116.061200
  157. 157. Alves MJM, Kawahara R, Viner R, Colli W, Mattos EC, Thaysen-Andersen M, et al. Comprehensive glycoprofiling of the epimastigote and trypomastigote stages ofTrypanosoma cruzi. Journal of Proteomics. 2017;151:182-192. DOI: 10.1016/j.jprot.2016.05.034
  158. 158. Atwood JA, Minning T, Ludolf F, Nuccio A, Weatherly DB, Alvarez-Manilla G, et al. Glycoproteomics ofTrypanosoma cruzitrypomastigotes using subcellular fractionation, lectin affinity, and stable isotope labeling. Journal of Proteome Research. 2006;5:3376-3384. DOI: 10.1021/pr060364b
  159. 159. de Godoy LMF, Marchini FK, Pavoni DP, Rampazzo R de CP, Probst CM, Goldenberg S, et al. Quantitative proteomics ofTrypanosoma cruziduring metacyclogenesis. Proteomics. 2012;12:2694-2703. DOI: 10.1002/pmic.201200078
  160. 160. Cordero EM, Nakayasu ES, Gentil LG, Yoshida N, Almeida IC, da Silveira JF. Proteomic analysis of detergent-solubilized membrane proteins from insect-developmental forms ofTrypanosoma cruzi. Journal of Proteome Research. 2009;8:3642-3652. DOI: 10.1021/pr800887u
  161. 161. Bayer-Santos E, Aguilar-Bonavides C, Rodrigues SP, Cordero EM, Marques AF, Varela-Ramirez A, et al. Proteomic analysis of Trypanosoma cruzi secretome: Characterization of two populations of extracellular vesicles and soluble proteins. Journal of Proteome Research. 2013;12:883-897. DOI: 10.1021/pr300947g
  162. 162. de Pablos Torró LM, Retana Moreira L, Osuna A. Extracellular vesicles in Chagas disease: A new passenger for an old disease. Frontiers in Microbiology. 2018;9:E1190. DOI: 10.3389/fmicb.2018.01190
  163. 163. Brunoro GVF, Caminha MA, Ferreira AT d S, Leprevost F d V, Carvalho PC, Perales J, et al. Reevaluating theTrypanosoma cruziproteomic map: The shotgun description of bloodstream trypomastigotes. Journal of Proteomics. 2015;115:58-65. DOI: 10.1016/j.jprot.2014.12.003
  164. 164. Brunoro GVF, Faça VM, Caminha MA, Ferreira AT d S, Trugilho M, de Moura KCG, et al. Differential gel electrophoresis (DIGE) evaluation of naphthoimidazoles mode of action: A study inTrypanosoma cruzibloodstream trypomastigotes. PLoS Neglected Tropical Diseases. 2016;10:e0004951. DOI: 10.1371/journal.pntd.0004951
  165. 165. Paba J, Santana JM, Teixeira ARL, Fontes W, Sousa MV, Ricart CAO. Proteomic analysis of the human pathogenTrypanosoma cruzi. Proteomics. 2004;4:1052-1059. DOI: 10.1002/pmic.200300637
  166. 166. Ritagliati C, Alonso VL, Manarin R, Cribb P, Serra EC. Overexpression of cytoplasmic TcSIR2RP1 and mitochondrial TcSIR2RP3 impacts onTrypanosoma cruzigrowth and cell invasion. PLoS Neglected Tropical Diseases. 2015;9:e0003725. DOI: 10.1371/journal.pntd.0003725
  167. 167. Moretti NS, da Silva Augusto L, Clemente TM, Antunes RPP, Yoshida N, Torrecilhas AC, et al. Characterization ofTrypanosoma cruziSirtuins as possible drug targets for Chagas disease. Antimicrobial Agents and Chemotherapy. 2015;59:4669-4679. DOI: 10.1128/AAC.04694-14
  168. 168. Gaspar L, Coron RP, KongThoo Lin P, Costa DM, Perez-Cabezas B, Tavares J, et al. Inhibitors ofTrypanosoma cruziSir2 related protein 1 as potential drugs against Chagas disease. PLoS Neglected Tropical Diseases. 2018;12:e0006180. DOI: 10.1371/journal.pntd.0006180
  169. 169. Wan X, Wen J-J, Koo S-J, Liang LY, Garg NJ. SIRT1-PGC1α-NFκB pathway of oxidative and inflammatory stress duringTrypanosoma cruziinfection: Benefits of SIRT1-targeted therapy in improving heart function in Chagas disease. PLoS Pathogens. 2016;12:e1005954. DOI: 10.1371/journal.ppat.1005954
  170. 170. Urbina JA. Specific chemotherapy of Chagas disease: Relevance, current limitations and new approaches. Acta Tropica. 2010;115:55-68. DOI: 10.1016/j.actatropica.2009.10.023
  171. 171. Filardi LS, Brener Z. Susceptibility and natural resistance ofTrypanosoma cruzistrains to drugs used clinically in Chagas disease. Transactions of the Royal Society of Tropical Medicine and Hygiene. 1987;81:755-759. DOI: 10.1016/0035-9203(87)90020-4
  172. 172. Chatelain E, Ioset J-R. Phenotypic screening approaches for Chagas disease drug discovery. Expert Opinion on Drug Discovery. 2018;13:141-153. DOI: 10.1080/17460441.2018.1417380
  173. 173. Moraes CB, Franco CH. Novel drug discovery for Chagas disease. Expert Opinion on Drug Discovery. 2016;11:447-455. DOI: 10.1517/17460441.2016.1160883
  174. 174. Sueth-Santiago V, Decote-Ricardo D, Morrot A, Freire-de-Lima CG, Lima MEF. Challenges in the chemotherapy of Chagas disease: Looking for possibilities related to the differences and similarities between the parasite and host. World Journal of Biological Chemistry. 2017;8:57. DOI: 10.4331/wjbc.v8.i1.57
  175. 175. Moraes CB, Giardini MA, Kim H, Franco CH, Araujo-Junior AM, Schenkman S, et al. Nitroheterocyclic compounds are more efficacious than CYP51 inhibitors againstTrypanosoma cruzi: Implications for Chagas disease drug discovery and development. Scientific Reports. 2014;4:4703. DOI: 10.1038/srep04703
  176. 176. Cal M, Ioset J-R, Fügi MA, Mäser P, Kaiser M. Assessing anti-T. cruzicandidates in vitro for sterile cidality. International Journal for Parasitology: Drugs and Drug Resistance. 2016;6:165-170. DOI: 10.1016/j.ijpddr.2016.08.003
  177. 177. Ferraz ML, Gazzinelli RT, Alves RO, Urbina JA, Romanha AJ. Absence of CD4+ T lymphocytes, CD8+ T lymphocytes, or B lymphocytes has different effects on the efficacy of posaconazole and benznidazole in treatment of experimental acuteTrypanosoma cruziinfection. Antimicrobial Agents and Chemotherapy. 2009;53:174-179. DOI: 10.1128/AAC.00779-08
  178. 178. Chatelain E, Ioset J-R. Drug discovery and development for neglected diseases: The DNDi model. Drug Design, Development and Therapy. 2011;5:175-181. DOI: 10.2147/DDDT.S16381
  179. 179. Friggeri L, Hargrove TY, Rachakonda G, Blobaum AL, Fisher P, de Oliveira GM, et al. Sterol 14α-demethylase structure-based optimization of drug candidates for human infections with the protozoan trypanosomatidae. Journal of Medicinal Chemistry. 2018;61:10910-10921. DOI: 10.1021/acs.jmedchem.8b01671
  180. 180. Machado-Silva A, Cerqueira PG, Grazielle-Silva V, Gadelha FR, Peloso E de F, Teixeira SMR, et al. HowTrypanosoma cruzideals with oxidative stress: Antioxidant defence and DNA repair pathways. Mutation Research. Reviews in Mutation Research. 2016;767:8-22. DOI: 10.1016/j.mrrev.2015.12.003
  181. 181. Tomás AM, Castro H. Redox metabolism in mitochondria of trypanosomatids. Antioxidants & Redox Signaling. 2013;19:696-707. DOI: 10.1089/ars.2012.4948
  182. 182. Sykes ML, Avery VM. Approaches to protozoan drug discovery: Phenotypic screening. Journal of Medicinal Chemistry. 2013;56:7727-7740. DOI: 10.1021/jm4004279
  183. 183. Peña I, Pilar Manzano M, Cantizani J, Kessler A, Alonso-Padilla J, Bardera AI, et al. New compound sets identified from high throughput phenotypic screening against three kinetoplastid parasites: An open resource. Scientific Reports. 2015;5:8771. DOI: 10.1038/srep08771
  184. 184. Chatelain E, Konar N. Translational challenges of animal models in Chagas disease drug development: A review. Drug Design, Development and Therapy. 2015;9:4807-4823. DOI: 10.2147/DDDT.S90208

Written By

Marcos André Vannier-Santos, Giselle V. Brunoro, Maria de Nazaré C. Soeiro, Solange L. DeCastro and Rubem F.S. Menna-Barreto

Submitted: September 6th, 2018 Reviewed: January 16th, 2019 Published: March 15th, 2019