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
- drug targets
- oxidative metabolism
Chagas disease, or American trypanosomiasis, was described in 1909 by the Brazilian physician Carlos Chagas, who identified the causative agent—
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 , 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 .
Chagas disease results from the establishment of
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 . 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 . 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 . 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].
There are significant drawbacks on the use of these drugs, mostly related to the limited efficacy in the chronic phase , 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-
Trypanosoma cruziand drug targets
One important point to be addressed in the search of alternative molecular targets in
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 . During electron leakage, ROS were produced that interfere with different biological processes . 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 . 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 . 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 . Many studies pointed to the susceptibility of
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
2.1.1 Mitochondrial ETC
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
2.1.3 ROS inducers
Varying the dose or the time of drug treatment, injury to the mitochondrion usually leads to ROS production . 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.
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 . 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 . 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) . 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 . 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 .
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 . Alternative enzymes have been associated with the reduction of nitro compounds in
Polyamines (PA) are ubiquitous organic polycations that play a plethora of ubiquitous biological roles in most cell types, including bacteria, protozoa, and higher organisms , with significant metabolic differences, therefore comprising promising drug targets for protozoal diseases . PA metabolism among parasitic protozoa is defective in a number of pathways as compared to mammalian cells.
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
2.2 Biosynthesis of sterols
Sterols are essential lipid molecules, performing numerous cellular roles associated with membrane and signal functions . 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 .
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 . SQS is a membrane-bound enzyme in
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 .
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 . CYP51s are the most conserved cytochrome P450 enzymes . 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
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 ; whereas in experiments with other parasite strains, no complete parasitological clearance was achieved . 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 . 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 . VT-1161, a 1-tetrazole-based drug undergoing phase II antifungal clinical trials, is active
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.
Another group of compounds that has been studied as cruzipain reversible inhibitors are those containing a nitrile head: purine nitriles , nitrile analogs of odanacatib [122, 123], and nonpeptidic nitriles . Salas-Sarduy et al.  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 . 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 , 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 .
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,
2.4 Nuclear and kinetoplast DNA
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 . 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 . Medicinal chemistry studies pointed to arylimidamides (AIAs) as the most promising antimicrobial diamidines . DB702, DB786, DB811, and DB889 presented anti-
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 . Recently, voacamine and an isobenzofuranone derivative induced important morphological alterations in different trypanosomatids, including
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 . 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 , 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.
In metacyclic trypomastigotes, Atwood and co-workers  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 . Plasma membrane subproteome pointed to a large repertoire of surface proteins in this parasite stage, including trans-sialidases, mucins, and GP63 protease . 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 . 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 . 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 . 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 . 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 .
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
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 . However, undesirable side effects and the resistance of some parasite strains , together with the limited efficacy in symptomatic chronic cases, drive the continuous search for novel chemotherapeutic agents . 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 .
High throughput screening complemented by whole-cell phenotypic assays represents the more feasible option in the search for novel anti-
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
In the current scenario, the CYP51 still represents one the most promising alternatives. Large-scale screening pointed to the high activity of CYP51 inhibitors
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 . However, no active inhibitors of this enzyme were described up to now . The hypothesis that
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 . In case of obligatory intracellular parasites as
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.