Identification of di-substituted ureas that prevent growth of trypanosomes through inhibition of translation initiation

Some 1,3-diarylureas and 1-((1,4-trans)−4-aryloxycyclohexyl)−3-arylureas (cHAUs) activate heme-regulated kinase causing protein synthesis inhibition via phosphorylation of the eukaryotic translation initiation factor 2 (eIF2) in mammalian cancer cells. To evaluate if these agents have potential to inhibit trypanosome multiplication by also affecting the phosphorylation of eIF2 alpha subunit (eIF2α), we tested 25 analogs of 1,3-diarylureas and cHAUs against Trypanosoma cruzi, the agent of Chagas disease. One of them (I-17) presented selectivity close to 10-fold against the insect replicative forms and also inhibited the multiplication of T. cruzi inside mammalian cells with an EC50 of 1–3 µM and a selectivity of 17-fold. I-17 also prevented replication of African trypanosomes (Trypanosoma brucei bloodstream and procyclic forms) at similar doses. It caused changes in the T. cruzi morphology, arrested parasite cell cycle in G1 phase, and promoted phosphorylation of eIF2α with a robust decrease in ribosome association with mRNA. The activity against T. brucei also implicates eIF2α phosphorylation, as replacement of WT-eIF2α with a non-phosphorylatable eIF2α, or knocking down eIF2 protein kinase-3 by RNAi increased resistance to I-17. Therefore, we demonstrate that eIF2α phosphorylation can be engaged to develop trypanosome-static agents in general, and particularly by interfering with activity of eIF2 kinases.

Parasitic diseases caused by protozoa such as Chagas disease and African Trypanosomiasis still lack effective, safe, and highly accessible chemotherapy [1][2][3][4] . The available drugs are toxic, the treatments show a high degree of abandon due to aggressive side effects 5 and resistance has been detected 6 . Currently, very few compounds are on clinical trials for Chagas disease and African Trypanosomiasis caused respectively by Trypanosoma cruzi and Trypanosoma brucei 7,8 .
Different approaches have been devised to identify new compounds to treat these diseases. Previous works have explored unique trypanosome biological processes to find new drug targets. Examples are studies targeting cysteine proteases, farnesyl pyrophosphatase synthase, glyceraldehyde 3-phosphate-dehydrogenase, 14α-demethylase and trans-sialidases [9][10][11] . The enzyme 14α-demethylase has gathered attention due to its essential role in biosynthesis of steroids. This enzyme is inhibited by azolic compounds originally developed as antifungal agents, which were subsequently repurposed for treatment of Chagas disease 12,13 . Although, the treatment with a 14α-demethylase inhibitor, called posaconazole, has shown promising results, this treatment did not eliminate the parasite in most cases, probably due to differences in parasite strains 14,15 and prompted combinatory therapies 16 . 1 A second approach has been to perform high-throughput screenings to identify new active compounds. For example, natural product libraries were used to identify new lead molecules against T. cruzi, T. brucei and Leishmania 17 . It is also noteworthy that a set of 1.8 million compounds from GlaxoSmithKline tested against these parasites revealed possibly new therapeutic molecules, including kinase inhibitors 18 .
Since drug exposure is an environmental challenge for parasites and considering that, for most cases, differences in strains susceptibility are also a problem 15 , global molecular responses to different stresses, such as the drug treatment, could be explored as a tool to improve the chemotherapy of parasitic diseases. A similar approach has been proposed for pathogenic fungi, such as Candida albicans 19 , where a genome-wide transcriptional factor has a dual role during stress fine-tuning responses to drug exposure and resistance. The phosphorylation of eIF2α has been recognized as the "integrated stress response hub" for various organisms, arresting general translation and promoting the accumulation of proteins and enzymes that cope with different types of damaging situations 20 . However, when the phosphorylation is sustained for long periods, apoptosis and cell death are induced 21 .
Urea derivatives, particularly those substituted by cyclic rings, provide significant opportunities for drug development and some of them have been previously shown to trigger eIF2α phosphorylation in mammalian cells [22][23][24] , a key event in the stress response and cell survival, especially for cancer cells chemotherapy 25 . Some urea derivatives were found to activate the heme-regulated kinase, one of the four mammalian kinases that phosphorylate eIF2α, arresting cancer cell proliferation 26 . In addition, other substituted ureas could inhibit several enzymes 27 , prevent cellular growth 28 and interfere with activation of intracellular complexes 29 .
Here, to exam the possible use of 1,3-di-substituted ureas in Trypanosomatids and to check whether they would also interfere with stress response and phosphorylation of eIF2α, we initially evaluated the activity of 25 urea derivatives against T. cruzi epimastigotes, which are the free proliferating form found in the midgut of the insect vector. Three compounds were found effective in preventing parasite growth and one of them, named I-17, showed a better selectivity index, controlling the intracellular replicative form at 17-fold lower concentrations than the detected toxicity to human host cells. Therefore, we further evaluate the biological effects of I-17 treatment and used T. brucei, which has more available genetic tools, to investigate how eIF2α phosphorylation and which eIF2α kinases were involved in growth inhibition by I-17.

T. cruzi epimastigotes replication was inhibited by 1,3-diarylureas and cHAUs. T. cruzi epimas-
tigotes were incubated in culture medium for three days with 25 different compounds at 10 µM. Seventeen inhibited parasite multiplication, while other 8 (3b, 3n, 3d, 3j, 3k, 3 l, 3t and NCPdCPU) were unable to produce any visible effect at 10 µM (data not shown). Therefore, we titrated the concentrations that caused 50% (EC 50 ) and 90% (EC 90 ) viability inhibition for the 17 more active compounds using an Alamar Blue assay, which is based on the capacity of viable cells to reduce resazurin to resofurin. Nine compounds (3p, 6a, I-17, 3e, 6b, I-18, 3 g, 3r and 3q) were most active against epimastigotes with EC 50 concentrations ranging from 1.3 to 3.7 ± 2.5 µM ( Table 1). The other eight presented EC 50 values higher than 5 µM. We also tested benznidazole (BZN), a reference compound for Chagas disease treatment, using the same Y strain and protocol to obtain an EC 50 of 211 ± 3 µM. This is remarkable since even the less active of the 17 1,3-di-substituted ureas presented at least 25-fold higher activity compared to BZN using a resistant strain, demonstrating the susceptibility of T. cruzi to these compounds. To further select useful compounds, we determined the selectivity index by measuring viability of monkey kidney cells (LLC-MK 2 ) for the most active compounds, also using Alamar Blue. As shown in Table I, the compounds  were usually toxic for mammalian cells, except 3p, 6a and I-17, which were at least 5-fold more effective in damaging parasites than host cells.

I-17 inhibited multiplication of different stages of T. brucei and T. cruzi parasites. As I-17 was
the best among the 25 tested compounds, with a selectivity index of 9.5-fold over LLC-MK 2 cells, we decided to further characterize its effects on different proliferative stages of T. cruzi and T. brucei. T. cruzi epimastigotes multiplication was diminished by 50% at ~3 µM I-17 (Fig. 1A), a value close to 3.4 µM, the EC 50 concentration obtained by the Alamar Blue assay (Table I). In contrast, minimal inhibition was observed for all tested concentrations of NCPdCPU, one inactive 1,3-diarylureas (EC 50 >10 µM). We also observed that up to 10 µM, I-17 could be washed out and the parasites would re-start to multiply (data not shown), indicating that it has a trypanostatic effect on epimastigotes. We also tested the effect of I-17 in cultures of T. brucei bloodstream form (BSF) that corresponds to the proliferative stage in mammalian host blood, and cultures that contained the procyclic form (PCF), the stage found in the gut of the insect vector. The multiplication of both forms was inhibited by I-17 but not by NCPdCPU (Fig. 1B). The inhibitory effect was more pronounced in PCF, starting to be detected at concentrations as low as 3 µM.
We observed that L6 rat myoblasts were also marginally affected after 72 hours incubation with up to 5 µM I-17, using a lactate dehydrogenase activity assay as a biomarker for cytotoxicity and cytolysis (Fig. 1C). Furthermore, we tested whether I-17 could also inhibit the proliferation of T. cruzi amastigotes in infected L6 cells. As shown in Fig. 1D, amastigote replication was strongly impaired when cells were treated with 3 µM I-17 for 72 h. These results indicated that I-17 prevented replication of different stages of the two Trypanosoma species.
To further validate the selectivity in T. cruzi intracellular forms, we infected U-2 OS cells plated on 384 well-microplates. The cells were treated with different concentrations of I-17 for 96 hours and the number of infected, non-infected cells and amastigotes was determined through high content analysis, which enables automated quantification of cells and parasites based on DNA staining (Fig. 1E). We found that I-17 caused a decrease in the numbers of T. cruzi amastigotes inside infected cells, with an EC 50 of 1.2 ± 0.5 µM (Fig. 1F). In the same cultures, the host cell cytotoxicity was evaluated by counting the total number of cells in treated wells relative to the number of cells in non-treated wells, showing that I-17 was mildly toxic to host cells (EC 50 = 21 ± 7.5 µM). I-17 changes trypanosomatids morphology and cell cycle. We observed aberrant cellular morphology of epimastigotes incubated with I-17. After 4 hours incubation with 3 µM of I-17, the parasites rounded up, as visualized by immunofluorescence for tubulin, which is mainly found in the parasite subpellicular array of microtubules ( Fig. 2A). This morphology was observed in 40% of the population treated with 3 µM I-17 and close to 90% in parasites incubated with 10 µM I-17 (Fig. 2B). Morphology changes also occurred in T. brucei PCF incubated with I-17 (data not show). Flow cytometry analysis indicated that I-17 treatment increased the percentage of T. cruzi epimastigote ( Fig. 2C and D) and T. brucei PCF ( Fig. 2E and F) population in the G1 cell cycle phase, suggesting an arrest of replication in both species.
I-17 induced eIF2α phosphorylation and protein synthesis arrest. Phosphorylation of eIF2α at serine 51 decreases protein synthesis in eukaryotic cells submitted to different types of stress 20 . This phosphorylation allows cells to translate preferentially proteins that act in stress remedial response 30 . We have previously found that T. cruzi epimastigotes phosphorylate eIF2α when submitted to nutritional stress 31 . The parasite eIF2α is phosphorylated in the threonine 169 (T169 [P] ), which corresponds to the serine 51 of most eukaryotes. Therefore, we tested whether eIF2α was phosphorylated when parasites were exposed to I-17. As shown in Fig. 3A, I-17 treatment at 10 µM for 4 hours caused an increased phosphorylation of eIF2α detected by an specific antibody to T169 [P] , in comparison to non-treated cells (NT), in a similar manner to nutritional stress caused by incubating parasites in a saline medium (TAU), mimicking the urine of the insect vector 32 . The increase of eIF2α phosphorylation was observed and quantified in three independent experiments, showing similar results (Fig. 3B). An increase in T169 [p] signal was also detected in PCF treated with I-17 for 24 hours (Fig. 3C).
These results suggest that I-17 could reduce general translation triggered by eIF2α phosphorylation. In fact, polysome profiling confirmed that less polysomes were present in cells treated with 10 µM I-17 in comparison with non-treated parasites (Fig. 3D). There was a large increase in RNA associated with the monosome (80 S) fractions compared to polysome fractions. The polysome to monosome ration (P/M) changed from 5.3 to 0.6 in    the parental strain (+/+) containing original alleles (Fig. 4A). This indicates that eIF2α phosphorylation is one, albeit possible not an exclusive, mediator of the anti-trypanosome inhibition induced by I-17.
The phosphorylation of eIF2α is regulated by four kinases in mammals 33 . Trypanosomatids genome contains three eIF2α kinases; K1, K2 and K3 34 . To identify which of these kinases could be involved in eIF2α phosphorylation by I-17, we generated knockdown lineages of PCF by RNAi for these three T. brucei kinases. We were able to obtain PCF lineages showing reduction of TbK1 and TbK3 RNA levels as seen by RT-PCR after induction of RNAi with tetracycline for 4 days (Fig. 4B). We could not obtain a conclusive knockdown of TbK2 (data not shown). As control, we found no changes in mRNA levels of enoyl CoA, a non-related enzyme. TbK1 and TbK3 knockdown lineages showed similar multiplication until 3 days after knockdown induction, but from the third to the fourth day of induction, TbK3 knockdowns replicated slightly less (Fig. 4C). Therefore, TbK1 and TbK3 lineages were tetracycline-induced for 2 or for 4 days previously to the treatment with 3 µM of I-17 for two more days. The different effect of I-17 was noticed by expressing the relative number of treated divided by non-treated cells (relative cell number), for TbK1 and TbK3 RNAi lineages induced (Ind) or not-induced (NI) (Fig. 4D). We observed that the relative cell number was 30% higher 2 or 4 days after induction of the TbK3 knockdown compared to the same lineage without RNAi induction. In contrast, TbK1 knockdown induction did not interfere with the inhibition of parasite multiplication caused by I-17. With these data, we demonstrated that, at least partially, TbK3 has a role on inducing inhibition of multiplication in T. brucei treated with I-17.

Discussion
In this work, we tested 1,3-di-substituted urea analogs designed and synthesized to activate mammalian heme regulated inhibitor and found that among 25 different compounds, I-17, 3p and 6a inhibited T. cruzi epimastigotes proliferation (EC 50 below 5 µM) with toxicity lower than mammalian cells. Compound I-17 also inhibited the intracellular replication of T. cruzi and the proliferative stages of T. brucei, BSF and PCF, with similar EC 50 values. The selectivity to inhibit intracellular multiplication of T. cruzi was around 17-fold higher than host cell toxicity and occurred simultaneously with reversible parasitic cell cycle arrest and inhibition of translation, at least partially, in an eIF2α phosphorylation-dependent manner.  As these compounds were shown to present inhibitory activity against melanoma cells 26 , they could be targeting relevant molecules that would allow growth and resistance of transformed cancerous cells as well as parasites. Interestingly, cancer cells have to deal with oxidative stresses 35 , a capacity also critical for T. cruzi virulence 36 . It is noticeable that changes on the 4-aryloxycyclohexyl moiety bearing an N-(3-trifluoromethoxy) phenyl ring, designed in 3p, 6a, 3p, 6b di-substituted ureas showed better results against epimastigotes but at the cost of a lower selectivity relative to mammalian cells. I-17 was effective at concentration slightly smaller for intracellular amastigotes (1.2 ± 0.5 µM) compared to epimastigotes (3.4 ± 1.3 µM) and the host cell toxicity was variable among different cell lines and employed methodology, resulting in a selectivity index ranging from 17 to 9.5. Despite this relatively moderate selectivity, the action of I-17 could serve to probe the role of eIF2α in controlling T. cruzi infection.
Observations that I-17 promotes formation of enlarged epimastigotes with increasing numbers of cells in G1 phase could indicate that I-17 triggered a blockage in the DNA replication (S phase of cell cycle), arresting T. cruzi proliferation. Inhibition of replication and a shift in the percentage of G1:G2 cell cycle phases ratio also occurred in a similar way for PCF of T. brucei. Nevertheless, this is a well-known phenotype generated by several gene knockdowns obtained by RNAi in T. brucei, some of them affecting cell cycle 37 . Therefore, it is possible that changes in cell shape and cell cycle might have arisen as consequence of a general trypanosome stress response [38][39][40] .
We found that I-17 induced phosphorylation of T. cruzi and T. brucei eIF2α at threonine 169 residue (T169), which is functionally homologous to the Ser51 of eIF2α in mammalian and yeast cells. On those organisms, it is relatively well described how Ser51 eIF2α phosphorylation is responsible for delaying binding of methionine initiator tRNA to ribosomes and regulating protein synthesis during stressful conditions 41 . The T169 phosphorylation signal increased in both T. cruzi and T. brucei with time and was associated with decreased levels of polysomes, correlating growth inhibition with protein translation decrease. Consistently, T. brucei lineages containing eIF2α with T169 mutated to non-phosphorylatable alanine (T169A/-) 42 were relatively more resistant to I-17 than control cells containing threonine, a phosphorylatable residue. There is a formal possibility that phosphorylation of eIF2α was consequence of stress induced by the compound rather than activation of parasite eIF2α kinase, as described for mammalian cells 21 . For example, a modified urea derivative was shown to interact with mammalian CXCR2 receptor preventing signaling 43 , and fluorinated 1,3-diarylureas were found to activate AMP-activated protein kinase, inhibiting cell-cycle progression and proliferation in colorectal and stem cells cancers 44 . This, however, is unlikely to occur in T. cruzi as eight closely related analogs (3b, 3n, 3d, 3j, 3k, 3l, 3t and NCPdCPU) which failed to activate mammalian eIF2α kinases also have no activity against parasites 23,26 .
We noticed that T. brucei BSF T169A/− was only partially resistant to I-17. This partial increase in resistance, i.e. cell numbers in presence of I-17 was closer but not equal to the number of cells from untreated cultures, implicates that I-17 may be interfering with other molecular targets mechanisms that affect T. brucei and possibly T. cruzi proliferation and/or survival. This partial protection in the non-phosphorylatable lineage (T169A/−) could also be explained if parasite cells required some level of eIF2α phosphorylation for stress protection triggered by I-17. Alternatively, T169A/− eIF2α lineage could have a different set of expressed proteins, as consequence of different translational regulation. Nevertheless, parasite survival could additionally be affected by preventing eIF2α phosphorylation, which is known to affect fate decisions in cancerous cells 21 .
Three eIF2α kinases homologues have been described in Trypanosomatids 34,45-47 . Here, we further demonstrated that RNAi knockdown of T. brucei eIF2α kinase 3 (TbK3) 33 , increased resistance of PCF to I-17. This suggests a role for TbK3 kinase as responsible for eIF2α phosphorylation triggered due to I-17 treatment, at least in T. brucei. TbK3 has a conserved eIF2α kinase domain 34 and it was shown to be essential in regulating spliced leader expression by affecting the transcription factor TRF4 after endoplasmic reticulum stress 47 . Whether TbK3 can directly phosphorylate eIF2α or targets TRF4 remains to be demonstrated for both trypanosomes, as the linkage between these pathways is still obscure. Furthermore, we could speculate that the presence of I-17 could activate TbK3, directly or indirectly, inducing eIF2α phosphorylation and causing translation arrest, further preventing spliced leader transcription. This would cause a general growth arrest since spliced leader is added to most of the pre-mRNA messengers to allow nuclear export and translation 48 . This type of response was described as an unfolded protein response-like that upon persistence could lead to annexin binding to the parasite and DNA fragmentation, independently of caspase activations 49 , further leading to cell death. Depletion of TbK1, a homologous of GCN2 kinase involved in recognizing amino acid starvation on different organisms, including Leishmania parasites 46 , did not change the resistance to I-17. We were not able to obtain knockdowns of TbK2, the other eIF2α kinase of T. brucei, so its role in I-17 could not be evaluated.
It is relevant that some 1,3-diarylureas have been tested with some success against flatworm parasites such as Schistosoma japonicum 50 and Schistosoma mansoni 51 , although no targets were identified in those cases. Also, a family of 1,3-diarylureas with a 4-aminoquinaldinyl as one of the aryl rings was shown to be active against chloroquine sensitive and resistant strains of Plasmodium falciparum 52 , the causative agent of malaria. 1,3-diarylureas have two available cyclic rings that can be readily substituted by various functional groups, allowing the synthesis of several improved compounds 53 . The structure of I-17 could be used to design Trypanosoma kinases activators capable of interfering with eIF2α stress responses, potentially useful in combinatory therapies with current drugs, such as benznidazole and nifurtimox, known to be potent oxidative stress inducers 8,54 .
using both TbK1 and TbK3 lineages non-induced with tetracycline (NI, empty bars) or after RNAi induction (Ind, grey bars). I-17 treatment was maintained for 48 hours and initiated 2 or 4 days after tetracycline addition. The values in (D) are means ± standard deviation of triplicate and independent experiments. *Indicates p < 0.05 calculated using the Student's t-test.

Conclusion
An 1,3-di-substituted urea (I-17) is cytostatic to trypanosomatids partially by promoting phosphorylation of eIF2α which causes protein synthesis arrest. Parasites lacking a phosphorylatable eIF2α site or with reduced levels of eIF2α-kinase 3 were more resistant to treatment. The compound I-17 also generates an aberrant cell morphology and cell cycle arrest. These findings indicate that further exploring the stress response through eIF2α regulation could help provide basis to obtain more selective compounds against Trypanosoma parasites compared to mammalian cells, especially through quantitative structure activity relationship studies, for example by elucidating the atomic structure of parasite eIF2α kinases. Parasites and cell cultures. The bloodstream form (BSF) of T. brucei Lister 427 strain was cultivated in HMI-9 medium in the presence of 10% fetal bovine serum (FBS) and Serum Plus (SAFC Biosciences) containing 59 mg/mL penicillin and 133 mg/mL streptomycin at 37 °C in a CO 2 incubator 55 . We used the T. brucei EATRO 1125 parental lineage (+/+), or derivatives in which one of the alleles of endogenous gene coding for eIF2α was replaced by blasticidin resistance coding sequence and the other allele by either the wild type eIF2α gene (T169T/−), or a mutated version in which threonine 169 was replaced by alanine (T169A/−) in a cassette containing the aminoglycoside 3′-phosphotransferase coding sequence that allow resistance to Geneticin G418 56 . These strains were maintained as described above by replacing the FBS by rabbit serum (Sigma-Aldrich) and the modified lineages were growth in the presence of 2.5 µg/mL Geneticin-G418 and 5 µg/mL blasticidin (both from Thermo Fisher Fisher). The procyclic form (PCF) of T. brucei Lister 427 and its derivative 29-13 57 were cultivated at 28 °C in SDM-79 medium 58 supplemented with 10% FBS and 7.5 µg/mL hemin. For 29-13 PCF, 50 µg/mL hygromycin B and 15 µg/mL Geneticin-G418 were also added. BSF and PCF were diluted to 2 × 10 5 /mL and incubated in medium containing the indicated concentrations of compounds previously diluted in DMSO. Epimastigotes of T. cruzi (Y strain) were cultivated at 28 °C in autoclaved liver infusion tryptose (LIT) medium supplemented with 1 µg/mL hemin, 10% FBS, 4 mg/mL glucose, 59 mg/mL penicillin and 133 mg/mL streptomycin 59 . The epimastigotes were diluted to 2 × 10 6 /mL and incubated with the indicated concentrations of compounds. Multiplication of T. brucei and T. cruzi were assessed by counting the number of parasites after 24, 48 and 72 hours respectively, using hemocytometer in three independent experiments.

Material and Methods
Tissue culture derived T. cruzi trypomastigotes (TCT) were obtained from supernatants of infected LLC-MK 2 (Rhesus monkey kidney epithelial cells, ATCC CCL-7) cultivated in low glucose Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific) supplemented with 3.7 g/L sodium bicarbonate, 10% FBS, 59 mg/mL penicillin and 133 mg/mL streptomycin at 37 °C in a CO 2 incubator. The parasites were collected by centrifugation at 1500 g for 5 min, incubated for at least 60 min at 37 °C and the TCT enriched supernatants were collected to be used for new infections. LLC-MK 2 , L6 rat myoblasts (ATCC CRL-1458) and U-2 OS (Banco de Células do Rio de Janeiro, Brazil) maintained in the same medium were used for evaluating the progression of T. cruzi infection. The cells (1 × 10 5 ) were seeded onto 13 mm glass coverslips in 24 well plates and incubated at 37 °C. The next day, 5 × 10 5 TCT in 500 µL were added to each well and incubations proceeded for 2 hours. Wells were washed twice with PBS and incubation continued in the presence of culture medium with the indicated amounts of each compound. After 72 hours, the cells were fixed in Bouin solution (acetic acid 5%, formaldehyde 9% and picric acid 0.9%) for 5 min and washed four times with PBS before staining for 1 hour with Giemsa solution (previously diluted 1:10 in tap water and filtered). Glass coverslips were dehydrated in acetone, acetone with xylol, and only xylol before mounted in glass slides covered with Entellan mounting medium (Sigma-Aldrich). Coverslips were visualized under an optical microscope and the number of intracellular amastigotes assessed in one hundred infected cells.
Parasite viability assays and EC 50 determination. T. cruzi epimastigotes at 1 × 10 7 /mL in 50 µL were incubated in 96-wells plates with the same volume of compounds pre-diluted in 50 µL of LIT medium for 48 hours at 28 °C. LLC-MK 2 cells (3 × 10 3 ) in 100 µL of medium were added to 96 well plates. After 5 hours, the medium was replaced by fresh medium containing serial dilutions of test compounds and incubations proceeded for 72 hours at 37 °C. In both cases, 10 µL of AlamarBlue (Thermo Fisher Scientific) was added and the plates were further incubated for 18 hours at 28 °C for epimastigotes and 4 hours at 37 °C for mammalian cells. Absorbance at 570 nm and 600 nm were measured by using a SpectraMax M3 (Molecular Devices). The differences between absorbance at 570 nm and 600 nm for each well was normalized against a blank value for medium without parasites and lysed cells. Alternatively, L6 rat myoblast cell death and lysis were determined using the cytotoxicity Detection Kit Plus LDH Version 6 (Roche) after 72 hours incubation with the indicated compounds using the manufacturer protocol. Data was analyzed using GraphPad Prism 6.01 software with logarithmic nonlinear regression to generate a response curve fitting and determine the effective concentration that affects 50% of cells (EC 50  with exceptions appointed on Table 1. Selective index was estimated by the ratio between LLC-MK 2 EC 50 and epimastigotes EC 50 on Table 1.

High content analysis of T. cruzi infection.
To evaluate resistance to I-17 during intracellular amastigote replication, we used a system of high content imaging analysis that allowed processing of thousands of cells to robustly evaluate inhibitory effects on intracellular T. cruzi multiplication simultaneously to evaluation of host cell toxicity. Stock solution of I-17 was diluted to 5.7 mM in DMSO. Before use, the compound was transferred onto 384-well polypropylene stock plates (Greiner Bio-One) and further diluted into 10-point dose response curve (2-fold serial dilution) in neat DMSO. These stock plates were kept frozen at −20 °C and protected from light.
U-2 OS cells were plated at 800 cells per well in 40 μL of medium and incubated for 24 hours at 37 °C. The cells were then infected with 10 μL of trypomastigotes suspension at 1.5 × 10 6 /mL, harvested from the supernatant of LLC-MK 2 cultures. The next day, serially diluted I-17 solutions were transferred into intermediate plate containing culture medium in order to decrease DMSO and I-17 highest concentration to 6% (v/v) and 342 µM, respectively. Finally, ten microliters of compound solution were transferred onto assay plates, yielding a final concentration of 1% DMSO (v/v) in a final volume of 60 μL, starting from 57 µM of I-17. DMSO-treated wells containing non-infected and infected cells were used as positive and negative controls, respectively. After 4 days at 37 °C, the cells were fixed with 4% paraformaldehyde diluted in PBS (PFA), stained with Draq5 (Biostatus) for 30 minutes at room temperature in the dark, and six images per well, totalizing more than a thousand cells, were acquired using the high content imaging system INCell 2200 (General Electric) at 20× agnification. Images were analyzed by using Investigator, version 1.6 (General Electric) for identification, segmentation and quantification of host cell nucleus, cytoplasm and intracellular parasites. The analysis provided output data for all images as the total number of cells, total number of infected cells and the total number of intracellular parasites. The infection ratio was obtained by considering the number of infected cells relative to the total number of cells and was normalized to the infections obtained in the presence of 1% DMSO (negative control) and non-infected cells to calculate the normalized antiparasitic activity, according to the following formula: Normalized Activity Immunofluorescence. Exponentially growing T. cruzi epimastigotes were diluted in medium to 8 × 10 6 / mL and incubated with the indicated compounds. At the end of treatments, cells were collected by centrifugation at 2000 g for 2 min, washed and resuspended in PBS before addition to glass slides (Tekdon Inc.) previously coated with 0.01% poly-L-lysine (Sigma-Aldrich) for 20 min. After 5 min, the unattached parasites were removed, and the slides were incubated with PFA, washed with PBS and permeabilized with 0.1% Triton X-100 in PBS for 5 min. The slides were then incubated 20 min with 1% BSA in PBS, followed by 1 hour incubation with a mouse antibody against β-tubulin C-terminal peptide 45 diluted 1:1000 in blocking solution. The slides were washed with PBS, incubated 1 hour in the dark with anti-rabbit IgG antibody coupled to Alexa Fluor 488 (Thermo Fisher Scientific) at 1:1000 in PBS and 10 µg/mL 4′,6-diamidino-2-phenylindole (DAPI). Slides were washed twice with PBS and mounted with Prolong Gold Antifade Reagent (Thermo Fisher Scientific). Images were acquired using a Hamamatsu Orca R2 CCD camera coupled to an Olympus BX-61 microscope equipped with an x100 plan Apo-oil objective (NA 1.4). AutoQuant X2.2 software (Media Cybernetics) was used to perform blind deconvolution.
Cell cycle analysis. After the indicated treatments, parasites were collected by centrifugation at 2000 g for 2 min, washed in PBS, fixed with 50% cold methanol for 10 min and washed in PBS again. To eliminate RNA background staining, the cells were incubated with RNAse (10 µg/mL) for 30 min at 37 °C, washed once more with PBS and diluted in 1 mL of PBS containing 10 µg of propidium iodide for 5 min. Parasites were quickly washed twice with PBS and analyzed in a BD Accuri C6 (BD Sciences) flow cytometry using FL-2 channel for fluorescence detection.
Immunoblotting. Parasites treated as indicated were collected by centrifugation at 2000 g for 5 min and washed once in TBS (50 mM Tris-HCl pH 7.5, 150 mM NaCl) before addition of Laemmli buffer and heat denaturation at 95 °C for 5 min. Electrophoresis in polyacrylamide gels containing SDS and transfer to nitrocellulose membranes were performed by standard procedures. The membranes were stained with 0.5% Ponceau S in 3% acetic acid and cut separating the portion containing proteins with mass above and below 60 and 40 kDa. All membranes pieces were blocked with TBS containing 0.1% Tween 20 (TBS-T) and 5% BSA overnight. Membrane pieces from 40 kDa to 60 kDa were incubated for 3 hours with affinity purified antibodies reacting with the phosphorylated T169 (T169 [P] ) of T. cruzi eIF2α 31 diluted 1:125 in blocking buffer. The primary antibody was washed 3 times in TBS-T for 10 min and incubated for 1 hour with anti-rabbit IgG peroxidase-conjugated antibody (Thermo Fisher Scientific) diluted 1:20000 in TBS. Membranes were washed three times in TBS-T for 10 min and bound antibodies detected by ECL (EMD Millipore) by using an Odyssey Fc System (LI-COR Biosciences). The same membrane pieces were re-probed with an antiserum (anti-TceIF2α) obtained by immunization of mice with a recombinant T. cruzi eIF2α expressed in Escherichia coli 31  5% non-fat dry milk and after 1 hour incubation the membranes were washed three times with TBS-T followed by 1 hour incubation with anti-mouse IgG IRDye 800 (LI-COR Biosciences) 1:10000 in PBS. Membranes were washed three times with TBS-T and bound antibodies detected as described above. The nitrocellulose pieces from 60 kDa to the membrane upper edge were incubated with rabbit serum anti-HSP70 60 diluted 1:10000 in blocking buffer for 1 h, washed three times with TBS-T and incubated with secondary anti-rabbit IgG IRDye 680 (LI-COR Biosciences) 1:10000 on TBS for 1 hour before another three washes in TBS-T and detection with Odyssey Fc System. Polysome profiling. At least 1 × 10 9 of exponentially growing parasites were treated with the indicated concentrations of inhibitors. After 24 h, cycloheximide was added to 100 µg/mL and the cells maintained at 28 °C for 10 min before further incubation on ice for 10 min, centrifuged 5 min at 2000 g at 4 °C, washed in ice-cold PBS containing the same concentration of cycloheximide and diluted in 300 µL of ice-cold Buffer A (10 mM Tris-HCl, pH. 7.4, 300 mM KCl, 10 mM MgCl 2 , 1 mM dithiothreitol and 100 µg/mL cycloheximide). A small drop of Triton X-100 was added on the lateral side of tube to reach 1% (v/v) and the tubes were repeatedly mixed by inversion for about 5 min. The lysates were centrifuged for 3 min at 5000 g at 4 °C and the supernatant collected and added to a new tube. Stocks of heparin at 100 mg/mL and NaCl at 5 M were added to 1 mg/mL and 150 mM respectively. The corresponding volume of the samples containing the equivalent of ten absorbance units at 260 nm were then loaded on the top of a 7% to 47% sucrose gradient in Buffer A prepared using a Gradient Master (Biocomp). The tubes were centrifuged at 200,000 g for 2.5 hours in a Beckman SW41 rotor at 4 °C. The gradient was collected from the top by injecting in the bottom a continuous flow of 60% sucrose at 1 mL/min using an Econo Gradient Pump (Bio-Rad) and the fractions monitored by reading the absorbance at 254 nm.

RT-PCR.
Total RNA was extracted from the parasites using Trizol (Thermo Fisher Scientific) and treated with 2 U of DNAse I per µL for 30 min at 37 °C. The enzyme was inactivated by 10 min incubation at 75 °C and 10 µg of the denatured RNA was incubated with 1 µM of oligo-dT, cooled on ice, and then incubated for 50 min at 50 °C with Superscript III reverse transcriptase (Thermo Fisher Scientific) in the presence of the recommend buffer, 5 mM dNTP, 2 mM DTT and 40 U RNase out (Thermo Fisher Scientific) in a final volume of 50 µL. The enzyme was inactivated at 70 °C for 15 min and the produced cDNA treated with RNAse H (2U) at 37 °C for 20 min. A PCR reaction was performed using 25 cycles at 62 °C for annealing using the following pair of oligonucleotides: TbK1Fq (5′-TCCGGAGAGTGTCAGCGGGT)/TbK1Rq (5′-TGTGGCTGAGAGCGGCCAAAC), and TbK3Fq (5′-GGGGTGAGTGGGAAAGGACGTG)/TbK3Rq 5′-GCCGAGGCACCGAAAGTCCC) to amplify the residues 788 to 985 of TbK1Fq and residues 424 to 619 of TbK3, respectively. Amplification of enoyl-CoA, a non-related protein of T. brucei 62 , was performed as a control using Enoyl_CR3_F (5′CGCATATGTTACGCTCCTGTGCACTTTTA) and Enoyl-CR3_R (5′CGCTCGAGCTACGAATTGGTGAAGAACGGTTT).