Acrylonitrile Derivatives against Trypanosoma cruzi: In Vitro Activity and Programmed Cell Death Study

The neglected infection known as Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, results in more than 7000 deaths per year, with an increasing number of cases in non-endemic areas such as Europe or the United States. Moreover, with the current available therapy, only two compounds which are active against the acute phase of the disease are readily available. In addition, these therapeutic agents display multiple undesired side effects such as high toxicity, they are expensive, the treatment is lengthy and the resistant strain has emerged. Therefore, there is a need to find new compounds against Chagas disease which should be active against the parasite but also cause low toxicity to the patients. In the present work, the activity of novel acrylonitriles against Trypanosoma cruzi was evaluated as well as the analysis of the physiological events induced in the treated parasites related to the cell death process. Hence, the characteristic features of an apoptosis-like process such as chromatin condensation and mitochondrial membrane potential, among others, were studied. From the 32 compounds tested against the epimastigote stage of T. cruzi, 11 were selected based on their selectivity index to determine if these compounds were able to induce programmed cell death (PCD) in the treated parasites. Furthermore, acrylonitriles Q5, Q7, Q19, Q27 and Q29 were shown to trigger physiological events related in the PCD. Therefore, this study highlights the therapeutic potential of acrylonitriles as novel trypanocidal agents.


Introduction
Chagas disease also known as American trypanosomiasis is a neglected infection caused by the protozoan parasite Trypanosoma cruzi, This disease is endemic to 21 Central and South American countries, affecting around 7 million infected people worldwide and causing more than 7000 deaths per year [1,2]. In addition, the number of reported cases in non-endemic areas has increased in recent years, reaching more than 100,000 persons in Europe and over 300,000 in the United States [3]. This increase is due to migration To assess the selectivity of these compounds, their cytotoxicity was measured in a J774A.1 murine macrophage cell line, obtaining CC 50 values, shown in Table 2. The selectivity index (SI) was calculated as the ratio of CC 50 value for J774A.1 and the IC 50 value for epimastigotes of T. cruzi. The results are represented in Table 3.

Chromatin Condensation Analysis
The Vybran ® kit uses two reagents: one of them is Hoechst 33342, which shows blue fluorescence when the chromatin is condensed; the other reagent is propidium iodide, which shows red fluorescence when the parasite is dead. Figure 1 shows an intense blue fluorescence in the nucleus, indicating chromatin condensation; the images correspond to parasites treated with acrylonitriles Q5, Q7, Q19 and Q29. Some of the parasites showed red fluorescence, indicating already dead cells, which correspond with the parasites treated with acrylonitriles Q5, Q7, Q18, Q19, Q27 and Q29.

Chromatin Condensation Analysis
The Vybran ® kit uses two reagents: one of them is Hoechst 33342, which shows blue fluorescence when the chromatin is condensed; the other reagent is propidium iodide, which shows red fluorescence when the parasite is dead. Figure 1 shows an intense blue fluorescence in the nucleus, indicating chromatin condensation; the images correspond to parasites treated with acrylonitriles Q5, Q7, Q19 and Q29. Some of the parasites showed red fluorescence, indicating already dead cells, which correspond with the parasites treated with acrylonitriles Q5, Q7, Q18, Q19, Q27 and Q29.

Mitochondrial Membrane Potential Analysis
The results of the fluorescence obtained with the JC-1 ® reagent are shown in Figure 2. These results are expressed in percentage of variations in the mitochondrial membrane potential relative to the negative control, without treatment.

Mitochondrial Membrane Potential Analysis
The results of the fluorescence obtained with the JC-1 ® reagent ar 2. These results are expressed in percentage of variations in the mitoch potential relative to the negative control, without treatment. The results showed that all the compounds, except Q29, presented tions in the mitochondrial membrane potential. These variations are h Q3, Q7, Q18 and Q27, which have a similar effect decreasing the inter-m of the mitochondria as the reference drug, benznidazole.

ATP Level Analysis
The results of the luminescence obtained with CellTiter-Glo ® rea Figure 3. These results are expressed in percentage of production of A The results showed that all the compounds, except Q29, presented significant variations in the mitochondrial membrane potential. These variations are highly significant in Q3, Q7, Q18 and Q27, which have a similar effect decreasing the inter-membrane potential of the mitochondria as the reference drug, benznidazole.

ATP Level Analysis
The results of the luminescence obtained with CellTiter-Glo ® reagent are shown in Figure 3. These results are expressed in percentage of production of ATP relative to the negative control, without treatment.
The results show that all of the tested acrylonitriles, except Q1 and Q18, have significant variations in the ATP levels of the parasites, demonstrating that Q27 and Q29 decrease the ATP levels under the 1%, continuing with Q7 and Q19, which have a percentage of

Plasmatic Membrane Permeability Analysis
The use of the dye SYTOX ® Green reagent means that when the plasmatic permeability is altered, it is possible for the reagent to go inside the cells, joining the parasitic DNA and emitting an intense green fluorescence.
The images reflect that almost all acrylonitriles altered the plasma membrane permeability, demonstrated by the intense green fluorescence, and compared to the negative control which does not present this event. This intensity is more remarkable for compounds Q5, Q7, Q19, Q27 and Q29 as shown in Figure 4

Reactive Oxygen Species Analysis
The CellROX ® reagent emits red fluorescence in the presence of reactive oxygen species. The pictures show that the most intense red fluorescence corresponds with the parasites treated with acrylonitrile Q3, while less intense fluorescence is shown in parasites treated with acrylonitriles Q5, Q7, Q8, Q18, Q27 and Q29 as shown in Figure 5. , x FOR PEER REVIEW entage of ATP level relative to untreated control. Benz: benznidazole. A Tukey te M ® 7.0a soft-ware test was carried out to test the statistical differences between means. (p < .0001 [****]).

Plasmatic Membrane Permeability Analysis
The use of the dye SYTOX ® Green reagent means that when the pla

Reactive Oxygen Species Analysis
The CellROX ® reagent emits red fluorescence in the presence of reactive oxygen species. The pictures show that the most intense red fluorescence corresponds with the parasites treated with acrylonitrile Q3, while less intense fluorescence is shown in parasites treated with acrylonitriles Q5, Q7, Q8, Q18, Q27 and Q29 as shown in Figure 5.

Discussion
A limited group of compounds such as butanolides, butyrolactones, thiocarbazones, chalcones, and hydrazine derivatives, have recently been reported to present activity against Trypanosma cruzi, showing IC 50 values ranging from 10.09 ± 1.5 to 590.96 ± 34.96 µM against epimastigotes [31][32][33]. Although it has been difficult to find molecules with IC 50 values lower than 10 µM in this type of study, in the present work we obtained five compounds that achieved this goal.
From the 32 compounds evaluated in this work, 25 of them were active against the epimastigote stage of Trypanosoma cruzi. As shown in Table 1, the derivatives Q8, Q11, Q16, Q21 and Q32 were the most active compounds when compared to the reference drug, benznidazole, presenting IC 50 values ranging from 3.73 ± 0.41 to 7.65 ± 1.51 µM.
The results of the present work showed that some of these compounds exhibited certain toxic effects [34,35]. However, their strong trypanocidal activities, represented by low IC 50 values, translate into acceptable selectivity indexes, and, hence, low toxicity at the concentrations needed to eliminate the parasite. For this reason, it would be necessary to mitigate against their side effects with methodologies focusing on reducing the cytotoxicity of these compounds. These methods could include the combination of these acrylonitrile derivatives with protective drugs that present the capacity of protection against the cytotoxic damage of the acrylonitriles [36,37] or even the use of nanoparticles to reduce the cytotoxic effects of acrylonitriles [38].
Regarding the present study, acrylonitriles Q2, Q25, Q27, Q28 and Q29 were the less toxic ones, as shown in Table 2. The compounds with the optimal ratio values of cytotoxicity/activity were Q1, Q2, Q3, Q5, Q7, Q8, Q18, Q19, Q25, Q27 and Q29. Hence, the compounds with better SI values were chosen to further evaluate the induction of cell death in treated parasites.
Different assays were performed to elucidate which pathways could be triggered by these acrylonitriles. The most probable cellular process induced by our compounds is the apoptosis or programmed cell death (PCD), in which the inflammatory response is weakly activated, in contrast to a necrotic event, where the inflammatory response is strong [39]. Acrylonitriles Q5, Q7, Q19, Q27 and Q29 presented robust evidence of inducing all the physiological features of an apoptosis-like process that probably starts with a ROS accumulation, which activates the loss of mitochondrial membrane potential, and, hence, the collapse of the ATP levels, also corroborated by the chromatin condensation. On the contrary, acrylonitriles Q1, Q2, Q3 and Q25 showed good trypanocidal activity, but they did not show evidence of causing a programmed cell death mechanism [40][41][42].
Comparing the molecular structure of the acrylonitriles and their activity, we could observe different behaviors. On one hand, the acrylonitriles that have a good selectivity index are mostly E isomers, and only two of them are Z isomers. The isomers E and Z of methyl-3-cyanoacrylate (Q1 and Q2, respectively) presented a similar selectivity index, and both did not show enough evidence of induction of programmed cell death mechanisms in the parasite. However, a different phenomenon occurred when isomers E and Z of phenyl-3-cyanoacrylate (Q7 and Q8, respectively) were checked, since both types of molecules were shown to induce an apoptosis-like cell death in the treated parasites. However, the selectivity index of the Z isomer is more than four times higher than the one for the E isomer.
Interestingly, the acrylonitriles with an amide group (Q23-Q32), presented the highest values of IC 50 , representing the less active compounds of the study. In addition, the acrylonitriles with a ketone group (Q10-Q17) seemed to show the highest cytotoxic profile compared to other groups. In addition, the most selective compounds presented ester or phosphorous substituents on the acrylonitrile derivative.

Compounds
The compounds tested in this work (Table 4) were synthesized by our group, as previously described [30]. They were all dissolved in dimethyl sulfoxide (DMSO) (Merck, Darmstadt, Germany) and stored at −20 • C in the dark.

Compounds
The compounds tested in this work (Table 4) were synthesized by our group, as previously described [30]. They were all dissolved in dimethyl sulfoxide (DMSO) (Merck, Darmstadt, Germany) and stored at −20 °C in the dark.

ID
Compound

Compounds
The compounds tested in this work (Table 4) were synthesized by our group, as previously described [30]. They were all dissolved in dimethyl sulfoxide (DMSO) (Merck, Darmstadt, Germany) and stored at −20 °C in the dark.

ID
Compound

Compounds
The compounds tested in this work (Table 4) were synthesized by our group, as previously described [30]. They were all dissolved in dimethyl sulfoxide (DMSO) (Merck, Darmstadt, Germany) and stored at −20 °C in the dark.

ID
Compound

Compounds
The compounds tested in this work (Table 4) were synthesized by our group, as previously described [30]. They were all dissolved in dimethyl sulfoxide (DMSO) (Merck, Darmstadt, Germany) and stored at −20 °C in the dark.

ID
Compound

Compounds
The compounds tested in this work (Table 4) were synthesized by our group, as previously described [30]. They were all dissolved in dimethyl sulfoxide (DMSO) (Merck, Darmstadt, Germany) and stored at −20 °C in the dark.

ID
Compound

Compounds
The compounds tested in this work (Table 4) were synthesized by our group, as previously described [30]. They were all dissolved in dimethyl sulfoxide (DMSO) (Merck, Darmstadt, Germany) and stored at −20 °C in the dark.

ID
Compound

Compounds
The compounds tested in this work (Table 4) were synthesized by our group, as previously described [30]. They were all dissolved in dimethyl sulfoxide (DMSO) (Merck, Darmstadt, Germany) and stored at −20 °C in the dark.

ID
Compound

Compounds
The compounds tested in this work (Table 4) were synthesized by our group, as previously described [30]. They were all dissolved in dimethyl sulfoxide (DMSO) (Merck, Darmstadt, Germany) and stored at −20 °C in the dark.

ID
Compound

Compounds
The compounds tested in this work (Table 4) were synthesized by our group, as previously described [30]. They were all dissolved in dimethyl sulfoxide (DMSO) (Merck, Darmstadt, Germany) and stored at −20 °C in the dark.

ID
Compound

Compounds
The compounds tested in this work (Table 4) were synthesized by our group, as previously described [30]. They were all dissolved in dimethyl sulfoxide (DMSO) (Merck, Darmstadt, Germany) and stored at −20 °C in the dark.

ID
Compound

Compounds
The compounds tested in this work (Table 4) were synthesized by our group, as previously described [30]. They were all dissolved in dimethyl sulfoxide (DMSO) (Merck, Darmstadt, Germany) and stored at −20 °C in the dark.

ID
Compound

Compounds
The compounds tested in this work (Table 4) were synthesized by our group, as previously described [30]. They were all dissolved in dimethyl sulfoxide (DMSO) (Merck, Darmstadt, Germany) and stored at −20 °C in the dark.

ID
Compound

Compounds
The compounds tested in this work (Table 4) were synthesized by our group, as previously described [30]. They were all dissolved in dimethyl sulfoxide (DMSO) (Merck, Darmstadt, Germany) and stored at −20 °C in the dark.

ID
Compound

Compounds
The compounds tested in this work (Table 4) were synthesized by our group, as previously described [30]. They were all dissolved in dimethyl sulfoxide (DMSO) (Merck, Darmstadt, Germany) and stored at −20 °C in the dark.

ID
Compound

Compounds
The compounds tested in this work (Table 4) were synthesized by our group, as previously described [30]. They were all dissolved in dimethyl sulfoxide (DMSO) (Merck, Darmstadt, Germany) and stored at −20 °C in the dark.

ID
Compound

Compounds
The compounds tested in this work (Table 4) were synthesized by our group, as previously described [30]. They were all dissolved in dimethyl sulfoxide (DMSO) (Merck, Darmstadt, Germany) and stored at −20 °C in the dark.

ID
Compound

Compounds
The compounds tested in this work (Table 4) were synthesized by our group, as previously described [30]. They were all dissolved in dimethyl sulfoxide (DMSO) (Merck, Darmstadt, Germany) and stored at −20 °C in the dark.

ID
Compound

Compounds
The compounds tested in this work (Table 4) were synthesized by our group, as previously described [30]. They were all dissolved in dimethyl sulfoxide (DMSO) (Merck, Darmstadt, Germany) and stored at −20 °C in the dark.

ID
Compound

Compounds
The compounds tested in this work (Table 4) were synthesized by our group, as previously described [30]. They were all dissolved in dimethyl sulfoxide (DMSO) (Merck, Darmstadt, Germany) and stored at −20 °C in the dark.

ID
Compound

Compounds
The compounds tested in this work (Table 4) were synthesized by our group, as previously described [30]. They were all dissolved in dimethyl sulfoxide (DMSO) (Merck, Darmstadt, Germany) and stored at −20 °C in the dark.

ID
Compound

Parasite Cultures
Epimastigotes of Trypanosoma cruzi (Y strain) were used in this study and cultured in liver infusion tryptose (LIT) medium supplemented with 10% of foetal bovine serum (FBS) at 26 °C. For the cytotoxicity assays, a murine macrophage (J774A.1) cell line was used, which was cultured in Roswell Park Memorial Institute medium (RPMI 1640, Gibco), supplemented with 10% of foetal bovine serum (FBS) at 37 °C and 5% CO2 atmosphere.

Antiparasitic Activity
In a sterile 96-well plate, a serial dilution of acrylonitriles was carried out in LIT medium supplemented with 10% FBS with a final volume of 100 µL. Parasites were added to wells to reach a concentration of 10 5 parasites per well. Finally, a 10% of alamarBlue Cell Viability Reagent ® (ThermoFisher Scientific, Waltham, MA, USA) was added into each well, and the plate was incubated for 72 h at 26°C. After the incubation, the EnSpire Multimode Plate Reader ® (PerkinElmer, Thermo Fischer Scientific, Madrid, Spain) was used to determinate the fluorescence of each well (544 nm excitation, 590 nm emission). The concentration that inhibits 50% of the parasite population (IC50) was calculated by nonlinear regression analysis with 95% confidence limits [43].

Cytotoxicity Activity
The cytotoxicity was evaluated against murine macrophages (J774A.1) using the same method mentioned in the antiparasitic assay, based on the alamarBlue Cell Viability Reagent ® . In a sterile 96-well plate, 10 4 cells per well were added, then, after the complete adhesion, serial dilutions of the 32 compounds, using a deep well plate, and a 10% of alamarBlue ® were joined. After 24 h of incubation at 37 °C and 5% CO2 environment, the fluorescence was determined with the EnSpire Multimode Plate Reader ® to calculate the CC50, the concentration that inhibits 50% of the cell population [44].

Parasite Cultures
Epimastigotes of Trypanosoma cruzi (Y strain) were used in this study and cultured in liver infusion tryptose (LIT) medium supplemented with 10% of foetal bovine serum (FBS) at 26 °C. For the cytotoxicity assays, a murine macrophage (J774A.1) cell line was used, which was cultured in Roswell Park Memorial Institute medium (RPMI 1640, Gibco), supplemented with 10% of foetal bovine serum (FBS) at 37 °C and 5% CO2 atmosphere.

Antiparasitic Activity
In a sterile 96-well plate, a serial dilution of acrylonitriles was carried out in LIT medium supplemented with 10% FBS with a final volume of 100 µL. Parasites were added to wells to reach a concentration of 10 5 parasites per well. Finally, a 10% of alamarBlue Cell Viability Reagent ® (ThermoFisher Scientific, Waltham, MA, USA) was added into each well, and the plate was incubated for 72 h at 26°C. After the incubation, the EnSpire Multimode Plate Reader ® (PerkinElmer, Thermo Fischer Scientific, Madrid, Spain) was used to determinate the fluorescence of each well (544 nm excitation, 590 nm emission). The concentration that inhibits 50% of the parasite population (IC50) was calculated by nonlinear regression analysis with 95% confidence limits [43].

Cytotoxicity Activity
The cytotoxicity was evaluated against murine macrophages (J774A.1) using the same method mentioned in the antiparasitic assay, based on the alamarBlue Cell Viability Reagent ® . In a sterile 96-well plate, 10 4 cells per well were added, then, after the complete adhesion, serial dilutions of the 32 compounds, using a deep well plate, and a 10% of alamarBlue ® were joined. After 24 h of incubation at 37 °C and 5% CO2 environment, the fluorescence was determined with the EnSpire Multimode Plate Reader ® to calculate the CC50, the concentration that inhibits 50% of the cell population [44].

Parasite Cultures
Epimastigotes of Trypanosoma cruzi (Y strain) were used in this study and cultured in liver infusion tryptose (LIT) medium supplemented with 10% of foetal bovine serum (FBS) at 26 °C. For the cytotoxicity assays, a murine macrophage (J774A.1) cell line was used, which was cultured in Roswell Park Memorial Institute medium (RPMI 1640, Gibco), supplemented with 10% of foetal bovine serum (FBS) at 37 °C and 5% CO2 atmosphere.

Antiparasitic Activity
In a sterile 96-well plate, a serial dilution of acrylonitriles was carried out in LIT medium supplemented with 10% FBS with a final volume of 100 µL. Parasites were added to wells to reach a concentration of 10 5 parasites per well. Finally, a 10% of alamarBlue Cell Viability Reagent ® (ThermoFisher Scientific, Waltham, MA, USA) was added into each well, and the plate was incubated for 72 h at 26°C. After the incubation, the EnSpire Multimode Plate Reader ® (PerkinElmer, Thermo Fischer Scientific, Madrid, Spain) was used to determinate the fluorescence of each well (544 nm excitation, 590 nm emission). The concentration that inhibits 50% of the parasite population (IC50) was calculated by nonlinear regression analysis with 95% confidence limits [43].

Cytotoxicity Activity
The cytotoxicity was evaluated against murine macrophages (J774A.1) using the same method mentioned in the antiparasitic assay, based on the alamarBlue Cell Viability Reagent ® . In a sterile 96-well plate, 10 4 cells per well were added, then, after the complete adhesion, serial dilutions of the 32 compounds, using a deep well plate, and a 10% of alamarBlue ® were joined. After 24 h of incubation at 37 °C and 5% CO2 environment, the fluorescence was determined with the EnSpire Multimode Plate Reader ® to calculate the CC50, the concentration that inhibits 50% of the cell population [44].

Parasite Cultures
Epimastigotes of Trypanosoma cruzi (Y strain) were used in this study and cultured in liver infusion tryptose (LIT) medium supplemented with 10% of foetal bovine serum (FBS) at 26 °C. For the cytotoxicity assays, a murine macrophage (J774A.1) cell line was used, which was cultured in Roswell Park Memorial Institute medium (RPMI 1640, Gibco), supplemented with 10% of foetal bovine serum (FBS) at 37 °C and 5% CO2 atmosphere.

Antiparasitic Activity
In a sterile 96-well plate, a serial dilution of acrylonitriles was carried out in LIT medium supplemented with 10% FBS with a final volume of 100 µL. Parasites were added to wells to reach a concentration of 10 5 parasites per well. Finally, a 10% of alamarBlue Cell Viability Reagent ® (ThermoFisher Scientific, Waltham, MA, USA) was added into each well, and the plate was incubated for 72 h at 26°C. After the incubation, the EnSpire Multimode Plate Reader ® (PerkinElmer, Thermo Fischer Scientific, Madrid, Spain) was used to determinate the fluorescence of each well (544 nm excitation, 590 nm emission). The concentration that inhibits 50% of the parasite population (IC50) was calculated by nonlinear regression analysis with 95% confidence limits [43].

Cytotoxicity Activity
The cytotoxicity was evaluated against murine macrophages (J774A.1) using the same method mentioned in the antiparasitic assay, based on the alamarBlue Cell Viability Reagent ® . In a sterile 96-well plate, 10 4 cells per well were added, then, after the complete adhesion, serial dilutions of the 32 compounds, using a deep well plate, and a 10% of alamarBlue ® were joined. After 24 h of incubation at 37 °C and 5% CO2 environment, the fluorescence was determined with the EnSpire Multimode Plate Reader ® to calculate the CC50, the concentration that inhibits 50% of the cell population [44].

Parasite Cultures
Epimastigotes of Trypanosoma cruzi (Y strain) were used in this study and cultured in liver infusion tryptose (LIT) medium supplemented with 10% of foetal bovine serum (FBS) at 26 °C. For the cytotoxicity assays, a murine macrophage (J774A.1) cell line was used, which was cultured in Roswell Park Memorial Institute medium (RPMI 1640, Gibco), supplemented with 10% of foetal bovine serum (FBS) at 37 °C and 5% CO2 atmosphere.

Antiparasitic Activity
In a sterile 96-well plate, a serial dilution of acrylonitriles was carried out in LIT medium supplemented with 10% FBS with a final volume of 100 µL. Parasites were added to wells to reach a concentration of 10 5 parasites per well. Finally, a 10% of alamarBlue Cell Viability Reagent ® (ThermoFisher Scientific, Waltham, MA, USA) was added into each well, and the plate was incubated for 72 h at 26°C. After the incubation, the EnSpire Multimode Plate Reader ® (PerkinElmer, Thermo Fischer Scientific, Madrid, Spain) was used to determinate the fluorescence of each well (544 nm excitation, 590 nm emission). The concentration that inhibits 50% of the parasite population (IC50) was calculated by nonlinear regression analysis with 95% confidence limits [43].

Cytotoxicity Activity
The cytotoxicity was evaluated against murine macrophages (J774A.1) using the same method mentioned in the antiparasitic assay, based on the alamarBlue Cell Viability Reagent ® . In a sterile 96-well plate, 10 4 cells per well were added, then, after the complete adhesion, serial dilutions of the 32 compounds, using a deep well plate, and a 10% of alamarBlue ® were joined. After 24 h of incubation at 37 °C and 5% CO2 environment, the fluorescence was determined with the EnSpire Multimode Plate Reader ® to calculate the CC50, the concentration that inhibits 50% of the cell population [44].

Parasite Cultures
Epimastigotes of Trypanosoma cruzi (Y strain) were used in this study and cultured in liver infusion tryptose (LIT) medium supplemented with 10% of foetal bovine serum (FBS) at 26 °C. For the cytotoxicity assays, a murine macrophage (J774A.1) cell line was used, which was cultured in Roswell Park Memorial Institute medium (RPMI 1640, Gibco), supplemented with 10% of foetal bovine serum (FBS) at 37 °C and 5% CO2 atmosphere.

Antiparasitic Activity
In a sterile 96-well plate, a serial dilution of acrylonitriles was carried out in LIT medium supplemented with 10% FBS with a final volume of 100 µL. Parasites were added to wells to reach a concentration of 10 5 parasites per well. Finally, a 10% of alamarBlue Cell Viability Reagent ® (ThermoFisher Scientific, Waltham, MA, USA) was added into each well, and the plate was incubated for 72 h at 26°C. After the incubation, the EnSpire Multimode Plate Reader ® (PerkinElmer, Thermo Fischer Scientific, Madrid, Spain) was used to determinate the fluorescence of each well (544 nm excitation, 590 nm emission). The concentration that inhibits 50% of the parasite population (IC50) was calculated by nonlinear regression analysis with 95% confidence limits [43].

Cytotoxicity Activity
The cytotoxicity was evaluated against murine macrophages (J774A.1) using the same method mentioned in the antiparasitic assay, based on the alamarBlue Cell Viability Reagent ® . In a sterile 96-well plate, 10 4 cells per well were added, then, after the complete adhesion, serial dilutions of the 32 compounds, using a deep well plate, and a 10% of alamarBlue ® were joined. After 24 h of incubation at 37 °C and 5% CO2 environment, the fluorescence was determined with the EnSpire Multimode Plate Reader ® to calculate the CC50, the concentration that inhibits 50% of the cell population [44].

Parasite Cultures
Epimastigotes of Trypanosoma cruzi (Y strain) were used in this study and cultured in liver infusion tryptose (LIT) medium supplemented with 10% of foetal bovine serum (FBS) at 26 °C. For the cytotoxicity assays, a murine macrophage (J774A.1) cell line was used, which was cultured in Roswell Park Memorial Institute medium (RPMI 1640, Gibco), supplemented with 10% of foetal bovine serum (FBS) at 37 °C and 5% CO2 atmosphere.

Antiparasitic Activity
In a sterile 96-well plate, a serial dilution of acrylonitriles was carried out in LIT medium supplemented with 10% FBS with a final volume of 100 µL. Parasites were added to wells to reach a concentration of 10 5 parasites per well. Finally, a 10% of alamarBlue Cell Viability Reagent ® (ThermoFisher Scientific, Waltham, MA, USA) was added into each well, and the plate was incubated for 72 h at 26°C. After the incubation, the EnSpire Multimode Plate Reader ® (PerkinElmer, Thermo Fischer Scientific, Madrid, Spain) was used to determinate the fluorescence of each well (544 nm excitation, 590 nm emission). The concentration that inhibits 50% of the parasite population (IC50) was calculated by nonlinear regression analysis with 95% confidence limits [43].

Cytotoxicity Activity
The cytotoxicity was evaluated against murine macrophages (J774A.1) using the same method mentioned in the antiparasitic assay, based on the alamarBlue Cell Viability Reagent ® . In a sterile 96-well plate, 10 4 cells per well were added, then, after the complete adhesion, serial dilutions of the 32 compounds, using a deep well plate, and a 10% of alamarBlue ® were joined. After 24 h of incubation at 37 °C and 5% CO2 environment, the fluorescence was determined with the EnSpire Multimode Plate Reader ® to calculate the CC50, the concentration that inhibits 50% of the cell population [44].

Parasite Cultures
Epimastigotes of Trypanosoma cruzi (Y strain) were used in this study and cultured in liver infusion tryptose (LIT) medium supplemented with 10% of foetal bovine serum (FBS) at 26 °C. For the cytotoxicity assays, a murine macrophage (J774A.1) cell line was used, which was cultured in Roswell Park Memorial Institute medium (RPMI 1640, Gibco), supplemented with 10% of foetal bovine serum (FBS) at 37 °C and 5% CO2 atmosphere.

Antiparasitic Activity
In a sterile 96-well plate, a serial dilution of acrylonitriles was carried out in LIT medium supplemented with 10% FBS with a final volume of 100 µL. Parasites were added to wells to reach a concentration of 10 5 parasites per well. Finally, a 10% of alamarBlue Cell Viability Reagent ® (ThermoFisher Scientific, Waltham, MA, USA) was added into each well, and the plate was incubated for 72 h at 26°C. After the incubation, the EnSpire Multimode Plate Reader ® (PerkinElmer, Thermo Fischer Scientific, Madrid, Spain) was used to determinate the fluorescence of each well (544 nm excitation, 590 nm emission). The concentration that inhibits 50% of the parasite population (IC50) was calculated by nonlinear regression analysis with 95% confidence limits [43].

Cytotoxicity Activity
The cytotoxicity was evaluated against murine macrophages (J774A.1) using the same method mentioned in the antiparasitic assay, based on the alamarBlue Cell Viability Reagent ® . In a sterile 96-well plate, 10 4 cells per well were added, then, after the complete adhesion, serial dilutions of the 32 compounds, using a deep well plate, and a 10% of alamarBlue ® were joined. After 24 h of incubation at 37 °C and 5% CO2 environment, the fluorescence was determined with the EnSpire Multimode Plate Reader ® to calculate the CC50, the concentration that inhibits 50% of the cell population [44].

Parasite Cultures
Epimastigotes of Trypanosoma cruzi (Y strain) were used in this study and cultured in liver infusion tryptose (LIT) medium supplemented with 10% of foetal bovine serum (FBS) at 26 °C. For the cytotoxicity assays, a murine macrophage (J774A.1) cell line was used, which was cultured in Roswell Park Memorial Institute medium (RPMI 1640, Gibco), supplemented with 10% of foetal bovine serum (FBS) at 37 °C and 5% CO2 atmosphere.

Antiparasitic Activity
In a sterile 96-well plate, a serial dilution of acrylonitriles was carried out in LIT medium supplemented with 10% FBS with a final volume of 100 µL. Parasites were added to wells to reach a concentration of 10 5 parasites per well. Finally, a 10% of alamarBlue Cell Viability Reagent ® (ThermoFisher Scientific, Waltham, MA, USA) was added into each well, and the plate was incubated for 72 h at 26°C. After the incubation, the EnSpire Multimode Plate Reader ® (PerkinElmer, Thermo Fischer Scientific, Madrid, Spain) was used to determinate the fluorescence of each well (544 nm excitation, 590 nm emission). The concentration that inhibits 50% of the parasite population (IC50) was calculated by nonlinear regression analysis with 95% confidence limits [43].

Cytotoxicity Activity
The cytotoxicity was evaluated against murine macrophages (J774A.1) using the same method mentioned in the antiparasitic assay, based on the alamarBlue Cell Viability Reagent ® . In a sterile 96-well plate, 10 4 cells per well were added, then, after the complete adhesion, serial dilutions of the 32 compounds, using a deep well plate, and a 10% of alamarBlue ® were joined. After 24 h of incubation at 37 °C and 5% CO2 environment, the fluorescence was determined with the EnSpire Multimode Plate Reader ® to calculate the CC50, the concentration that inhibits 50% of the cell population [44].

Parasite Cultures
Epimastigotes of Trypanosoma cruzi (Y strain) were used in this study and cultured in liver infusion tryptose (LIT) medium supplemented with 10% of foetal bovine serum (FBS) at 26 °C. For the cytotoxicity assays, a murine macrophage (J774A.1) cell line was used, which was cultured in Roswell Park Memorial Institute medium (RPMI 1640, Gibco), supplemented with 10% of foetal bovine serum (FBS) at 37 °C and 5% CO2 atmosphere.

Antiparasitic Activity
In a sterile 96-well plate, a serial dilution of acrylonitriles was carried out in LIT medium supplemented with 10% FBS with a final volume of 100 µL. Parasites were added to wells to reach a concentration of 10 5 parasites per well. Finally, a 10% of alamarBlue Cell Viability Reagent ® (ThermoFisher Scientific, Waltham, MA, USA) was added into each well, and the plate was incubated for 72 h at 26°C. After the incubation, the EnSpire Multimode Plate Reader ® (PerkinElmer, Thermo Fischer Scientific, Madrid, Spain) was used to determinate the fluorescence of each well (544 nm excitation, 590 nm emission). The concentration that inhibits 50% of the parasite population (IC50) was calculated by nonlinear regression analysis with 95% confidence limits [43].

Cytotoxicity Activity
The cytotoxicity was evaluated against murine macrophages (J774A.1) using the same method mentioned in the antiparasitic assay, based on the alamarBlue Cell Viability Reagent ® . In a sterile 96-well plate, 10 4 cells per well were added, then, after the complete adhesion, serial dilutions of the 32 compounds, using a deep well plate, and a 10% of alamarBlue ® were joined. After 24 h of incubation at 37 °C and 5% CO2 environment, the fluorescence was determined with the EnSpire Multimode Plate Reader ® to calculate the CC50, the concentration that inhibits 50% of the cell population [44].

Parasite Cultures
Epimastigotes of Trypanosoma cruzi (Y strain) were used in this study and cultured in liver infusion tryptose (LIT) medium supplemented with 10% of foetal bovine serum (FBS) at 26 °C. For the cytotoxicity assays, a murine macrophage (J774A.1) cell line was used, which was cultured in Roswell Park Memorial Institute medium (RPMI 1640, Gibco), supplemented with 10% of foetal bovine serum (FBS) at 37 °C and 5% CO2 atmosphere.

Antiparasitic Activity
In a sterile 96-well plate, a serial dilution of acrylonitriles was carried out in LIT medium supplemented with 10% FBS with a final volume of 100 µL. Parasites were added to wells to reach a concentration of 10 5 parasites per well. Finally, a 10% of alamarBlue Cell Viability Reagent ® (ThermoFisher Scientific, Waltham, MA, USA) was added into each well, and the plate was incubated for 72 h at 26°C. After the incubation, the EnSpire Multimode Plate Reader ® (PerkinElmer, Thermo Fischer Scientific, Madrid, Spain) was used to determinate the fluorescence of each well (544 nm excitation, 590 nm emission). The concentration that inhibits 50% of the parasite population (IC50) was calculated by nonlinear regression analysis with 95% confidence limits [43].

Cytotoxicity Activity
The cytotoxicity was evaluated against murine macrophages (J774A.1) using the same method mentioned in the antiparasitic assay, based on the alamarBlue Cell Viability Reagent ® . In a sterile 96-well plate, 10 4 cells per well were added, then, after the complete adhesion, serial dilutions of the 32 compounds, using a deep well plate, and a 10% of alamarBlue ® were joined. After 24 h of incubation at 37 °C and 5% CO2 environment, the fluorescence was determined with the EnSpire Multimode Plate Reader ® to calculate the CC50, the concentration that inhibits 50% of the cell population [44].

Parasite Cultures
Epimastigotes of Trypanosoma cruzi (Y strain) were used in this study and cultured in liver infusion tryptose (LIT) medium supplemented with 10% of foetal bovine serum (FBS) at 26 °C. For the cytotoxicity assays, a murine macrophage (J774A.1) cell line was used, which was cultured in Roswell Park Memorial Institute medium (RPMI 1640, Gibco), supplemented with 10% of foetal bovine serum (FBS) at 37 °C and 5% CO2 atmosphere.

Antiparasitic Activity
In a sterile 96-well plate, a serial dilution of acrylonitriles was carried out in LIT medium supplemented with 10% FBS with a final volume of 100 µL. Parasites were added to wells to reach a concentration of 10 5 parasites per well. Finally, a 10% of alamarBlue Cell Viability Reagent ® (ThermoFisher Scientific, Waltham, MA, USA) was added into each well, and the plate was incubated for 72 h at 26°C. After the incubation, the EnSpire Multimode Plate Reader ® (PerkinElmer, Thermo Fischer Scientific, Madrid, Spain) was used to determinate the fluorescence of each well (544 nm excitation, 590 nm emission). The concentration that inhibits 50% of the parasite population (IC50) was calculated by nonlinear regression analysis with 95% confidence limits [43].

Cytotoxicity Activity
The cytotoxicity was evaluated against murine macrophages (J774A.1) using the same method mentioned in the antiparasitic assay, based on the alamarBlue Cell Viability Reagent ® . In a sterile 96-well plate, 10 4 cells per well were added, then, after the complete adhesion, serial dilutions of the 32 compounds, using a deep well plate, and a 10% of alamarBlue ® were joined. After 24 h of incubation at 37 °C and 5% CO2 environment, the fluorescence was determined with the EnSpire Multimode Plate Reader ® to calculate the CC50, the concentration that inhibits 50% of the cell population [44].

Parasite Cultures
Epimastigotes of Trypanosoma cruzi (Y strain) were used in this study and cultured in liver infusion tryptose (LIT) medium supplemented with 10% of foetal bovine serum (FBS) at 26 • C. For the cytotoxicity assays, a murine macrophage (J774A.1) cell line was used, which was cultured in Roswell Park Memorial Institute medium (RPMI 1640, Gibco), supplemented with 10% of foetal bovine serum (FBS) at 37 • C and 5% CO 2 atmosphere.

Antiparasitic Activity
In a sterile 96-well plate, a serial dilution of acrylonitriles was carried out in LIT medium supplemented with 10% FBS with a final volume of 100 µL. Parasites were added to wells to reach a concentration of 10 5 parasites per well. Finally, a 10% of alamarBlue Cell Viability Reagent ® (ThermoFisher Scientific, Waltham, MA, USA) was added into each well, and the plate was incubated for 72 h at 26 • C. After the incubation, the EnSpire Multimode Plate Reader ® (PerkinElmer, Thermo Fischer Scientific, Madrid, Spain) was used to determinate the fluorescence of each well (544 nm excitation, 590 nm emission). The concentration that inhibits 50% of the parasite population (IC 50 ) was calculated by nonlinear regression analysis with 95% confidence limits [43].

Cytotoxicity Activity
The cytotoxicity was evaluated against murine macrophages (J774A.1) using the same method mentioned in the antiparasitic assay, based on the alamarBlue Cell Viability Reagent ® . In a sterile 96-well plate, 10 4 cells per well were added, then, after the complete adhesion, serial dilutions of the 32 compounds, using a deep well plate, and a 10% of alamarBlue ® were joined. After 24 h of incubation at 37 • C and 5% CO 2 environment, the fluorescence was determined with the EnSpire Multimode Plate Reader ® to calculate the CC 50 , the concentration that inhibits 50% of the cell population [44].

Mitochondrial Membrane Potential Analysis
The detection of variations in the mitochondrial membrane potential was evaluated with the JC-1 Mitochondrial Membrane Potential Assay Kit ® (Cayman Chemical, Ann Arbor, MI, USA). In a 24-well plate, 500 µL of epimastigotes at 10 6 parasites per ml were added with the inhibitory concentration 90 (IC 90 ) of the compounds. After 24 h of incubation at 26 • C, the epimastigotes were centrifuged (3000 rpm, 10 min, 4 • C), resuspended in 50µL of JC-1 buffer and added to a black 96-well plate with 5 µL of JC-1. After 30 min of incubation at 26 • C, the green and red fluorescence were measured using an EnSpire Multimode Plate Reader ® (PerkinElmer). Results were expressed in percentage relative to negative control (without treatment) of the ratio 595/535 nm (J-aggregates/Jmonomers) [46].

ATP Level Analysis
Variations in the level of ATP were measured with the CellTiter-Glo ® Luminescent Cell Viability Assay (Promega, WI, USA). After 24 h of incubation with the IC 90 of the compounds, the epimastigotes were centrifuged (3000 rpm, 10 min, 4 • C). Then, in a white 96-well plate, the epimastigotes were resuspended in 25µL of culture media and mixed with 25 µL of the kit. After 10 min of incubation at room temperature, the luminescence was measured using an EnSpire Multimode Plate Reader ® (PerkinElmer). The results were expressed in percentage of production of ATP level relative to the negative control (without any treatment) [47].

Plasmatic Membrane Permeability Analysis
To determine the plasmatic membrane permeability, SYTOX ® Green nucleic acid stain fluorescent dye (ThermoFisher Scientific, MA, USA) was used. A suspension of 10 6 cells per mL of the parasites with the IC 90 of the molecules were incubated (24 h at 26 • C) and centrifuged (3000 rpm, 10 min, 4 • C). The pellet was resuspended in a 50 µL staining buffer and the Sytox ® Green at 1 µM (final concentration). After 15 min of incubation at room temperature, parasites were analyzed. Fluorescence pictures were taken with the EVOS ® FL Cell Imaging System (ThermoFisher Scientific, MA, USA) [48].

Reactive Oxygen Species Analysis
The oxidative stress was measured with the CellROX ® Deep Red Reagent (Ther-moFisher Scientific, MA, USA). After 24 h of incubation with the IC 90 of the molecules, the parasites were centrifuged (3000 rpm, 10 min, 4 • C) and incubated with 5 µM of CellROX reagent for 30 min at 26 • C. Finally, the fluorescence was analyzed using the EVOS ® FL Cell Imaging System (ThermoFisher Scientific, MA, USA) with the Cy5 light cube [49].

Statistic Methods
The results of IC 50 and CC 50 are presented as the mean ± the standard deviation (SD). All the experiments were performed in triplicate on different days. The analysis were carried out using a Tukey test with the GraphPad.PRISM ® 7.0a software. Values of p < 0.05 were considered significant.

Conclusions
The studied acrylonitrile derivatives presented trypanocidal activity against Trypanosoma cruzi with moderate selectivity indexes, due to their moderate cytotoxic effect against murine macrophages. Several ultrastructural and morphological changes such as mitochondrial membrane potential changes, decrease in ATP levels, reactive oxygen species accumulation and chromatin condensation were observed in Trypanosoma cruzi epimastigotes 24 h after treatment with the mentioned acrylonitriles. The present results highlight the potential use of acrylonitrile derivatives as programmed cell death inducers on T. cruzi, sharing several phenotypic characteristics with other cases of programmed cell death in metazoans. However, further studies are needed to reveal the target and to decrease the cytotoxic effect of these acrylonitriles in T. cruzi parasites, to establish them as novel and safe trypanocidal agents.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.