Quinoxaline derivatives T-001 (Methyl 2-acetyl-3-methyl-quinoxaline-7-carboxylate 1,4-di-N-oxide) and T-017 (Ethyl 2-benzoyl-3-methylquinoxaline-7-carboxylate 1,4-di-N-oxide) were synthesized as described (Gómez-Caro et al., 2011). Stock solutions were prepared in DMSO (the concentration used in the experiments did not exceed 0.01%) and each compound was prepared at a final concentration of 1 mg/mL. Metronidazole (Sigma-Aldrich) was used as a reference drug.

E. histolytica trophozoites (HM1: IMSS strain) were axenically cultured in 75 cm² culture flasks at 37°C in TYIS-33 medium supplemented with 16.8% (v/v) heat-inactivated adult bovine serum (Diamond et al., 1978), 3.2% (v/v) Diamond Vitamin-Tween 80 solution (Sigma-Aldrich), 100 U/mL penicillin, and 100 μg/mL streptomycin. Trophozoites were treated for 48 h at 37°C with the IC₅₀ of T-001 (1.41 µM) or T-017 (1.93 µM), previously reported by Duque-Montaño et al. (2013). Parasites without treatment were included as controls. This experiment was carried out in triplicate.

Trophozoites were centrifuged and washed with PBS pH 6.8 at 4°C and resuspended in 1 ml buffer lysis, (50 mM Tris pH 7.4, 0.25% SDS, protease inhibitors: 0.04 mM E-64, 100 µM Leupeptin, 7 mM PMSF, 8.75 mM iodoacetamide and protease inhibitor cocktail [Complete, Roche]). After homogenization, trophozoites were lysed by freeze-thaw cycles; the solution was centrifuged at 16,000 g for 5 min at 4°C and the supernatant was distributed in aliquots in Eppendorf tubes and stored at -80°C, until use.

Protein cleaning and purification were carried out by acetone precipitation, the proteins were obtained by centrifugation at 21,000 g for 5 min at 4°C, the pellet was allowed to air dry and resuspended in rehydration buffer (7 M urea, 2 M thiourea, 2% CHAPS, 40 mM DTT, 0.5% ampholytes 4-7, and traces of bromophenol blue).

Protein extract (500 µg) were applied to acrylamide gel strips with an immobilized linear gradient of pH 4.0-7.0 (ReadyStrip ™ IPG strips pH 4.0-7.0, BioRad). Isoelectric focusing was performed with the Protean IEF i12 ™ cell system (BioRad), following the recommendations of the commercial company, under the following conditions: step 1: 250 V for 20 min, step 2: 8000 V for 1h, step 3: 8000 V for 26 h, step 4: 1500 V sustained. For the second dimension, the IPG strips were placed directly on the 12% polyacrylamide gel, along with 7 µL of pre-stained molecular weight marker (Bio-Rad). Electrophoresis was carried out at 80 V for 15 min, and at 200 V until the end of the run. The gel was immersed for 1 h in fixing solution (50% methanol, 10% acetic acid), washed three times with Milli-Q water for 10 min and stained with Sypro^® Ruby Protein Gel Stain (Invitrogen ™, Molecular Probes) according to the supplier recommendations. Finally, the stained gel was visualized with UV light through the ChemiDocs XRS system (Bio-Rad Laboratories), images were acquired using the Image lab 5.2.1 software (Bio-Rad Laboratories) and spots were detected with the PDQuest 8.0.1 software (Bio-Rad Laboratories). The protein profiles of the biological triplicates for each condition were normalized using a synthetic image called MatchSet master. Subsequently, the intensity of the spots was normalized according to the total intensity of the valid spots, in order to minimize possible errors due to differences in the amount of protein and intensity of the staining. The Student´s t test was used to determine any significant differences in spot intensity (p<0.05).

The protein profiles corresponding to the treatment with T-001 and T-017 were compared with that of trophozoites without treatment. Only those spots that showed a Fold change> 2-fold between control and treatments were considered as differentially expressed proteins and selected for subsequent analysis by mass spectrometry (MS), according to Student´s t test (p<0.05).

A total of 45 spots were selected and excised with the EXQuest spot cutter (Bio-Rad Laboratories). They were bleached using acetonitrile (C₂H₃N) and 50 mM ammonium bicarbonate (NH₄CO₃) in a 1:1 ratio for 15 min, and then washed with 50 µL acetonitrile for 5 min. All the liquid was removed, and samples were dried in a vacuum centrifuge. Proteins were reduced with 10 mM DTT and 50 mM ammonium bicarbonate for 45 min at 56°C and alkylated with 55 mM iodoacetamide for 30 min at room temperature. Later, they were washed with sodium bicarbonate and acetronitrile, and digested with 0.1 µg/µL trypsin in 50 mM ammonium bicarbonate, overnight at 37°C. The peptides were extracted with 0.1% trifluoroacetic acid and 25 µL of acetronitrile for 30 min, the solvent was removed by lyophilization and peptides were submitted to a MALDI-TOF/TOF analyzer (Ultraflex III, Bruker, Germany). Mass spectra were acquired in positive ion mode and automatically submitted to Mascot software v.2.1 (http://www.matrixscience.com) for protein identification against the NCBI database for non-redundant proteins (http://www.ncbi.nlm.nih.gov/). As defined by Mascot probability analysis, only significant scores greater than “identity” (95% level of confidence) were considered to assign protein identity. All positive protein identification scores were significant (p < 0.05).

The identity of the proteins was corroborated in the amoeba (https://amoebadb.org/amoeba/) and Uniprot (https://www.uniprot.org) databases. Subsequently, they were categorized and classified according with their function, using the DAVID Bioinformatics Resources 6.8 (https://david.ncifcrf.gov/) and the PANTHER classification system (Protein Annotation Through Evolutionary Relationship) (http://www.pantherdb.org/). The proteins with unknow function were searched in the Argot 2 program (http://www.medcomp.medicina.unipd.it/Argot2/index.php). Finally, information related to the involvement of these proteins in various biological processes in E. histolytica was documented.

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al., 2022) partner repository with the dataset identifier PXD032322.

Total RNA was obtained from trophozoites previously treated with the respective quinoxaline derivatives T-001 and T-017, using TRIzol reagent (Thermo Fisher Scientific). RNA from trophozoites growing in standard conditions was used as control. Following DNase 1 treatment, cDNA synthesis was carried out using 5 µg of RNA and the SuperScript III First-Strand kit (Invitrogen). Specific primers for Peroxiredoxin putative (Forward, 5′-agcatggtgtgaagcagataa-3′; Reverse, 5′-cctgcttcgacatttaacattcc-3′), Actin 2 putative (Forward, 5′-atgggagacgaagaagttcaag-3′; Reverse, 5′-ttgacccataccagccataac-3′), Tyrosine kinase (Forward, 5′-cagagctgatggtcctccaa-3′; Reverse, 5′-tgaccaaatgacccagctcc-3′), Thioredoxin putative (Forward, 5′-ttgtgaatggtgtaaggaaatgag-3′; Reverse, 5′-gcactggctattaaagaatccatga-3′), Adapter protein (Forward, 5′-agcctcvtcttgattttgctcca-3′; Reverse, 5′-acacattgagaagccaaactagg-3′) and Cysteinyl-tRNA synthetase (Forward, 5′-caaaagtcggtgtacgtgttga-3′; Reverse, 5′-ttggatctgaagcccaactct-3′) were used in qPCR assays. Primers to amplify the RNA polymerase II gene (Forward, 5′-gatccaacatatcctaaaacaaca-3′; Reverse, 5′-tcaattattttctgacccgtcttc-3′) were used as internal control (Penuliar et al., 2012). The reactions were carried out in Fast Optical 48-Well Reaction Plates using 500 ng of cDNA, 5 µM of primers and 5 µL of Sensi FAST SYBR Hi-ROX master mix (Bioline). To ensure the presence of specific products, the denaturation curve (melt curve) was carried out and the relative expression of the genes was calculated by the ΔΔCT method (Livak & Schmittgen, 2001). Three independent and triplicate experiments were done. Data were analyzed using the Tukey test with the Sigma Stat version 2 software.

Trophozoites previously treated for 48 h with the IC₅₀ of T-001 or T-017 (as mentioned above), were incubated in TYI-S-33 medium without serum for 3 h at 37°C (Serum starvation) (Franco et al., 1997). They were chilled on ice for 5 min, centrifuged at 320 x g for 5 min and washed three times in TBS-CaCl₂ buffer (50 mM Tris, 150 mM NaCl and 1 mM CaCl₂, pH 7.2). Then, 5x10⁴ trophozoites were placed in the upper part of a transwell chamber (8 µm, Corning), while 600 µL of TYI-S-33 medium with serum was placed in the lower compartment. Cells were incubated at 37°C for 3 h and the number of trophozoites that migrated to the lower compartment was determined by counting in a Neubauer chamber and staining with 0.4% trypan blue (Gilchrist et al., 2008). The tests were carried out three times in triplicate. Untreated trophozoites and parasites treated with DMSO 0.01% were included as controls. The results were expressed as the mean of migration percentage ± standard deviation (SD). The comparisons between groups were carried out with the one way ANOVA test.

Trophozoites previously treated with the selected QdNOs were washed and resuspended in 1 mL of TYI-S-33 medium without serum; later they were seeded on monolayers of SW-480 cells previously cultured in 24-well plates, in a ratio of 1 trophozoite per 4 cells. The plates were incubated at 37°C for 15 min, after which the unattached trophozoites were removed from the culture medium, centrifuged at 320 x g for 5 min and counted using a Neubauer chamber. The tests were carried out in triplicate (Kobiler and Mirelman, 1981). Untreated trophozoites and parasites treated with DMSO 0.01% were included as controls. The results were expressed as the mean of adhesion percentage ± standard deviation (SD). The comparisons between groups were carried out with the one way ANOVA test.

SW-480 cells were seeded in a 24-well plate (1.8x10⁵ cells/well) in supplemented RPMI-1640 medium and incubated 48 h at 37°C with 5% CO₂. The cell monolayers were washed three times with PBS pH 6.8 and incubated at 37°C for 45 min with trophozoites (previously treated with selected QdNOs, untreated or DMSO treated, and resuspended in TYI-S-33 medium without serum) in 1:10 ration (amoeba: SW-480 cells). After incubation, the supernatant was removed, and lactate dehydrogenase activity was determined using the CytoTox 96^® Non-Radioactive Cytotoxicity Assay Kit, following the manufacturer instructions. Plates were read at 492 nm using a Multiskan FC reader (Thermo Scientific). SW-480 cells or trophozoites only with TYI-S-33 culture medium were used as negative controls; as lysis control, SW-480 cells lysed for 45 min with 9% X-100 triton. All conditions were tested in triplicate. Each experiment was repeated three times. The comparisons between groups were carried out with the one way ANOVA test.

Human erythrocytes (0.5 x 10⁸ cells/mL) were mixed with an equal volume of E. histolytica trophozoites (0.5x10⁶ cells/ml). Both cell suspensions were prepared with serum-free medium. Cell mixture was incubated for 0, 5, 10 or 15 min at 37°C, subsequently fixed with 4% paraformaldehyde (Trissl et al., 1978), and washed three times with PBS 6.8 to remove non-phagocytosed erythrocytes. Ingested erythrocytes were contrasted by the Novikoff staining method (Novikoff et al., 1972) using fresh diaminobenzidine solution (3,3'-diaminobenzidine 2 mg/mL, 0.048% H₂O₂ and 50 Mm Tris-HCL, pH 9.7). The number of ingested erythrocytes was quantified and documented under an optical microscope. The results were expressed as the mean of ingested erythrocyte ratio ± standard deviation (SD). The comparisons between groups were carried out with the one way ANOVA test.

Erythrophagocytosis capacity was evaluated by quantitative determination of hemoglobin within trophozoites. After 10 min of interaction of the amoeba trophozoites with human erythrocytes as described above, the trophozoites were recovered by washing three times with 1 mL of cold distilled water; then they were lysed with 1 mL formic acid and the amount of hemoglobin was measured by spectrophotometric analysis at 400 nm, using formic acid as blank (Voigt et al., 1999). The hemoglobin quantitation was obtained in relation to the untreated parasites. The results were expressed as the mean of hemoglobin ± standard deviation (SD). The comparisons between groups were carried out with the one way ANOVA test.

To categorize the function of the modulated proteins, the DAVID Bioinformatics Resources 6.8 bionformatic program (https://david.ncifcrf.gov/) and the PantherDB database (http://www.pantherdb.org/) were used. Those proteins whose function was not found were searched in the Argot 2 program (Functional annotation of proteins using the semantic similarity in the Gene Ontology). Table 3 shows the function of proteins deregulated by compound T-001. Interestingly, these include proteins related with translation (guanine nucleotide-binding protein subunit beta 2-like 1, 40S ribosomal protein S24 and cysteinyl-tRNA synthetase), intracellular traffic (TBC domain containing protein, vesicle-fusing ATPase and adapter protein), phosphorylation (hypothetical protein [3] and [16], and tyrosine kinase), cytoskeletal organization (actin 2 and calmodulin), energy generation (pyruvate phosphate dikinase and fumarate hydratase class I), nucleic acid binding (hypothetical protein [8] and PCI domain containing protein), redox homeostasis (peroxiredoxin), stress response (truncated hsp70 family protein), ubiquitination (hypothetical protein [12]), kinase activity (hypothetical protein [1]), transport (hypothetical protein [7]) and proteins with unknown function (hypothetical proteins [9] and [18], and F-box/WD domain containing protein). On the other hand, proteins deregulated by compound T-017 were related with intracellular traffic (Rab GTPase family protein, ADP-ribosylation factor 1, importin beta-3 family protein and adapter protein), nucleic acid binding (hypothetical protein [9] and [1]), translation (60S ribosomal protein L27 and cysteinyl-tRNA synthetase), binding to calcium ion (grainin), hydrolase activity (hypothetical protein [15]), redox homeostasis (thioredoxin), rRNA processing (ribosome biogenesis protein), stress response (AIG1 family protein), energy generation (pyruvate phosphate dikinase), post-traslational modifications (hypothetical protein [17]), cytoskeletal organization (adenylylcyclase-associated protein), post-transcriptional processing (hypothetical protein [12]) and proteins with unknown function (hypothetical protein [3], [7] and [13], and F-box/WD domain containing protein) ( Table 4 ).

Interestingly, both quinoxaline derivatives affect proteins that participate in the dynamics of the actin cytoskeleton and intracellular trafficking. Notably, Actin 2 and calmodulin overexpression was evidenced in trophozoites treated with T-001, while the protein associated with adenylyl cyclase were found to be downregulated by T-017. On the other hand, proteins associated with trafficking as TBC domain containing protein, vesicle-fusing ATPase and adapter protein were deregulated by compound T-001, while T-017 deregulated Rab GTPase family protein, ADP-ribosylation factor 1, importin beta-3 family protein and adapter protein. The cytoskeleton is particularly relevant for the pathogenicity mechanisms of amoeba. Adhesion is one of the first steps in the pathogenesis of E. histolytica and a prerequisite for host cell destruction. Together with the motility and ability to engulf host cells, adhesion allows trophozoites to colonize, invade and destroy different human tissues. Therefore, we decided to evaluate the impact of T-001 and T-017 treatment on migration, phagocytosis, and adhesion in E. histolytica; changes in the cytopathic effect of this parasite were also evaluated.

To evaluate the effect of quinoxaline derivatives on the migration of amoeba trophozoites, an assay was carried out using transwell chambers. The trophozoites treated with T-001 showed a decrease of 27.14% in their migration with respect to untreated amoebae; similarly, compound T-017 produced a 23.77% decrease in the migration of trophozoites ( Figure 3A ). Likewise, to evaluate if quinoxaline derivatives could affect the adhesion of ameba trophozoites, an assay was carried out to evaluate trophozoite adhesion to SW-480 cell cultures. As shown in Figure 3B , both compounds caused a reduction in trophozoites adhesion, being compound T-001 the one that produced the greatest effect with a reduction of 49% compared to untreated trophozoites, while compound T-017 reduced adhesion in 27%. To evidence whether the decrease in adhesion caused by T-001 and T-017 compounds could affect the cytolytic activity of the amoeba trophozoites, we carried out interaction tests between SW-480 cell cultures and trophozoites at 37°C. After 45 min of interaction with untreated trophozoites, cell cultures showed an average of 75.66% destruction ( Figure 3C ). Similarly, amoebae treated with 0.01% DMSO showed 71.33% cell destruction. On the contrary, trophozoites treated with T-001 caused a lower degree of damage, since they only produced 52.66% of destruction. However, T-017 treatment did not significantly affect the ability of trophozoites to destroy SW-480 cell monolayers, since trophozoites treated with T-017 produced 67.66% of cell destruction.

The effect of quinoxaline derivatives on the phagocytic capacity of amoeba trophozoites was carried out by interacting amoeba trophozoites with human erythrocytes. For this analysis, the number of erythrocytes ingested by amoeba trophozoites (previously treated with T-001 and T-017) was determined after 0, 5, 10 and 15 min of interaction. The results were visualized through staining with diaminobenzidine. As shown in Figure 4 , this is a time-dependent process in untreated trophozoites since there is a progressive increase in the number of ingested erythrocytes through time. Trophozoites treated with 0.01% DMSO presented no significant difference with respect to untreated trophozoites. Interestingly, in the case of T-001 treatment, the erythrophagocytosis rate was significantly decreased by 32% and 23% at 10 and 15 minutes of interaction, respectively in reference to untreated group. In contrast, T-017 treatment increased erythrophagocytosis by about 50% and 42% at the same interaction times, with respect to untreated trophozoites. ( Figure 4A ). These results were corroborated by measuring the hemoglobin contained in the trophozoites after ten minutes of interaction with red blood cells; as shown in Figure 4B , the amount of hemoglobin was decreased by 30% and increased by 32%, in trophozoites treated with T-001 and T-017, respectively ( Figure 4B ).