Identifying inhibitors of Trypanosoma cruzi nucleoside diphosphate kinase 1 as potential repurposed drugs for Chagas' disease.

Trypanosoma cruzi is the causative agent of Chagas' disease, an endemic and neglected disease. The treatment is limited to only two drugs, benznidazole (BZL) and nifurtimox (NFX), introduced more than fifty years ago and no new advances have been made since then. Nucleoside diphosphate kinases (NDPK) are key metabolic enzymes which have gained interest as drug targets of pathogen organisms. Taking advantage of the computer-assisted drug repurposing approaches, in the present work we initiate a search of potential T. cruzi nucleoside diphosphate kinase 1 (TcNDPK1) inhibitors over an âˆ¼ 12,000 compound structures database to find drugs targeted to this enzyme with trypanocidal activity. Four medicines were selected and evaluated in vitro, ketorolac (KET, an anti-inflamatory), dutasteride (DUT, used to treat benign prostatic hyperplasia), nebivolol and telmisartan (NEB and TEL, used to treat high blood pressure). The four compounds were weak inhibitors and presented different trypanocidal effect on epimastigotes, trypomastigotes and intracellular stages. NEB and TEL were the most active drugs with increased effect on intracellular stages, (IC50 = 2.25 µM and 13.21 µM respectively), and selectivity indexes of 13.01 and 8.59 respectively, showing comparable effect to BZL, the first line drug for Chagas' disease treatment. In addition, both presented positive interactions when combined with BZL. Finally, transgenic epimastigotes with increased expression of TcNDPK1 were more resistant to TEL and NEB, suggesting that TcNDPK1 is at least one of the molecular targets. In view of the results, NEB and TEL could be repurposed medicines for Chagas' disease therapy.

Revisiting trypanosomatid nucleoside diphosphate kinases.

BACKGROUND: An increasing amount of research has led to the positioning of nucleoside diphosphate kinases (NDPK/NDK) as key metabolic enzymes among all organisms. They contribute to the maintenance the intracellular di- and tri- phosphate nucleoside homeostasis, but they also are involved in widely diverse processes such as gene regulation, apoptosis, signal transduction and many other regulatory roles. OBJETIVE: Examine in depth the NDPKs of trypanosomatid parasites responsible for devastating human diseases (e.g., Trypanosoma cruzi, Trypanosoma brucei and Leishmania spp.) which deserve special attention. METHODS: The earliest and latest advances in the topic were explored, focusing on trypanosomatid NDPK features, multifunctionality and suitability as molecular drug targets. FINDINGS: Trypanosomatid NDPKs appear to play functions different from their host counterparts. Evidences indicate that they would perform key roles in the parasite metabolism such as nucleotide homeostasis, drug resistance, DNA damage responses and gene regulation, as well as host-parasite interactions, infection, virulence and immune evasion, placing them as attractive pharmacological targets. MAIN CONCLUSIONS: NDPKs are very interesting multifunctional enzymes. In the present review, the potential of trypanosomatid NDPKs was highlighted, raising awareness of their value not only with respect to parasite biology but also as molecular targets.

Trypanocidal activity of the anthocyanidin delphinidin, a non-competitive inhibitor of arginine kinase.

Arginine kinase from Trypanosoma cruzi (TcAK) catalyzes the interconversion of arginine and phosphoarginine to maintain the ATP/ADP cell balance, and is involved in the parasites' energetic homeostasis and stress responses. Using virtual screening approaches, some plant-derived polyphenolic pigments, such as anthocyanidins, were predicted to inhibit TcAK activity. Here, it was demonstrated that the anthocyanidin delphinidin showed a non-competitive inhibition mechanism of TcAK (Ki arginine = 1.32 µM and Ki ATP = 500 µM). Molecular docking simulations predicted that delphinidin occupies part of the ATP/ADP pocket, more specifically the one that binds the ribose phosphate, and molecular dynamics simulations confirmed the amino acids involved in binding. Delphinidin exerted trypanocidal activity over T. cruzi trypomastigotes with a calculated IC50 of 19.51 µM. Anthocyanidins are low-toxicity natural products which can be exploited for the development of trypanocidal drugs with less secondary effects than those currently used for the treatment of Chagas disease.

Revisiting trypanosomatid nucleoside diphosphate kinases

BACKGROUND An increasing amount of research has led to the positioning of nucleoside diphosphate kinases (NDPK/NDK) as key metabolic enzymes among all organisms. They contribute to the maintenance the intracellular di- and tri- phosphate nucleoside homeostasis, but they also are involved in widely diverse processes such as gene regulation, apoptosis, signal transduction and many other regulatory roles. OBJETIVE Examine in depth the NDPKs of trypanosomatid parasites responsible for devastating human diseases (e.g., Trypanosoma cruzi, Trypanosoma brucei and Leishmania spp.) which deserve special attention. METHODS The earliest and latest advances in the topic were explored, focusing on trypanosomatid NDPK features, multifunctionality and suitability as molecular drug targets. FINDINGS Trypanosomatid NDPKs appear to play functions different from their host counterparts. Evidences indicate that they would perform key roles in the parasite metabolism such as nucleotide homeostasis, drug resistance, DNA damage responses and gene regulation, as well as host-parasite interactions, infection, virulence and immune evasion, placing them as attractive pharmacological targets. MAIN CONCLUSIONS NDPKs are very interesting multifunctional enzymes. In the present review, the potential of trypanosomatid NDPKs was highlighted, raising awareness of their value not only with respect to parasite biology but also as molecular targets.

Effect of capsaicin on the protozoan parasite Trypanosoma cruzi.

Trypanosoma cruzi is the causative agent of Chagas disease. There are only two approved treatments, both of them unsuitable for the chronic phase, therefore the development of new drugs is a priority. Trypanosoma cruzi arginine kinase (TcAK) is a promising drug target since it is absent in humans and it is involved in cellular stress responses. In a previous study, possible TcAK inhibitors were identified through computer simulations resulting the best compounds capsaicin and cyanidin derivatives. Here, we evaluate the effect of capsaicin on TcAK activity and its trypanocidal effect. Although capsaicin produced a weak enzyme inhibition, it had a strong trypanocidal effect on epimastigotes and trypomastigotes (IC50 = 6.26 µM and 0.26 µM, respectively) being 20-fold more active on trypomastigotes than mammalian cells. Capsaicin was also active on the intracellular cycle reducing by half the burst of trypomastigotes at approximately 2 µM. Considering the difference between the concentrations at which parasite death and TcAK inhibition occur, other possible targets were predicted. Capsaicin is a selective trypanocidal agent active in nanomolar concentrations, with an IC50 57-fold lower than benznidazole, the drug currently used for treating Chagas disease.

Role of Trypanosoma cruzi nucleoside diphosphate kinase 1 in DNA damage responses.

BACKGROUND: NME23/NDPKs are well conserved proteins found in all living organisms. In addition to being nucleoside diphosphate kinases (NDPK), they are multifunctional enzymes involved in different processes such as DNA stability, gene regulation and DNA repair among others. TcNDPK1 is the canonical NDPK isoform present in Trypanosoma cruzi, which has nuclease activity and DNA-binding properties in vitro. OBJECTIVES: In the present study we explored the role of TcNDPK1 in DNA damage responses. METHODS: TcNDPK1 was expressed in mutant bacteria and yeasts and over-expressed in epimastigotes. Mutation frequencies, tolerance to genotoxic agents and activity of DNA repair enzymes were evaluated. FINDINGS: Bacteria decreased about 15-folds the spontaneous mutation rate and yeasts were more resistant to hydrogen peroxide and to UV radiation than controls. Parasites overexpressing TcNDPK1 were able to withstand genotoxic stresses caused by hydrogen peroxide, phleomycin and hidroxyurea. They also presented less genomic damage and augmented levels of poly(ADP)ribose and poly(ADP)ribose polymerase, an enzyme involved in DNA repair. MAIN CONCLUSION: These results strongly suggest a novel function for TcNDPK1; its involvement in the maintenance of parasite's genome integrity.

Computational approaches for drug discovery against trypanosomatid-caused diseases.

During three decades, only about 20 new drugs have been developed for malaria, tuberculosis and all neglected tropical diseases (NTDs). This critical situation was reached because NTDs represent only 10% of health research investments; however, they comprise about 90% of the global disease burden. Computational simulations applied in virtual screening (VS) strategies are very efficient tools to identify pharmacologically active compounds or new indications for drugs already administered for other diseases. One of the advantages of this approach is the low time-consuming and low-budget first stage, which filters for testing experimentally a group of candidate compounds with high chances of binding to the target and present trypanocidal activity. In this work, we review the most common VS strategies that have been used for the identification of new drugs with special emphasis on those applied to trypanosomiasis and leishmaniasis. Computational simulations based on the selected protein targets or their ligands are explained, including the method selection criteria, examples of successful VS campaigns applied to NTDs, a list of validated molecular targets for drug development and repositioned drugs for trypanosomatid-caused diseases. Thereby, here we present the state-of-the-art of VS and drug repurposing to conclude pointing out the future perspectives in the field.

Crystal violet structural analogues identified by in silico drug repositioning present anti-Trypanosoma cruzi activity through inhibition of proline transporter TcAAAP069.

BACKGROUND: Crystal violet (CV) was used for several years in blood banks to eliminate the parasite Trypanosoma cruzi in endemic areas in order to prevent transfusion-transmitted Chagas disease. One mechanism of action described for CV involves inhibition of proline uptake. In T. cruzi, proline is essential for host cell infection and intracellular differentiation among other processes, and can be obtained through the proline permease TcAAAP069. METHODOLOGY/PRINCIPAL FINDINGS: CV inhibited proline transporter TcAAAP069 and parasites overexpressing this permease were 47-fold more sensitive to this compound than control parasites. Using CV as reference molecule, loratadine, cyproheptadine, olanzapine and clofazimine were identified as structurally related compounds to CV (structural analogues) by in silico drug repurposing through a similarity-based virtual screening protocol. All these already-approved drugs for clinical use inhibited TcAAAP069 activity with different efficacies and also presented trypanocidal action in epimastigotes, trypomastigotes and amastigotes of the Y, CL Brener and Dm28c T. cruzi strains. Finally, a synergistic effect between benznidazole and the CV chemical analogues was evidenced by combination and dose-reduction indexes values in epimastigotes and trypomastigotes of the Y strain. CONCLUSIONS/SIGNIFICANCE: Loratadine, cyproheptadine and clofazimine inhibit TcAAAP069 proline transporter and also present trypanocidal effect against all T. cruzi life stages in strains from three different DTUs. These CV structural analogues could be a starting point to design therapeutic alternatives to treat Chagas disease by finding new indications for old drugs. This approach, called drug repurposing is a recommended strategy by the World Health Organization to treat neglected diseases, like Chagas disease, and combination therapy may improve the possibility of success of repositioned drugs.

In silico repositioning of etidronate as a potential inhibitor of the Trypanosoma cruzi enolase.

Enolase is a glycolytic enzyme that catalyzes the interconversion between 2-phosphoglycerate and phosphoenolpyruvate. In trypanosomatids, enolase was proposed as a key enzyme after in silico and in vivo analysis and it was validated as a protein essential for the survival of the parasite. Therefore, enolase constitutes an interesting enzyme target for the identification of drugs against Chagas disease. In this work, a combined virtual screening strategy was implemented, employing similarity virtual screening, molecular docking, and molecular dynamics. First, two known enolase inhibitors and the enzyme substrates were used as queries for the similarity screening on the Sweetlead database using five different algorithms. Compounds retrieved in the top 10 of at least three search algorithms were selected for further analysis, resulting in six compounds of medical use (etidronate, pamidronate, fosfomycin, acetohydroxamate, triclofos, and aminohydroxybutyrate). Molecular docking simulations and pose re-scoring predicted that binding with acetohydroxamate and triclofos would be weak, while fosfomycin and aminohydroxybutyrate predicted binding is experimentally implausible. Docking poses obtained for etidronate, pamidronate, and PEP were used for molecular dynamics calculations to describe their mode of binding. From the obtained results, we propose etidronate as a potential TcENO inhibitor and describe molecular motifs to be taken into account in the repurposing or design of drugs targeting this enzyme active site.

Role of Trypanosoma cruzi nucleoside diphosphate kinase 1 in DNA damage responses

BACKGROUND NME23/NDPKs are well conserved proteins found in all living organisms. In addition to being nucleoside diphosphate kinases (NDPK), they are multifunctional enzymes involved in different processes such as DNA stability, gene regulation and DNA repair among others. TcNDPK1 is the canonical NDPK isoform present in Trypanosoma cruzi, which has nuclease activity and DNA-binding properties in vitro. OBJECTIVES In the present study we explored the role of TcNDPK1 in DNA damage responses. METHODS TcNDPK1 was expressed in mutant bacteria and yeasts and over-expressed in epimastigotes. Mutation frequencies, tolerance to genotoxic agents and activity of DNA repair enzymes were evaluated. FINDINGS Bacteria decreased about 15-folds the spontaneous mutation rate and yeasts were more resistant to hydrogen peroxide and to UV radiation than controls. Parasites overexpressing TcNDPK1 were able to withstand genotoxic stresses caused by hydrogen peroxide, phleomycin and hidroxyurea. They also presented less genomic damage and augmented levels of poly(ADP)ribose and poly(ADP)ribose polymerase, an enzyme involved in DNA repair. MAIN CONCLUSION These results strongly suggest a novel function for TcNDPK1; its involvement in the maintenance of parasite's genome integrity.

Identification of Trypanosoma cruzi Polyamine Transport Inhibitors by Computational Drug Repurposing.

Trypanosoma cruzi is the causative agent of Chagas disease, a parasitic infection endemic in Latin America. In T. cruzi the transport of polyamines is essential because this organism is unable to synthesize these compounds de novo. Therefore, the uptake of polyamines from the extracellular medium is critical for survival of the parasite. The anthracene-putrescine conjugate Ant4 was first designed as a polyamine transport probe in cancer cells. Ant4 was also found to inhibit the polyamine transport system and produced a strong trypanocidal effect in T. cruzi. Considering that Ant4 is not currently approved by the FDA, in this work we performed computer simulations to find trypanocidal drugs approved for use in humans that have structures and activities similar to Ant4. Through a similarity ligand-based virtual screening using Ant4 as reference molecule, four possible inhibitors of polyamine transport were found. Three of them, promazine, chlorpromazine, and clomipramine, showed to be effective inhibitors of putrescine uptake, and also revealed a high trypanocidal activity against T. cruzi amastigotes (IC50 values of 3.8, 1.9, and 2.9 µM, respectively) and trypomastigotes (IC50 values of 3.4, 2.7, and 1.3 µM, respectively) while in epimastigotes the IC50 were significantly higher (34.7, 41.4, and 39.7 µM, respectively). Finally, molecular docking simulations suggest that the interactions between the T. cruzi polyamine transporter TcPAT12 and all the identified inhibitors occur in the same region of the protein. However, this location is different from the site occupied by the natural substrates. The value of this effort is that repurposing known drugs in the treatment of other pathologies, especially neglected diseases such as Chagas disease, significantly decreases the time and economic cost of implementation.

Amino Acid and Polyamine Membrane Transporters in Trypanosoma cruzi: Biological Function and Evaluation as Drug Targets.

Amino acids and polyamines are involved in relevant processes for the parasite Trypanosoma cruzi, like protein synthesis, stress resistance, life cycle progression, infection establishment and redox balance, among others. In addition to the biosynthetic routes of amino acids, T. cruzi possesses transport systems that allow the active uptake from the extracellular medium; and in the case of polyamines, the uptake is the unique way to obtain these compounds. The TcAAAP protein family is absent in mammals and its members are responsible for amino acid and derivative uptake, thus the TcAAAP permeases are not only interesting and promising therapeutic targets but could also be used to direct the entry of toxic compounds into the parasite. Although there is a treatment available for Chagas disease, its limited efficacy in the chronic stage of the disease, as well as the side effects reported, highlight the urgent need to develop new therapies. Discovery of new drugs is a slow and cost-consuming process, and even during clinical trials the drugs can fail. In this context, drug repositioning is an interesting and recommended strategy by the World Health Organization since costs and time are significantly reduced. In this article, amino acids and polyamines transport and their potential as therapeutic targets will be revised, including examples of synthetic drugs and drug repurposing.

Repurposing of terconazole as an anti Trypanosoma cruzi agent.

Trypanosoma cruzi is the causative agent of Chagas disease, a parasitic infection endemic in Latin America. Currently there are no effective treatments for the chronic phase of the disease, when most patients are diagnosed, therefore the development of new drugs is a priority area. Several triazoles, used as fungicides, exhibit trypanocidal activity both in vitro and in vivo. The mechanism of action of such drugs, both in fungi and in T. cruzi, relies in the inhibition of ergosterol biosynthesis affecting the cell viability and growth. Among them, terconazole was the first triazole antifungal drug for human use. In this work, the trypanocidal activity of terconazole was evaluated using in vitro assays. In epimastigotes of two parasites strains from different discrete typing units (Y and Dm28c) the calculated IC50 were 25.7 µM and 21.9 µM, respectively. In trypomastigotes and amastigotes (the clinically relevant life-stages of T. cruzi) a higher drug susceptibility was observed with IC50 values of 4.6 µM and 5.9 µM, respectively. Finally, the molecular docking simulations suggest that terconazole inhibits the T. cruzi cytochrome P450 14-α-demethylase, interacting in a similar way that other triazole drugs. Drug repurposing to Chagas disease treatment is one of the recommended approach according to the criterion of international health organizations for their application in neglected diseases.

Targeting polyamine transport in Trypanosoma cruzi.

Polyamines play critical roles as regulators of cell growth and differentiation. In contrast with other protozoa, the human parasite Trypanosoma cruzi, the etiological agent of Chagas disease, is auxotrophic for polyamines. Therefore, their intracellular availability depends exclusively on polyamine transport and inhibition of these uptake processes can alter the viability of the parasite. The polyamine analogues used in this work were successfully tested as antiproliferative agents in cancer cells, bacteria, fungi and also showed a potent antiplasmodial effect. We evaluated the activity of these compounds on polyamine transport in T. cruzi and assessed the effects on parasite viability. Three polyamine derivatives, AMXT1501, Ant4 and Ant44, inhibited the putrescine transport in epimastigotes (the insect stage of T. cruzi) with calculated IC50 values of 2.43, 5.02 and 3.98 µM, respectively. In addition, only Ant4 and Ant44 inhibited spermidine transport with IC50 of 8.78 µM and 13.34 µM, respectively. The Ant4 analogue showed a high trypanocidal effect on trypomastigotes (the bloodstream stage of T. cruzi) with an IC50 of 460 nM, (SI = 12.7) while in epimastigotes the IC50 was significantly higher (16.97 µM). In addition, we studied the effect of the combination of benznidazole, a drug used in treating Chagas disease, with Ant4 on the viability of epimastigotes. The combined treatment produced a significant increase on the inhibition of parasites growth compared with individual treatments. In summary, these results suggest that Ant4, a putrescine conjugate, is a promising compound for the treatment of Chagas disease because it showed a potent trypanocidal effect via its inhibition of polyamine import.

Techniques Used for Functional Characterization of Polyamine Transporters.

Transport systems are key processes in every living organism: they allow the entry of all essential nutrients into the cell and its compartments and regulate the intracellular concentrations of metabolites. The transport of cell nutrients represents the first step of many metabolic routes and may also regulate such processes. They are also responsible for reaching the effective intracellular concentration of therapeutic drugs and some mechanisms of resistance and tolerance also depend on them. However, the common techniques used to evaluate the metabolites transport in different cells types are not easy to carry out and require extensive training. In this chapter, we report detailed protocols and tips about the expression of transporters, different activity assays and transporter kinetics determination.

Trypanocidal Effect of Isotretinoin through the Inhibition of Polyamine and Amino Acid Transporters in Trypanosoma cruzi.

Polyamines are essential compounds to all living organisms and in the specific case of Trypanosoma cruzi, the causative agent of Chagas disease, they are exclusively obtained through transport processes since this parasite is auxotrophic for polyamines. Previous works reported that retinol acetate inhibits Leishmania growth and decreases its intracellular polyamine concentration. The present work describes a combined strategy of drug repositioning by virtual screening followed by in vitro assays to find drugs able to inhibit TcPAT12, the only polyamine transporter described in T. cruzi. After a screening of 3000 FDA-approved drugs, 7 retinoids with medical use were retrieved and used for molecular docking assays with TcPAT12. From the docked molecules, isotretinoin, a well-known drug used for acne treatment, showed the best interaction score with TcPAT12 and was selected for further in vitro studies. Isotretinoin inhibited the polyamine transport, as well as other amino acid transporters from the same protein family (TcAAAP), with calculated IC50 values in the range of 4.6-10.3 µM. It also showed a strong inhibition of trypomastigote burst from infected cells, with calculated IC50 of 130 nM (SI = 920) being significantly less effective on the epimastigote stage (IC50 = 30.6 µM). The effect of isotretinoin on the parasites plasma membrane permeability and on mammalian cell viability was tested, and no change was observed. Autophagosomes and apoptotic bodies were detected as part of the mechanisms of isotretinoin-induced death indicating that the inhibition of transporters by isotretinoin causes nutrient starvation that triggers autophagic and apoptotic processes. In conclusion, isotretinoin is a promising trypanocidal drug since it is a multi-target inhibitor of essential metabolites transporters, in addition to being an FDA-approved drug largely used in humans, which could reduce significantly the requirements for its possible application in the treatment of Chagas disease.

Pentamidine antagonizes the benznidazole's effect in vitro, and lacks of synergy in vivo: Implications about the polyamine transport as an anti-Trypanosoma cruzi target.

Benznidazole is the first-line drug used in treating Chagas disease, which is caused by the parasite Trypanosoma cruzi (T. cruzi). However, benznidazole has limited efficacy and several adverse reactions. Pentamidine is an antiprotozoal drug used in the treatment of leishmaniasis and African trypanosomiasis. In T. cruzi, pentamidine blocks the transport of putrescine, a precursor of trypanothione, which constitutes an essential molecule in the resistance of T. cruzi to benznidazole. In the present study, we describe the effect of the combination of benznidazole and pentamidine on isolated parasites, mammalian cells and in mice infected with T. cruzi. In isolated trypomastigotes, we performed a dose-matrix scheme of combinations, where pentamidine antagonized the effect of benznidazole, mainly at concentrations below the EC50 of pentamidine. In T. cruzi-infected mammalian cells, pentamidine reversed the effect of benznidazole (measured by qPCR). In comparison, in infected BALB/c mice, pentamidine failed to get synergy with benznidazole, measured on mice survival, parasitemia and amastigote nest quantification. To further explain the in vitro antagonism, we explored whether pentamidine affects intracellular trypanothione levels, however, pentamidine produced no change in trypanothione concentrations. Finally, the T. cruzi polyamine permease (TcPAT12) was overexpressed in epimastigotes, showing that pentamidine has the same trypanocidal effect, independently of transporter expression levels. These results suggest that, in spite of the high potency in the putrescine transport blockade, TcPAT12 permease is not the main target of pentamidine, and could explain the lack of synergism between pentamidine and benznidazole.

Resveratrol inhibits Trypanosoma cruzi arginine kinase and exerts a trypanocidal activity.

Arginine kinase catalyzes the reversible transphosphorylation between ADP and phosphoarginine which plays a critical role in the maintenance of cellular energy homeostasis. Arginine kinase from the protozoan parasite Trypanosoma cruzi, the etiologic agent of Chagas disease, meets the requirements to be considered as a potential therapeutic target for rational drug design including being absent in its mammalian hosts. In this study a group of polyphenolic compounds was evaluated as potential inhibitors of arginine kinase using molecular docking techniques. Among the analyzed compounds with the lowest free binding energy to the arginine kinase active site (<-6.96kcal/mol), resveratrol was chosen for subsequent assays. Resveratrol inhibits 50% of recombinant arginine kinase activity at 325µM. The trypanocidal effect of resveratrol was evaluated on the T. cruzi trypomastigotes bursting from infected CHO K1 cells, with IC50=77µM. Additionally epimastigotes overexpressing arginine kinase were 5 times more resistant to resveratrol compared to controls. Taking into account that: (1) resveratrol is considered as completely nontoxic; (2) is easily accessible due to its low market price; and (3) has as a well-defined target enzyme which is absent in the mammalian host, it is a promising compound as a trypanocidal drug for Chagas disease.

Trypanosoma cruzi Polyamine Transporter: Its Role on Parasite Growth and Survival Under Stress Conditions.

Trypanosoma cruzi is the etiological agent of Chagas disease, a major health problem in Latin America. Polyamines are polycationic compounds that play a critical role as regulators of cell growth and differentiation. In contrast with other protozoa, T. cruzi is auxotrophic for polyamines because of its inability to synthesize putrescine due to the lack of both, arginine and ornithine decarboxylase; therefore, the intracellular availability of polyamines depends exclusively on transport processes. In this work, the polyamine transporter TcPAT12 was overexpressed in T. cruzi epimastigotes demonstrating that growth rates at different concentrations of polyamines strongly depend on the regulation of the polyamine transport. In addition, parasites overexpressing TcPAT12 showed a highly increased resistance to hydrogen peroxide and the trypanocidal drugs nifurtimox and benznidazole, which act by oxidative stress and interfering the synthesis of polyamine derivatives, respectively. Finally, the presence of putative polyamine transporters was analyzed in T. cruzi, Trypanosoma brucei, and Leishmania major genomes identifying 3-6 genes in these trypanosomatids.