International Journal for Parasitology: Drugs and Drug Resistance 14 (2020) 17–27 Contents lists available at ScienceDirect International Journal for Parasitology: Drugs and Drug Resistance journal homepage: www.elsevier.com/locate/ijpddr Repurposing bioenergetic modulators against protozoan parasites responsible for tropical diseases Alba Martínez-Flórez a, 1, Melina Galizzi b, Luis Izquierdo c, Juan M. Bustamante d, Ana Rodriguez e, Fernando Rodriguez f, Alhelí Rodríguez-Cortés a, **, 1, Jordi Alberola a, *, 1 a Departament de Farmacologia, de Terapèutica i de Toxicologia, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain b Complex Carbohydrate Research Center, University of Georgia, Athens, GA, USA c ISGlobal, Barcelona Centre for International Health Research (CRESIB), Hospital Clínic, Universitat de Barcelona, Barcelona, Spain d Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA, USA e Department of Microbiology, New York University School of Medicine, New York, NY, USA f Centre de Recerca en Sanitat Animal (CReSA)-Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Bellaterra, Barcelona, Spain A R T I C L E I N F O A B S T R A C T Keywords: Malaria, leishmaniasis and trypanosomiasis are arthropod-borne, parasitic diseases that constitute a major global Bionergetic modulators health problem. They are generally found in developing countries, where lack of access to preventive tools and Protozoan parasites treatment hinders their management. Because these parasites share an increased demand on glucose consump­ Repurposing tion with most cancer cells, six compounds used in anti-tumoral research were selected to be tested as anti­ Sirolimus Dichloroacetate parasitic agents in in vitro models of Leishmania infantum, Trypanosoma brucei, T. cruzi, and Plasmodium 3-bromopyruvate falciparum: dichloroacetic acid (DCA), 3-bromopyruvic acid (3BP), 2-deoxy-D-glucose (2DG), lonidamine (LND), 2-deoxy-D-glucose metformin (MET), and sirolimus (SIR). No parasite-killing activity was found in L. infantum promastigotes, Lonidamine whereas DCA and 3BP reduced the burden of intra-macrophagic amastigotes. For T. brucei all selected com­ Metformin pounds, but 2DG, decreased parasite survival. DCA, 2DG, LND and MET showed parasite-killing activity in T. cruzi. Finally, anti-plasmodial activity was found for DCA, 2DG, LND, MET and SIR. These results reinforce the hypothesis that drugs with proven efficacy in the treatment of cancer by interfering with ATP production, proliferation, and survival cell strategies might be useful in treating threatening parasitic diseases and provide new opportunities for their repurposing. 1. Introduction treatment. The existence of both wild and domestic reservoirs makes controlling the spread of these diseases even more difficult. Despite Arthropod-borne parasitic infections are responsible for a number of considerable efforts undertaken throughout the last decades, which have severe diseases found around the world. They include primarily malaria, resulted in a marked decline in their incidence, parasite diseases remain visceral leishmaniasis (VL), and American and African trypanosomiasis a major public health problem. The latest World Health Organization (Chagas disease and Human African Trypanosomiasis (HAT), also (WHO) report shows that these parasites cause diseases in more than known as sleeping sickness, respectively). Malaria is caused by parasites 200 million people annually. In 2018, there were an estimated 228 belonging to the Plasmodium genus with P. falciparum being the most million cases of malaria worldwide, around 90,000 people were diag­ pathogenic for humans. VL, Chagas disease and HAT are brought about nosed with VL, almost 1000 with HAT, and roughly 7 million people by Leishmania spp., Trypanosoma cruzi, and T. brucei, respectively, all were estimated to be chronically affected by Chagas disease. For the belonging to the Trypanosomatidae family. These diseases are currently same year, more than 400,000 individuals, many of them children, died considered neglected because they mainly affect developing countries due to these parasitic infections (World Health Organization, 2020). where there is a lack of access to tools and drugs for prevention and No vaccines have yet been commercialized for humans against these * Corresponding author. ** Corresponding author. E-mail addresses: Alheli.Rodriguez@uab.cat (A. Rodríguez-Cortés), Jordi.Alberola@uab.cat (J. Alberola). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.ijpddr.2020.07.002 Received 25 February 2020; Received in revised form 17 July 2020; Accepted 20 July 2020 Available online 22 July 2020 2211-3207/© 2020 The Authors. Published by Elsevier Ltd on behalf of Australian Society for Parasitology. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). A. Martínez-Flórez et al. International Journal for Parasitology: Drugs and Drug Resistance 14 (2020) 17–27 pathogens, thus the main approach for their management is based on interesting targets for fighting protozoan diseases (Verlinde et al., 2001; pharmacological treatment. Developing new drugs involves both large Jacobs et al., 2011; Saldivia et al., 2013). Likewise, some energy mod­ budgets and long lead-times. In contrast, drug repurposing is a rapid and ulators are already in use in cancer diagnosis, or in advanced phases of cheaper alternative because it employs validated molecular targets and clinical trials (Scatena et al., 2008; Granchi and Minutolo, 2013), thus drugs for new indications. This is especially helpful for neglected dis­ facilitating repurposing. eases since only a small percentage of total investment in health research The aim of the present study was to test the hypothesis that drugs goes to drug discovery for tropical diseases (Nwaka et al., 2009). acting on these metabolic pathways, and whose efficacy in cancer Because of their changing life conditions, these pathogens exhibit research has been demonstrated, could be useful in the management of a different metabolic profiles during their biological cycle, which are number of parasitic diseases, including Chagas disease, HAT, VL and associated with major changes in central carbon metabolism. Of special malaria. The following compounds were selected: 3-bromopyruvic acid interest is the energetic metabolism that these parasites exhibit within (3BP), 2-deoxy-D-glucose (2DG), lonidamine (LND), dichloroacetic acid the host where they undergo rapid multiplication. (DCA), metformin (MET), and sirolimus (SIR) (Table 1). LND, 3BP, and Bloodstream stages of T. brucei show the simplest metabolism of 2DG block early stages of the glycolytic route as they interfere with the trypanosomatids, where the sole source of ATP is glucose via glycolysis, activity of hexokinase (HK) or cannot be further metabolized (Paggi being pyruvate the main end product (Coley et al., 2011). T. cruzi and et al., 1988). Initially, 3BP was described as inhibiting HKII activity (Ko Leishmania spp amastigotes can degrade other metabolites than glucose, et al., 2001), however, it has been shown to also exhibit inhibitory ac­ such as amino acids, and glycolysis is extended to a reversible succinate tivity on GAPDH, another enzyme of the same pathway (Ganapathy-­ production pathway within the glycosome (Besteiro et al., 2002; Mau­ Kanniappan et al., 2009). DCA and the biguanide MET were chosen geri et al., 2011). The resulting pyruvate is transformed into alanine or because of their ability to modulate ATP and biomass production transported to the mitochondrion, instead of being excreted to the pathways at mitochondrial level. DCA switches cell metabolism from external milieu. These parasites show a much more complex mito­ aerobic glycolysis to oxidative phosphorylation by restoring mitochon­ chondrial metabolism that is strongly connected to anabolic processes drial activity (Michelakis et al., 2008). MET blocks the mitochondrial and OXPHOS, with a cytochrome-containing respiratory chain (Tielens respiratory chain complex I thus provoking unbalanced ATP production and van Hellemond, 2009). Notwithstanding, the high metabolic de­ (Owen et al., 2000) and AMPK activation (Fryer et al., 2002). Finally, mand to support rapid multiplication in the mammalian host, turns SIR was included because of its direct inhibiting activity on mTOR (Mita glycolysis into an essential process for these amastigote-stage trypano­ et al., 2003). somatids (Verlinde et al., 2001; Subramanian et al., 2015; Shah-Simpson et al., 2017), with special interest for NADPH regeneration and for the 2. Material and methods correct functioning of their PPP (Maugeri et al., 2003, 2011). As it is the case for trypanosomatids, P. falciparum intraerythrocytic asexual forms To initially assess the aptitude of the known compounds 2-deoxy-D- are highly dependent on glucose (Sherman, 1998), and preliminary re­ Glucose, 3-bromopyruvic acid, lonidamine, sirolimus, dichloroacetic sults indicate that these parasites are aerobic glycolytic organisms with a acid and metformin to manage malaria, visceral leishmaniasis, and Af­ modified TCA mainly fueled by glutamine and glutamate and low rican and American trypanosomiasis, we first studied their associated in OXPHOS (Salcedo-Sora et al., 2014). Same as T. brucei bloodstream vitro effect. For that purpose, we tested the cytotoxicity of these com­ stage, P. falciparum glycolysis products are pyruvate and alanine. pounds to host cells of each in vitro culture model based on cancer However, P. falciparum has a functional lactate dehydrogenase which research literature, determining the range of possible therapeutic con­ transforms some of the generated pyruvate into lactate that is excreted centrations. Once established, these compounds were assayed on in vitro to the external milieu (Salcedo-Sora et al., 2014; Penkler et al., 2015). As cultures of L. infantum promastigotes, intra-macrophagic L. infantum a result, glycolysis appears to be a particularly suitable target to be amastigotes, intra-cellular T. cruzi amastigotes, bloodstream T. brucei blocked, as these parasites tend to be more dependent on this pathway trypomastigotes, and P. falciparum-infected red blood cells. Parasites than the quiescent cells from differentiated tissues of their hosts (Renslo were co-cultured for 48–72 h with the studied compounds and their and McKerrow, 2006). efficacy was recorded as the ability of each one to reduce the number of Interestingly, an increased glucose consumption and glycolytic ac­ parasite forms. IC50 values were calculated and compared between tivity is also a common feature in some studied disease models, as it is models and with first-line drugs. Each test was performed three times. the case of cancer. Most cancer cell types are known to have an enhanced and modified glycolytic metabolism, even in the presence of oxygen 2.1. Parasites and mammalian cell cultures (aerobic glycolysis), resulting in glucose transformation to lactate (Warburg et al., 1924; Warburg, 1956). In addition, there is a reduced L. infantum JPC strain (MCAN/ES/1998/LLM-724) promastigotes flux of carbon to the mitochondrion which is highly involved in anabolic were cultured at 26 ◦ C in R15 medium [RPMI 1640 medium (Gibco®) processes (Vander Heiden et al., 2009). Hence, glycolysis turns into a supplemented with 15% heat-inactivated fetal bovine serum (FBS) therapeutic target for proliferation-committed cells. (Gibco®), 2% HEPES 1M (Gibco®), 1% 10000 U/mL penicillin, and An imbalance in ATP production —either due to glucose deprivation, hypoxia, pharmacological disruptors, such as glycolysis inhibitors, or bioenergetic modulators— leads to activation of AMP-activated protein Table 1 Selected drugs and their energetic and biomass production-related targets. kinase (AMPK), an upstream protein of the mechanistic target of rapa­ mycin (mTOR) (Hardie, 2011). As in the case of glycolysis, the mTOR Compound Energy and biomass production-related target route could be also a main therapeutic target for 3-Bromopyruvic acid GAPDH (Ganapathy-Kanniappan et al., 2009); proliferation-committed cells, since it acts as a key homeostatic sensor Hexokinase II (Ko et al., 2001) that integrates microenvironmental and proliferation signals with bio­ 2-Deoxy-D-glucose Glycolysis (Nirenberg and Hogg, 1958) Lonidamine Hexokinase I (Paggi et al., 1988) energetic metabolism to regulate biomass production and cell survival Sodium dichloroacetate PDK (Michelakis et al., 2008) (Sabatini and Laplante, 2012). Furthermore, the mTOR route is strongly Metformin Complex I of the OXPHOS (Owen et al., 2000); related to glycolysis as it is able to regulate the expression of many AMP-activated protein kinase (Fryer et al., 2002) glucose transporters and some glycolysis enzymes through the Sirolimus mTOR (Mita et al., 2003) Hypoxia-inducible Factor 1 (HIF-1), thus favoring aerobic glycolysis Acronyms: GAPDH (glyceraldehyde-3-phosphate dehydrogenase), PDK (pyru­ over mitochondrial respiration (Düvel et al., 2010). vate dehydrogenase kinase), OXPHOS (oxidative phosphorylation), AMP These bioenergetics pathways have been proposed as potential and (adenosine monophosphate), and mTOR (mechanistic target of rapamycin). 18 A. Martínez-Flórez et al. International Journal for Parasitology: Drugs and Drug Resistance 14 (2020) 17–27 10,000 μg/mL streptomycin (Gibco®)]. Weekly passages were per­ interferences (Ganapathy-Kanniappan et al., 2010). Alamar Blue was formed. Metacyclic promastigotes for in vitro infections were obtained added in an amount equal to 10% of the culture volume and plates were from a 6-day-old stationary culture. U937 human macrophages derived kept in the incubator for 24 h. Absorbance was read at 570 nm and from a Caucasian histiocytic lymphoma were obtained from the Euro­ 600 nm, and results were calculated following the manufacturer’s pean Collection of Cell Cultures (ECACC). Cells were cultured in RU937 recommendations. media (RPMI-1640 medium supplemented with 10% FBS and 1% For cytotoxicity assays on Vero cells, 1.7 × 104 cells were seeded in Penicillin/Streptomycin), and kept in a humid atmosphere at 37 ◦ C and black, clear-bottom 96-well plates and incubated overnight at 37 ◦ C and 5% CO2. Before carrying out each experiment, 1 × 106 U937 cells were in 5% CO2. Serial dilutions of the drugs were added and incubated at seeded in a 25 mm2 culture flask and differentiated in a monolayer by 37 ◦ C and in 5% CO2 for 72 h. Alamar Blue (AbD Serotec) was then adding Phorbol 12-myristate 13-acetate (PMA) at a final concentration added and the measurements performed 6 h later as described above. of 50 nM for 48 h and then washed twice with 1 × phosphate buffered In order to evaluate the compatibility of the selected drugs and saline (1 × PBS), and fresh media added (Maia et al., 2007). erythrocytes, hemolysis was studied in vitro by direct spectrophotom­ P. falciparum 3D7 (obtained from MR4-ATCC) chloroquine-sensitive etry. RBCs (3% hematocrit) were cultured in round-bottom 96-well parasites were cultured with human red blood cells (RBCs) obtained plates and incubated for 48 h with serial dilutions of the studied com­ from the Blood and Tissue Bank (Catalonia, Spain), after approval from pounds in a humid atmosphere with concentrations of 5% CO2, 2% O2, Hospital Clínic of Barcelona’s Clinical Research Ethics Committee. and 93% N2. Each sample was assayed in duplicate, and a positive Parasites were co-cultured with B + RBCs (3% hematocrit) in RPMI control for hemolysis was included by adding autoclaved distilled water medium with 10% human AB + type serum, and kept at 37 ◦ C in an to a well containing RBCs. The Harboe method was used to assess hae­ atmosphere of 93% N2, 2% O2, and 5% CO2. In order to select ring stage molysis (Harboe, 1959). parasites, a sorbitol synchronization protocol was followed, keeping infected erythrocytes at 37 ◦ C for 10 min with 10 times its volume of 2.4. Effect of bioenergetic modulators on L. infantum sorbitol 5% (Trager and Jensen, 1976). The T. brucei brucei 427 strain was maintained in HMI-9 culture The killing effect of these drugs was assessed in vitro against the two medium [Iscove’s modification of DMEM (IMDM; Cell Gro) supple­ stages of the biological cycle of Leishmania: motile promastigotes in the mented with 10% FBS, 10%, Serum plus (SAFC), 0.05 mM Bath­ sand-fly vector and intracellular amastigotes in the host. ocuproinesulfonate, 1.5 mM L-cysteine, 1 mM hypoxanthine, 0.2 mM β-mercaptoethanol, 0.16 mM thymidine 1 mM pyruvate)] and passages 2.4.1. Anti-L. infantum promastigotes drug test were performed twice weekly. Promastigotes from a 4-day culture were seeded at 1 × 105 parasites CL strain T. cruzi overexpressing a tdTomato red fluorescent protein per well in a 96-well microplate with increasing concentrations of drugs was used (Canavaci et al., 2010). Parasites were maintained in Vero cell for 48 h at 26 ◦ C. AmB at 0.25 μM was used as positive control. In order cultures through weekly passage. Fibroblast-like kidney cells from Af­ to determine parasite viability, the phosphatase activity assay was per­ rican green monkey (Vero cells, ECACC) were purchased from Sigma formed. Briefly, parasites were completely lysed using a solution made Aldrich (Ref 84113001). Vero cells were maintained in culture media of 1% Triton X-100 and 2.9% sodium citrate in distilled water. Then, [RPMI 1640 supplemented with 10% fetal bovine serum, gentamycin 5 mM of p-nitrophenyl phosphate was added to each well. In viable cells, (25 μg/mL), Penicillin/Streptomycin (0.1 mg/mL), L-glutamine (2 mM), p-nitrophenyl phosphate was hydrolyzed by intracellular acid phos­ sodium pyruvate 1 mM), and 2-mercaptoethanol (50 μM)] at 37 ◦ C and phatases to p-nitrophenol. After 2 h of incubation at 37 ◦ C, the reaction 5% CO2. Passages were performed twice weekly. After 4–5 days of was stopped by adding 60 μL of NaOH 1 N to each well. Data were culture, trypomastigotes released from Vero cells were collected to carry collected by reading the optic density at a wavelength of 405 nm (Yang out the drug screening assay. et al., 1996). 2.2. Drugs 2.4.2. Drug test on L. infantum intra-macrophage amastigotes Cell line U397 was cultured in 25 mm2 flasks and differentiated as 3BP (CAS N1113-59-3), DCA (CAS N 79-43-6), 2DG (CAS N 154-17- explained above. Metacyclic promastigotes (6 days’ culture) were added 6), LND (CAS N 50264-69-2), MET (CAS N 1115-70-4), and SIR (CAS N to the pre-washed differentiated cell culture at a parasite:cell ratio of 53123-88-9) (Table 1) were purchased from Sigma-Aldrich and freshly 10:1, and co-cultured for 24 h. They were then washed with 1 × PBS to prepared before use in the appropriate culture media. The following discard non-internalized promastigotes. Infected macrophages were re- reference drugs were selected as positive controls: chloroquine (CQ) for suspended in RU937 and incubated in 8 well chamber slides at a den­ P. falciparum, benznidazole (BZD) for T. cruzi, meglumine antimoniate sity of 5 × 104 with increasing, but non-toxic to human macrophages, (Glucantime®, Sanofi-Avensis) for L. infantum intracellular amastigotes, concentrations of 3BP, DCA, 2DG, SIR, LND, and MET for 48 h. Meglu­ amphotericyn B (AmB) (Sigma-Aldrich) for L. infantum promastigotes, mine antimoniate at a concentration of 273 μM was used as positive and suramin (SUR) for T. brucei in vitro experiments. control. Each concentration was tested in duplicate. Preparations were fixed with methanol and stained with Giemsa 11%. The numbers of 2.3. Cytotoxicity to mammalian cells infected macrophages and the intracellular parasites were recorded by direct microscopic count of 200 cells per sample. In order to test toxicity on human macrophages, 5 × 104 U937 differentiated cells per well were seeded in 96-well microplates, and 2.5. P. falciparum growth inhibition assay cultured with increasing concentrations of 3BP, DCA, 2DG, MET, LND, and SIR diluted in RU937 culture media. Each concentration was RBCs were cultured in RPMI and spiked P. falciparum ring infected assayed in triplicate. The cells were kept at 37 ◦ C in a 5% CO2 atmo­ erythrocytes to a final hematocrit of 3% and 0.8% infected blood cells. sphere. Cell viability was determined 48 h later by the MTT assay. Cultures were then transferred to 96 microwell plates and exposed to Briefly, Thiazolyl Blue Tetrazolium Bromide (MTT) was added to each increasing concentrations of 3BP, DCA, 2DG, MET, LND, and SIR pre­ well at a final concentration of 0.5 mg/mL. After 4 h of incubation in the pared in media culture. CQ (80 nM) was used as reference drug to dark, 100 μL of HCl 0.1 N in isopropanol +10% Triton X-100 was added achieve parasite growth inhibition values above 90% (>IC90). Each to each well to solubilize the resulting formazan crystals, and then read concentration was assayed in triplicate and kept at 37 ◦ C in a 5% CO2, at a wavelength of 570 nm (reference 690 nm). The Alamar Blue 93% N2, and 2% O2 atmosphere. After 48 h, parasitemia was determined (Invitrogen) assay was performed in the case of 3BP, in order to avoid by fluorescence-assisted cell sorting [FACS] (BD LSRFortessa™ cell 19 A. Martínez-Flórez et al. International Journal for Parasitology: Drugs and Drug Resistance 14 (2020) 17–27 analyzer, Becton Dickinson), after staining infected RBCs with SYTO 11 concentration of 2 μM. Each concentration was assayed in quadrupli­ (Molecular Probes, Life Technologies). Non-infected RBCs and infected cate. After 72 h, fluorescence was measured (Ex = 544 nm; non-treated RBCs were used as controls. Em = 612 nm). 2.6. Screening of drugs against T. brucei 2.8. Data analysis Cultured parasites were collected and spun for 10 min at 900 g to In all cases, results were expressed as the percentage inhibition of eliminate supernatant and were then cultivated at 5 × 105 parasites per parasite growth in relation to the infected but non-treated controls for well in a 96-well white, sterile plate in warm medium with serial di­ each assay (100 − [(mean parasitemia treated/mean parasitemia con­ lutions of the selected compounds. Suramin (SUR) at 100 μM was used as trol) × 100]). In case of Plasmodium viability assays, values were firstly positive control. Each concentration was tested in triplicate. After 24 h, standardized based on that obtained with CQ for eliminating technique- Alamar Blue (Sigma) was added as previously described, and plates were dependent variability (post-treatment presence of DNA debris in sam­ incubated at 37 ◦ C for 4 h before fluorescence was read at excitation (Ex) ples with death parasites). 530 nm and 590 nm emission (Em) wavelength. IC50 values were determined by using a sigmoidal dose-response with variable slope equation (Y = Bottom + ((Top - Bottom)/(1 + 10 2.7. Drug test on intracellular amastigotes of T. cruzi (LogIC50 - X) - Hill Slope))) (GraphPad Prism Software v5). Gamma-irradiated (2200 rad) Vero cells were plated in 96-well, 3. Results black, clear-bottom plates at 3.6 × 104 cells per well and left overnight at 37 ◦ C, in 5% CO2 atmosphere. Vero cells were then infected with 3.1. Leishmanicidal activity of bioenergetic modulators on L. infantum 3.6 × 105 trypomastigotes of the CL tdTomato strain of T. cruzi per well promastigotes and intramacrophagic amastigotes for 5 h at 37 ◦ C and 5% of CO2. After infection, the plates were washed once with Hanks’ solution to eliminate extracellular parasites and The activity of DCA, 3BP, 2DG, LND, MET, and SIR on Leishmania cultured again in the presence of serial dilutions of the selected com­ promastigotes was assessed after culturing parasites with the drugs for pounds. Benznidazole was used as a positive control drug at a 48 h. Only 3BP and 2DG showed a slight parasite-killing effect at doses Fig. 1. Efficacy of DCA and 3BP on L. infantum-infected macrophages. Dose-response curves showing the inhibition of L. infantum intracellular amastigotes growth (1) and L. infantum-infected macrophages (2). The percentage inhibition was determined by comparing with untreated controls after 48 h of treatment with (A) DCA and (B) 3BP. Each point represents the mean ± standard error of the mean (SEM) obtained from three independent tests where each concentration was tested in duplicate. 20 A. Martínez-Flórez et al. International Journal for Parasitology: Drugs and Drug Resistance 14 (2020) 17–27 of 37 μM and 0.2 mM, respectively, and did not reach the values ob­ 3.4. Effect of bioenergetic modulators on T. cruzi amastigotes tained by the control drug AmB (0.25 μM) (36% survival rate) (data not shown). SIR, DCA, MET, and LND did not detectably impair L. infantum LND and 2DG produced the highest reduction in T. cruzi amastigote promastigote survival. numbers, reaching inhibition values of 99.4% (IC50 = 82.41 μM) and When these compounds were tested in an infection assay of L. 89.3% (IC50 = 7.27 mM), respectively. DCA and MET also decreased infantum intra-macrophage amastigotes, an inhibitory, dose-dependent parasite survival but did not reach the level of the positive control. DCA effect was observed in infected and treated cells with 3BP and DCA. action required high concentrations (up to 100 mM) in order to reach the The percentages of infected macrophages after 48 h of treatment with highest value of 74.1% (IC50 = 25.82 mM), while MET only produced the highest, but non-cytotoxic doses of these compounds, were reduced mild inhibition with a maximum of 51.7% at 20 mM (IC50 = 18.48 mM) by 74.4% for 25 mM of DCA and 48.4% for 75 μM of 3BP. Concurrently, (Table 2). A sustained mild reduction in fluorescence was detected at the number of intracellular amastigotes decreased by 90.7% for DCA higher concentrations, which may suggest a trypanosomastatic effect and 78.3% for 3BP, values 31.1% and 18.6% higher than those obtained (Fig. 4). In contrast, 3BP and SIR did not show consistent anti­ with meglumine antimoniate at 273 μM, respectively (Fig. 1). Their trypanosomal activity (data not shown). parasite-killing activity is translated to IC50 values of 631.5 μM for DCA and 24.94 μM for 3BP (Table 2). The other tested drugs only showed a 4. Discussion slight effect, and their anti-parasitic activity was lower than that pro­ duced by meglumine antimoniate (data not shown). SIR did not show The aim of this study was to demonstrate and compare the in vitro any parasiticidal effect although a modest increase of the parasite effectiveness of different bioenergetic modulators previously used in burden and number of infected macrophages were observed at a con­ diagnostic and anticancer studies on L. infantum, P. falciparum, T. cruzi, centration of 10 μM (data not shown). and T. brucei. These drugs were selected because of their modulating activity on enzymatic pathways known to be related to ATP production, 3.2. Antiplasmodial activity of bioenergetic modulators proliferation, and cell survival strategies. To the best of our knowledge, this is the first time that some of these bioenergetic modulators have P. falciparum intraerythrocytic stages were sensitive in a dose- been specifically used for this purpose. DCA has demonstrated a dependent manner to DCA, 2DG, SIR, and MET and reached growth remarkable in vitro parasiticidal activity in models of highly replicative inhibition values close to those obtained by CQ. IC50 values (Table 2) stages of L. infantum, P. falciparum, T. cruzi, and T. brucei. Moreover, indicate P. falciparum is highly susceptible to SIR (IC50 = 2.50 μM) while although 2DG has been utilized several times to measure glucose uptake other assayed compounds as DCA (IC50 = 5.39 mM), 2DG in different cellular models, this is the first time it has been specifically (IC50 = 4.19 mM), and MET (IC50 = 1.32 mM) needed higher concen­ employed for a parasite-killing indication. It is also the first time that trations to inhibit parasite growth. A slight effect on parasite growth LND activity has been tested in L. infantum and P. falciparum in vitro inhibition was observed at high concentrations for 3BP. LND showed a models, and in the case of 3BP, additionally in a T. cruzi in vitro model. peak of antimalarial activity at 300 μM (IC50 = 209.13 μM) (Fig. 2). Although biguanides antiplasmodial properties are widely known, this is the first study where MET activity has been assayed in a L. infantum and 3.3. Anti-T. brucei activity of bioenergetic modulators T. brucei in vitro model. Our results show that bioenergetic modulators have greater efficacy DCA and SIR achieved complete clearance of cultured T. brucei brucei on those parasites that are more dependent on glycolysis, as is the case parasites at 12.5 mM (IC50 = 1.24 mM) and 6.85 μM (IC50 = 2.14 μM) for T. brucei and P. falciparum (Michels et al., 2006; Schalkwyk et al., concentrations, respectively. Similar values were also obtained for LND 2008; Salcedo-Sora et al., 2014). Apart from the specific efficacy of the (IC50 = 26.76 μM) and 3BP (IC50 = 76.57 μM), showing 97.7% and compounds in the inhibition of target enzymes, such differential para­ 93.2% parasite-killing activity, respectively. MET only produced 57% siticidal activity profile of the studied compounds may be due to the parasite elimination at 20 mM (IC50 = 17.30 mM) (Table 2). No parasite- contrasting life-condition adaptations and physiological particularities killing activity was detected for 2DG. SUR 100 μM was used as a control of each organism. Thus, whereas T. cruzi and Plasmodium spp. are hosted drug, inducing nearly complete parasite clearance (96%) (Fig. 3). in the cytoplasm of the mammalian host cell during their asexual intracellular stage, and T. brucei prefers multiplying in the host blood­ stream thereby exploiting its glucose serum levels, Leishmania spp. show Table 2 IC50 values obtained from selected drugs during protozoan parasites in vitro studies. COMPOUND IN VITRO PARASITE MODEL Range of tested concentrations P. falciparum T. cruzi intracelular T. brucei L. infantum intracelular L. infantum infected Intraerythrocytic stage amastigotes Bloodstream stage amastigotes macrophages IC50 IC50 IC50 IC50 IC50 5 5 5 3BP – – 7.66 ± 0.22 £ 10− 2.49 ± 0.23 × 10− >7.50 × 10− 7.50 × 10− 5 to 2.40 × 10− 8 3 2 3 4 3 DCA 5.39 ± 0.67 × 10− 2.58 ± 0.59 × 10− 1.24 ± 0.05 × 10− 6.32 ± 1.63 × 10− 2.69 ± 1.58 £ 10− 2.50 × 10− 2 to 1.00 × 10− 4 3 3 2DG 4.19 ± 0.21 × 10− 7.27 ± 0.47 £ 10− – – – 1.60 × 10− 2 to 2.00 × 10− 3 4 5 5 LND 2.09 ± 0.13 × 10− 8.24 ± 0.15 × 10− 2.68 ± 0.26 × 10− – – 3.00 × 10− 4 to 1.2 × 10− 6 3 2 2 MET 1.32 ± 0.18 × 10− 1.85 ± 0.18 × 10− 1.73 ± 0.15 × 10− – – 4.00 × 10− 2 to 6.4 × 10− 6 6 6 SIR 2.50 ± 0.47 × 10− – 2.14 ± 0.11 £ 10− – – 1.00 × 10− 5 to 0.10 × 10− 6 Abbreviations: IC50 values are given in molar (M) ± standard deviation. Acronyms: 3BP (3-bromopyruvic acid), DCA (dichloroacetic acid), 2DG (2-deoxy-D-glucose), LND (lonidamine), MET (metformin), and SIR (sirolimus). 21 A. Martínez-Flórez et al. International Journal for Parasitology: Drugs and Drug Resistance 14 (2020) 17–27 Fig. 2. Growth inhibition of P. falciparum. Dose-dependent effect of (A) dichloroacetic acid, (B) sirolimus, (C) metformin, (D) lonidamine, and (E) 2-deoxy-D-glucose on P. falciparum in vitro assays. Data was obtained by flow cytometry and the inhibition of parasite growth was standardized with CQ-treated results and then calculated with respect to infected but untreated RBCs. Each concentration was tested in triplicate and data represent mean ± SEM of three independent experiments. 22 A. Martínez-Flórez et al. International Journal for Parasitology: Drugs and Drug Resistance 14 (2020) 17–27 Fig. 3. Dose-response curves of anti-trypanosomal activity on T. brucei cultured parasites. Fluorescence values, obtained after culturing parasites with (A) dichloroacetic acid, (B) sirolimus, (C) metformin, (D) lonidamine, and (E) 3-bromopyruvic acid, were compared to non-treated controls to indicate their T. brucei- killing activity. Suramin was used as a positive control. Each point represents the mean ± standard error of the mean (SEM) obtained from three independent tests where each concentration was tested in triplicate. special metabolic features as a result of their adaptation to the low vacuole where Leishmania amastigotes reside. glucose concentration in PV (Naderer and McConville, 2008). Although DCA is the only bioenergetic modulator we have found to have glucose oxidation plays an important role in Leishmania spp. (McCon­ antiparasitic activity at the asexual replicative stages within the host of ville et al., 2015), these parasites were less susceptible to glycolysis in­ the three genera of parasites studied. It reduced the parasite burden of hibitors than other assayed parasites. The apparent lack of activity of infected cells in trypanosomatids and the number of infected cells for L. these compounds may be due to their low access to the parasitophorus infantum in a dose-dependent manner. The main target of DCA is 23 A. Martínez-Flórez et al. International Journal for Parasitology: Drugs and Drug Resistance 14 (2020) 17–27 Fig. 4. Parasite-killing activity on T. cruzi intracellular amastigotes. The dose-dependent reduction in emitted fluorescence with respect to non-treated controls represents the parasite-killing power of (A) dichloroacetic acid, (B) lonidamine, (C) metformin, and (D) 2-deoxy-D-glucose. Each concentration was assayed in quadruplicate in three independent tests and mean values and SEM are represented in the figure. pyruvate dehydrogenase kinase (PDK) (Stacpoole et al., 1998). PDK drug could, therefore, be enhanced by a lack of key proteins for parasite inhibits the activity of pyruvate dehydrogenase complex (PDH) which survival. Whilst several studies have shown the efficacy of DCA in regulates the entrance of pyruvate —the end-product of glycolysis— to rapidly reducing the concentration of lactic acidosis, a common the mitochondria and its transformation to acetyl-CoA to start the citric complication of severe malaria strongly related to death in children acid cycle. Thus, when DCA inhibits PDK it is redirecting pyruvate to (Agbenyega et al., 2003), this is the first time that its antiplasmodial mitochondria and restoring mitochondrial respiration. Genes encoding activity has been demonstrated. for PDK have been found in T. brucei, T. cruzi, and L. major (Parsons In addition, DCA activity may also be boosted through actions trig­ et al., 2005). However, bloodstream T. brucei is known to have an gered in the host cell. Many protozoans such as Leishmania and Toxo­ alternative mitochondrial oxidase non-dependent on pyruvate meta­ plasma activate and stabilize HIF1A in the host cell (Wiley et al., 2010; bolism, and therefore, PDK is not involved (Chaudhuri et al., 1998). This Singh et al., 2012). HIF1A controls the cellular response to low oxygen fact suggests there must be other possible targets of DCA still unknown stress inducing the transcription of genes related to the metabolic switch in this stage of T. brucei. The high IC50 observed for DCA in T. cruzi may from oxidative phosphorylation to glycolytic profile, angiogenesis, and suggest that this is not an essential target at this stage, however low apoptosis inhibition (Semenza, 2012). Such a strategy may help these accessibility of the compound to the parasite enzyme and or its speci­ parasites to survive by taking advantage of the increase in macromole­ ficity may also be a cause for its reduced efficacy. No PDK genetic se­ cules provided by glycolytic metabolism and the induced inhibition of quences have been identified in P. falciparum (Ward et al., 2004). host cell apoptosis. It has been recently described that the DCA However, our results show a dose-dependent inhibition of parasite anti-tumor effect also takes place by decreasing HIF1A activity in cancer growth by DCA on the intra-erythrocytic stage of P. falciparum, sug­ cells (Sutendra et al., 2013). As a consequence, the observed DCA gesting additional effects of this compound on other essential steps for parasite burden reduction may be related to a decreased HIF1A activity parasite survival in a non-host cell dependent manner due to the basic and, in turn, the loss of the metabolic advantages that the host cell metabolism of erythrocytes. The ability of this compound to restore provides to its undesirable guests. mitochondrial activity may also lead to a decrease of pyruvate avail­ We found that the glycolysis inhibitor 3BP showed antiparasitic ac­ ability for other biosynthetic pathways, such as alanine biosynthesis tivity on T. brucei and L. infantum intracellular amastigotes, with po­ (Tielens and van Hellemond, 2009). The parasite-killing activity of this tential for VL and African trypanosomiasis treatment. Previous studies 24 A. Martínez-Flórez et al. International Journal for Parasitology: Drugs and Drug Resistance 14 (2020) 17–27 have reported some activity on T. brucei, inhibiting motility and growth energetic stress, and activation of AMPK, thus causing down regulation at 13.4 μM (Barnard et al., 1993), which corroborates our results. of ATP-consuming processes and pushing the cell into a quiescent state Nevertheless, we found no efficacy of 3BP against either T. cruzi or P. (Fryer et al., 2002). However, if cells lack the ability to cope with en­ falciparum asexual replicative forms despite these parasites being also ergetic stress, as may be the case for tumor cells and highly glycolytic highly dependent on glycolysis. The main target of 3BP is GAPDH, an parasites, a metabolic crisis develops leading to death. In addition, the enzyme of the glycolytic pathway which, in the case of kinetoplastida, is inhibition of the mitochondrial respiratory-chain complex I by MET in localized mainly in the glycosomes. Some Leishmania species also have a host cells reduces O2 consumption as the final acceptor of electrons in cytosolic GAPDH isoenzyme shown to play a significant role in VL the electron transport chain. The subsequent redistribution of intracel­ (Zhang et al., 2013). The efficacy we observed on Leishmania may lular O2 promotes hydroxylation of HIF1A by procollagen-proline therefore be due to the inhibition of both isoenzymes. A predictive dioxygenase (P4HA) and its destruction in the proteasome (Takiyama model led to the conclusion that slight depletions of glycosomal GAPDH et al., 2011). As described above, HIF1A appears to be a key component result in complete impairment of growth and ATP synthesis in L. for parasite survival, perhaps opening new avenues for anti-parasite infantum amastigotes (Subramanian et al., 2015), thus reinforcing the therapeutic intervention. In the case of P. falciparum, there is an alter­ role of this enzyme for parasite-killing purposes. Furthermore, an in­ native NADH dehydrogenase in substitution of the classic complex I crease in GAPDH expression in Leishmania spp. has been related to a (Biagini et al., 2006). P. falciparum relies on its respiratory chain to resistance to NO (Rios et al., 2015), a basic molecule for intracellular support pyrimidine biosynthesis, and to maintain the essential mito­ parasite clearance (Mauël et al., 1991). Blocking GAPDH with 3BP chondrial membrane potential. Antiplasmodial activity of MET is would avoid this resistance and allow parasite-killing. Despite 3BP explained by the disruption on metabolic pathways related to folates, apparently being safe in rodents, where only a reduction in body weight responsible for purine and pyrimidine synthesis, for catabolism of was recorded (Schaefer et al., 2012), 3BP could not be a therapeutic several amino acids, and for glutathione synthesis, thus hindering option because of its toxicity in humans (Barnard et al., 1993). However, parasite survival (Bridges et al., 2014). its molecular target can still be considered for further research. The mTOR route is a highly conserved pathway in eukaryotic or­ 2DG has been used as a substrate to measure glucose intake in many ganisms, that regulates the balance between catabolism and anabolism cell systems due to its condition as a mannose analog that cannot be according to cellular needs by acting on ATP-generating routes, DNA further metabolized, and also in PET scanning for cancer diagnostic transcription, synthesis of biological molecules, autophagy, and cell purposes. It blocks glycolysis because it cannot be further metabolized death (Sabatini and Laplante, 2012). In trypanosomatids, TORC2 after phosphorylation by the first enzyme of the glycolysis pathway, orthologue is the only TOR complex susceptible to SIR inhibition. hexokinase (HK) (Nirenberg and Hogg, 1958). HK isotypes are overex­ TORC2 is involved in cytoskeletal actin reorganization, playing a key pressed in cancer cells and bound to the mitochondria, thus contributing role during host cell invasion and parasite survival (Barquilla et al., to the maintenance of the Warburg effect (Bustamante and Pedersen, 2008). In our model, low concentrations of SIR were needed to induce T. 1977). LND inhibits HK1 mitochondrial-bounding in tumor cells (Paggi brucei parasite depletion. The sensitivity of this parasite stage to SIR was et al., 1988), and has been reported to also inhibit that of T. brucei already known as a previous study pointed to an even lower IC50 value (Chambers et al., 2008). The efficacy of LND on trypanosomatids has than the one we obtained (IC50 = 152 nM) (Barquilla et al., 2008). been described and tested on several species, including T. cruzi epi­ However, this may be reflecting the different sensitivity of the different mastigotes (Turrens, 1986), T. brucei bloodstream and procyclic forms T. brucei strains used. Curiously enough, SIR did not affect the viability (Chambers et al., 2008) and L. mexicana promastigotes (Turrens and of the intracellular forms of the other trypanosomatids analysed.In Cazzulo, 1987). We found an IC50 for T. cruzi amastigotes very close to contrast, an increase of parasite load was observed in the case of Leish­ that previously observed in epimastigotes (IC50 = 80 μM) (Turrens, mania. Similar results have been reported in other intravesicular para­ 1986) and even lower for BS forms of T.brucei in comparison to former sites, such as different Leishmania species (Schaible et al., 1999; Pinheiro authors (IC50 = 50 μM) (Chambers et al., 2008). In contrast, no effect et al., 2009; Jaramillo et al., 2011) or even in P. berghei during its was detected on L. infantum promastigotes. Results previously presented intrahepatic stage when the parasite inhabits non-acidified PV (Hanson by other authors demonstrate that only a leishmaniostatic effect was et al., 2013). It has been suggested that mTOR inhibition by SIR triggers observed for L. mexicana promastigotes at concentrations as high as autophagy and other processes in the host cell, providing them with 0.5 mM (Turrens and Cazzulo, 1987). Both 2DG and LND showed anti­ nutrients for their survival and parasite replication (Brunton et al., plasmodial activity on P. falciparum. 2DG has been previously reported 2013). Such processes in the host cell may provide a feasible explanation to interfere with glucose uptake in P. falciparum, with parasite glycol­ since autophagy has been reported to promote the generation and ysis, and with the synthesis of glycosylated macromolecules that play a maturation of replicative vacuoles of other intracellular pathogens, such key role in the intraerythrocytic stage development of trophozoites, as Coxiella sp. (Gutierrez et al., 2005). Moreover, the mTOR route is therefore reducing their viability (Naik et al., 2000; Schalkwyk et al., related to the production of NO in innate immune cells (Weichhart et al., 2008). This is the first time LND has been used as an antiplasmodial 2008), thus impairing intracellular parasite clearance by blocking compound. Because it resulted in 50% reduction of P. falciparum mTOR activity when SIR is administered. viability at concentrations of 209.13 μM, it emerges as a potential In case of the intraerythrocytic stages of P. falciparum, our results candidate for future malaria treatment. concur with previous studies, obtaining an IC50 around 2,5 μM (Bell Whilst biguanides were initially developed as a potential treatments et al., 1994; Yap et al., 2014). Erythrocytes have high avidity for SIR, against malaria, MET’s ability to decrease blood sugar levels and the therefore most of it reaching the bloodstream is sequestered in RBCs and discovery of more powerful candidates to fight plasmodium, has resul­ bound to their immunophilin binding protein FKBP12 (Trepanier et al., ted in its employment for type II diabetes control. In this study, we have 1998). RBCs have an iron dependent mTOR pathway that regulates confirmed the previously described MET antiplasmodial activity hemoglobin production and erythrogenesis. However, SIR has shown (Sweeney et al., 2003) and report, for the first time, its efficacy against only modest effect on RBCs in contrast to other mTOR inhibitors T. cruzi and T. brucei. MET is thought to block the mitochondrial responsible for anemia and the decrease in different blood cell pop­ respiratory-chain complex I. The presence of mitochondrial complex I in ulations (Knight et al., 2014). Besides, whilst some authors have even Trypanosomatida has been predicted, and all of its components identi­ indicated the expression of mTORC1 and C2 proteins in the proteome of fied, but electron transfer through complex I in T. brucei BSF has been mature erythrocytes, there is no evidence of functional proteins (Ales­ demonstrated not essential for this parasite stage viability (Surve et al., sandro et al., 2010). 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