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Initial studies on mechanism of action and cell death of active N-oxide-containing heterocycles in Trypanosoma cruzi epimastigotes in vitro

Published online by Cambridge University Press:  27 January 2014

DIEGO BENÍTEZ ,
GABRIELA CASANOVA ,
GONZALO CABRERA ,
NORBEL GALANTI ,
HUGO CERECETTO  and
MERCEDES GONZÁLEZ
DIEGO BENÍTEZ
Affiliation:
Grupo de Química Medicinal, Laboratorio de Química Orgánica, Facultad de Ciencias-Facultad de Química, Universidad de la República, Montevideo, Uruguay
GABRIELA CASANOVA
Affiliation:
Unidad de Microscopía Electrónica de Transmisión, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay
GONZALO CABRERA
Affiliation:
Facultad de Medicina, Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Universidad de Chile, Santiago, Chile
NORBEL GALANTI
Affiliation:
Facultad de Medicina, Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Universidad de Chile, Santiago, Chile
HUGO CERECETTO*
Affiliation:
Grupo de Química Medicinal, Laboratorio de Química Orgánica, Facultad de Ciencias-Facultad de Química, Universidad de la República, Montevideo, Uruguay
MERCEDES GONZÁLEZ*
Affiliation:
Grupo de Química Medicinal, Laboratorio de Química Orgánica, Facultad de Ciencias-Facultad de Química, Universidad de la República, Montevideo, Uruguay
*
*Corresponding authors: Grupo de Química Medicinal, Laboratorio de Química Orgánica, Facultad de Ciencias-Facultad de Química, Universidad de la República, Iguá 4225, 11400 Montevideo, Uruguay. E-mail: megonzal@fq.edu.uy and hcerecetto@cin.edu.uy
*Corresponding authors: Grupo de Química Medicinal, Laboratorio de Química Orgánica, Facultad de Ciencias-Facultad de Química, Universidad de la República, Iguá 4225, 11400 Montevideo, Uruguay. E-mail: megonzal@fq.edu.uy and hcerecetto@cin.edu.uy
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Summary

Chagas disease, endemic in 21 countries across Latin America, kills more people in the region each year than any other parasite-borne disease. Therapeutic options have problems ranging from toxicity, poor efficacy, drug resistance and high cost. Thus, cheaper and less toxic treatments are necessary. From our in-house chemical library of agents against Trypanosoma cruzi the most relevant N-oxide-containing heterocycles were selected for mode of action and type of death studies. Also included in these studies were two active nitrofuranes. Epimastigotes of T. cruzi were used as the biological model in this study. The metabolic profile was studied by 1H NMR in association with the MTT assay. Excreted catabolites data, using 1H NMR spectroscopy, showed that most of the studied N-oxides were capable of decreasing both the release of succinate and acetate shedding, the compounds therefore possibly acting on mitochondria. Only quinoxalines and the nitrofurane Nf1 showed significant mitochondrial dehydrogenase inhibitions, but with different dose–time profiles. In the particular case of quinoxaline Qx2 the glucose uptake study revealed that the integrity of some pathways into the glycosome could be affected. Optic, fluorescence (TUNEL and propidium iodide) and transmission electron microscopy (TEM) were employed for type of death studies. These studies were complemented with 1H NMR to visualize mobile lipids. At low concentrations none of the selected compounds showed a positive TUNEL assay. However, both quinoxalines, one furoxan and one benzofuroxan showed a necrotic effect at high concentrations. Curiously, one furoxan, Fx1, one benzofuroxan, Bfx1, and one nitrofurane, Nf1, caused a particular phenotype, with a big cytoplasmatic vacuole being observed while the parasite was still alive. Studies of TEM and employing a protease inhibitor (3-methyladenine) suggested an autophagic phenotype for Bfx1 and Nf1 and a ‘BigEye’ phenotype for Fx1.

Keywords

Trypanosoma cruziChagas diseaseN-oxidesnecrosisautophagyNMR
Type
Research Article
Information
Parasitology , Volume 141 , Issue 5 , April 2014 , pp. 682 - 696
Copyright
Copyright © Cambridge University Press 2014 

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References

Allen, C. L., Goulding, D. and Field, M. C. (2003). Clathrin-mediated endocytosis is essential in Trypanosoma brucei . EMBO Journal 22, 4991–5002.Google Scholar
Alvarez, V. E., Kosec, G., Sant'Anna, C., Turk, V., Cazzulo, J. J. and Turk, B. (2008 a). Autophagy is involved in nutritional stress response and differentiation in Trypanosoma cruzi . Journal of Biological Chemistry 283, 3454–3464.Google Scholar
Alvarez, V. E., Kosec, G., Sant'Anna, C., Turk, V., Cazzulo, J. J. and Turk, B. (2008 b). Blocking autophagy to prevent parasite differentiation. A possible new strategy for fighting parasitic infections? Autophagy 4, 361–363.Google Scholar
Aravena, C. M., Olea-Azar, C., Cerecetto, H., González, M., Maya, J. D. and Rodríguez-Becerra, J. (2011). Potent 5-nitrofuran derivatives inhibitors of Trypanosoma cruzi growth: electrochemical, spectroscopic and biological studies. Spectrochimica Acta A Molecular Biomolecular Spectroscopy 79, 312–319.Google Scholar
Benítez, D., Cabrera, M., Hernández, P., Boiani, L., Lavaggi, M. L., Di Maio, R., Yaluff, G., Serna, E., Torres, S., Ferreira, M. E., Vera de Bilbao, N., Torres, E., Pérez-Silanes, S., Solano, B., Moreno, E., Aldana, I., López de Ceráin, A., Cerecetto, H., González, M. and Monge, A. (2011). 3-Trifluoromethylquinoxaline N,N′-dioxides as anti-trypanosomatid agents. Identification of optimal anti-T. cruzi agents and mechanism of action studies. Journal of Medicinal Chemistry 54, 3624–3636.Google Scholar
Benítez, D., Pezaroglo, H., Martínez, V., Casanova, G., Cabrera, G., Galanti, N., González, M. and Cerecetto, H. (2012). Study of Trypanosoma cruzi epimastigote cell death by NMR-visible mobile lipid analysis. Parasitology 139, 506–515.Google Scholar
Blankenberg, F. G., Storrs, R. W., Naumovski, L., Goralski, T. and Spielman, D. (1996). Detection of apoptotic cell death by proton nuclear magnetic resonance spectroscopy. Blood 87, 1951–1956.Google Scholar
Boiani, L., Aguirre, G., González, M., Cerecetto, H., Chidichimo, A., Cazzulo, J. J., Bertinaria, M. and Guglielmo, S. (2008). Furoxan-, alkylnitrate-derivatives and related compounds as anti-trypanosomatid agents: mechanism of action studies. Bioorganic and Medicinal Chemistry 16, 7900–7907.Google Scholar
Boiani, M., Boiani, L., Alicia, A., Hernández, P., Chidichimo, A., Cazzulo, J. J., Cerecetto, H. and González, M. (2009). Second generation of 2H-benzimidazole 1,3-dioxide derivatives as anti-trypanosomatid agents: synthesis, biological evaluation, and mode of action studies. European Journal of Medicinal Chemistry 44, 4426–4433.Google Scholar
Boiani, M., Piacenza, L., Hernández, P., Boiani, L., Cerecetto, H., González, M. and Denicola, A. (2010). Mode of action of nifurtimox and N-oxide-containing heterocycles against Trypanosoma cruzi: is oxidative stress involved? Biochemical Pharmacology 79, 1736–1745.CrossRefGoogle ScholarPubMed
Bringaud, F., Rivière, L. and Coustou, V. (2006). Energy metabolism of trypanosomatids: adaptation to available carbon sources. Molecular and Biochemical Parasitology 149, 1–9.CrossRefGoogle ScholarPubMed
Caterina, M. C., Perillo, I. A., Boiani, L., Pezaroglo, H., Cerecetto, H., González, M. and Salerno, A. (2008). Imidazolidines as new anti-Trypanosoma cruzi agents: biological evaluation and structure-activity relationships. Bioorganic and Medicinal Chemistry 16, 2226–2234.Google Scholar
Cazzulo, J. J. (1992). Aerobic fermentation of glucose by trypanosomatids. FASEB Journal 6, 3153–3161.Google Scholar
Cerecetto, H. and González, M. (2008). Anti-T. cruzi agents: our experience in the evaluation of more than five hundred compounds. Mini Review in Medicinal Chemistry 8, 1355–1383.Google Scholar
Cerecetto, H. and González, M. (2010). Synthetic medicinal chemistry in Chagas’ disease: compounds at the final stage of “Hit-to-Lead” phase. Pharmaceuticals 3, 810–838.Google Scholar
Coustou, V., Besteiro, S., Rivière, L., Biran, M., Biteau, N., Franconi, J. M., Boshart, M., Baltz, T. and Bringaud, F. (2005). A mitochondrial NADH-dependent fumarate reductase involved in the production of succinate excreted by procyclic Trypanosoma brucei . Journal of Biological Chemistry 280, 16559–16570.Google Scholar
De Castro, S. L. and Meirelles, M. N. (1990). Mechanism of action of a nitroimidazole thiadiazole derivative upon Trypanosoma cruzi tissue culture amastigotes. Memórias do Instituto Oswaldo Cruz 85, 95–99.Google Scholar
Fernandez-Ramos, C., Luque, F., Fernández-Becerra, C., Osuna, A., Jankevicius, S. I., Jankevicius, V., Rosales, M. J. and Sánchez-Moreno, M. (1999). Biochemical characterisation of flagellates isolated from fruits and seeds from Brazil. FEMS Microbiology Letters 170, 343–348.Google Scholar
Frearson, J. A., Brand, S., McElroy, S. P., Cleghorn, L. A. T., Smid, O., Stojanovski, L., Price, H. P., Guther, M. L. S., Torrie, L. S., Robinson, D. A., Hallyburton, I., Mpamhanga, C. P., Brannigan, J. A., Wilkinson, A. J., Hodgkinson, M., Hui, R., Qiu, W., Raimi, O. G., van Aalten, D. M. F., Brenk, R., Gilbert, I. H., Read, K. D., Fairlamb, A. H., Ferguson, M. A. J., Smith, D. F. and Wyatt, P. G. (2010). N-myristoyltransferase inhibitors as new leads to treat sleeping sickness. Nature 464, 728–734.Google Scholar
González, M. and Cerecetto, H. (2011). Novel compounds to combat trypanosomatid infections: a medicinal chemical perspective. Expert Opinion on Therapeutic Patents 21, 699–715.Google Scholar
Hakumäki, J. M., Poptani, H., Sandmair, A. M., Ylä-Herttuala, S. and Kauppinen, R. A. (1999). 1H MRS detects polyunsaturated fatty acid accumulation during gene therapy of glioma: implications for the in vivo detection of apoptosis. Nature Medicine 5, 1323–1327.Google Scholar
Hernández, P., Rojas, R., Gilman, R. H., Sauvain, M., Lima, L. M., Barreiro, E. J., González, M. and Cerecetto, H. (2013). Hybrid furoxanyl N-acylhydrazone derivatives as hits for the development of neglected diseases drug candidates. European Journal of Medicinal Chemistry 59, 64–74.Google Scholar
Irigoín, F., Inada, N. M., Fernandes, M. P., Piacenza, L., Gadelha, F. R., Vercesi, A. E. and Radi, R. (2009). Mitochondrial calcium overload triggers complement-dependent superoxide-mediated programmed cell death in Trypanosoma cruzi . Biochemical Journal 418, 595–604.Google Scholar
Jiménez, V., Paredes, R., Sosa, M. A. and Galanti, N. (2008). Natural programmed cell death in T. cruzi epimastigotes maintained in axenic cultures. Journal of Cellular Biochemistry 105, 688–698.Google Scholar
Klionsky, D. J., Abdalla, F. C., Abeliovich, H., Abraham, R. T., Acevedo-Arozena, A., Adeli, K., Agholme, L., et al. (2012). Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 8, 445–544.Google Scholar
Maarouf, M., De Kouchkovsky, Y., Brown, S., Petit, P. X. and Robert-Gero, M. (1997). In vivo interference of paromomycin with mitochondrial activity of Leishmania . Experimental Cell Research 232, 339–348.Google Scholar
MacKenzie, S. H. and Clark, A. C. (2008). Targeting cell death in tumors by activating caspases. Current Cancer Drug Targets 8, 98–109.Google Scholar
Menna-Barreto, R. F., Salomão, K., Dantas, A. P., Santa-Rita, R. M., Soares, M. J., Barbosa, H. S. and de Castro, S. L. (2009 a). Different cell death pathways induced by drugs in Trypanosoma cruzi: an ultrastructural study. Micron 40, 157–168.Google Scholar
Menna-Barreto, R. F., Corrêa, J. R., Cascabulho, C. M., Fernandes, M. C., Pinto, A. V., Soares, M. J. and de Castro, S. L. (2009 b). Naphthoimidazoles promote different death phenotypes in Trypanosoma cruzi . Parasitology 136, 499–510.Google Scholar
Menna-Barreto, R. F. S., Beghini, D. G., Ferreira, A. T. S., Pinto, A. V., De Castro, S. L. and Perales, J. (2010). A proteomic analysis of the mechanism of action of naphthoimidazoles in Trypanosoma cruzi epimastigotes in vitro . Journal of Proteomics 73, 2306–2315.CrossRefGoogle ScholarPubMed
Merlino, A., Benítez, D., Chavez, S., Da Cunha, J., Hernández, P., Tinoco, L. W., Campillo, N. E., Páez, J. A., Cerecetto, H. and González, M. (2010). Development of second generation amidinohydrazones, thio- and semicarbazones as Trypanosoma cruzi-inhibitors bearing benzofuroxan and benzimidazole 1,3-dioxide core scaffolds. Medicinal Chemistry Communications 1, 216–228.CrossRefGoogle Scholar
Mesa-Valle, C. M., Castilla-Calvente, J., Sánchez-Moreno, M., Moraleda-Lindez, V., Barbe, J. and Osuna, A. (1996). Activity and mode of action of acridine compounds against Leishmania donovani . Antimicrobial Agents and Chemotherapy 40, 684–690.Google Scholar
Mikhailenko, V. M., Philchenkov, A. A. and Zavelevich, M. P. (2005). Analysis of 1H NMR-detectable mobile lipid domains for assessment of apoptosis induced by inhibitors of DNA synthesis and replication. Cell Biology International 29, 33–39.Google Scholar
Milkevitch, M., Shim, H., Pilatus, U., Pickup, S., Wehrle, J. P., Samid, D., Poptani, H., Glickson, J. D. and Delikatny, E. J. (2005). Increases in NMR-visible lipid and glycerophosphocholine during phenylbutyrate-induced apoptosis in human prostate cancer cells. Biochimica et Biophysica Acta 1734, 1–12.Google Scholar
Opperdoes, F. R. and Coombs, G. H. (2007). Metabolism of Leishmania: proven and predicted. Trends in Parasitology 23, 149–158.Google Scholar
Rodrigues, J. C. and De Souza, W. (2008). Ultrastructural alterations in organelles of parasitic protozoa induced by different classes of metabolic inhibitors. Current Pharmaceutical Design 14, 925–938.Google Scholar
Ricci, M. S. and Zong, W. X. (2006). Chemotherapeutic approaches for targeting cell death pathways. Oncologist 11, 342–357.Google Scholar
Sánchez-Moreno, M., Fernández-Becerra, C., Castilla, J. and Osuna, A. (1995). Metabolic studies by 1H NMR of different forms of Trypanosoma cruzi as obtained by ‘in vitro’ culture. FEMS Microbiology Letters 133, 119–125.Google Scholar
Sánchez-Moreno, M., Gómez-Contreras, F., Navarro, P., Marín, C., Ramírez-Macías, I., Olmo, F., Sanz, A. M., Campayo, L., Cano, C. and Yunta, M. J. (2012). In vitro leishmanicidal activity of imidazole- or pyrazole-based benzo[g]phthalazine derivatives against Leishmania infantum and Leishmania braziliensis species. Journal of Antimicrobial Chemotherapy 67, 387–397.Google Scholar
Tan, T. T. and White, E. (2008). Therapeutic targeting of death pathways in cancer: mechanisms for activating cell death in cancer cells. Advances in Experimental Medicine and Biology 615, 81–104.Google Scholar
WHO. (2013). http://www.who.int/neglected_diseases/diseases/chagas/en/index.html.Google Scholar
Yorimitsu, T. and Klionsky, D. J. (2007). Eating the endoplasmic reticulum: quality control by autophagy. Trends in Cell Biology 17, 279–285.Google Scholar
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