The antidepressant clomipramine induces programmed cell death in Leishmania amazonensis through a mitochondrial pathway

  • Jean Henrique da Silva Rodrigues
  • Nathielle Miranda
  • Hélito Volpato
  • Tânia Ueda-Nakamura
  • Celso Vataru NakamuraEmail author
Protozoology - Original Paper


Despite many efforts, the currently available treatments for leishmaniasis are not fully effective. To discover new medications, drug repurposing arises as a promising strategy. We present data that supports the use of the antidepressant clomipramine against Leishmania amazonensis. The drug presented selective activity at micromolar range against both the parasite forms and stimulated nitric oxide production in host macrophages. Regarding the mechanism of action, clomipramine led parasites do mitochondrial depolarization, which coupled with the inhibition of trypanothione reductase induced strong oxidative stress in the parasites. The effects observed in promastigotes included lipoperoxidation, plasma membrane permeabilization, and apoptosis hallmarks (i.e., DNA fragmentation, phosphatidylserine exposure, and cell shrinkage). The mechanism of action in both parasitic forms was quite similar, but amastigotes also exhibited energetic stress, reflected by a reduction of adenosine triphosphate levels. Such differential effects might be attributable to the metabolic particularities of each form of the parasitic. Ultrastructural alterations of the endomembrane system and autophagy were also observed, possibly indicating an adaptive response to oxidative stress. Our results suggest that clomipramine interferes with the redox metabolism of L. amazonensis. In spite of the cellular responses to recover the cellular homeostasis, parasites underwent programmed cell death.


Leishmaniasis Repurposing Repositioning Apoptosis Intracellular amastigotes isolation Oxidative stress 



We thank all the staffs of the “Laboratório de Inovação Tecnológica no Desenvolvimento de Fármacos e Cosméticos” and the “Complexo de Centrais de Apoio à Pesquisa (COMCAP-UEM)”.


This study was supported by grants of Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes), Financiadora de Estudos e Projetos (FINEP) and Programa de Núcleos de Excelência (PRONEX/Fundação Araucária).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Andrews KT, Fisher G, Skinner-Adams TS (2014) Drug repurposing and human parasitic protozoan diseases. Int J Parasitol Drugs Drug Resist 4:95–111CrossRefGoogle Scholar
  2. Barral A, Pedral-Sampaio D, Grimaldi-Júnior G, Momen H, McMahon-Pratt D, Ribeiro de Jesus A, Almeida R, Badaro R, Barral-Netto M, Carvalho EM (1991) Leishmaniasis in Bahia, Brazil: evidence that Leishmania amazonensis produces a wide spectrum of clinical disease. Am J Trop Med Hyg 44:536–546CrossRefGoogle Scholar
  3. Benson TJ, McKie JH, Garforth J, Borges A, Fairlamb A, Douglas KT (1992) Rationally designed selective inhibitors of trypanothione reductase. Phenothiazines and related tricyclics as lead structures. Biochem J 286:9–11CrossRefGoogle Scholar
  4. Berman JD, Dwyer DM, Wyler DJ (1979) Multiplication of Leishmania in human macrophages in vitro. Infect Immun 26:375–379Google Scholar
  5. Blommaart EF, Krause U, Schellens JP, Vreeling-Sindelárová H, Meijer AJ (1997) The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 inhibit autophagy in isolated rat hepatocytes. Eur J Biochem 243:240–246CrossRefGoogle Scholar
  6. Bringaud F, Rivière L, Coustou V (2006) Energy metabolism of trypanosomatids: adaptation to available carbon sources. Mol Biochem Parasitol 149:1–9CrossRefGoogle Scholar
  7. Desoti VC, Lazarin-Bidóia D, Martins-Ribeiro F, Martins SC, Rodrigues JHS, Ueda-Nakamura T et al (2015) The combination of vitamin K3 and vitamin C has synergic activity against forms of Trypanosoma cruzi through a redox imbalance process. PLoS One 10:1–23Google Scholar
  8. Duszenko M, Figarella K, Macleod ET, Welburn SC (2006) Death of a trypanosome: a selfish altruism. Trends Parasitol 22:536–542CrossRefGoogle Scholar
  9. El Mansari M, Blier P (2006) Mechanisms of action of current and potential pharmacotherapies of obsessive-compulsive disorder. Prog Neuro-Psychopharmacol Biol Psychiatry 30:362–373CrossRefGoogle Scholar
  10. Field MC, Carrington M (2009) The trypanosome flagellar pocket. Nat Rev Microbiol 7:775–786CrossRefGoogle Scholar
  11. Galluzzi L, Bravo-San Pedro JM, Vitale I, Aaronson S, Abrams JM, Adam D et al (2015) Essential versus accessory aspects of cell death: recommendations of the NCCD 2015. Cell Death Differ 22:58–73CrossRefGoogle Scholar
  12. Georgiadou SP, Makaritsis KP, Dalekos GN (2015) Leishmaniasis revisited: current aspects on epidemiology, diagnosis and treatment. J Transl Int Med 3:43–50CrossRefGoogle Scholar
  13. Hammond DJ, Cover B, Gutteridge WE (1984) A novel series of chemical structures active in vitro against trypomastigote form of Trypanosoma cruzi. Trans R Soc Trop Med Hyg 78:91–95CrossRefGoogle Scholar
  14. Hicks SW, Machamer CE (2005) Golgi structure in stress sensing and apoptosis. Biochim Biophys Acta 1744:406–414CrossRefGoogle Scholar
  15. Higgins SC, Pilkington GJ (2010) The in vitro effects of tricyclic drugs and dexamethasone on cellular respiration of malignant glioma. Anticancer Res 398:391–397Google Scholar
  16. Holzmuller P, Bras-Gonçalves R, Lemesre J (2006) Phenotypical characteristics, biochemical pathways, molecular targets and putative role of nitric oxide-mediated programmed cell death in Leishmania. Parasitology 132:19–32CrossRefGoogle Scholar
  17. Kaiser M, Mäser P, Tadoori LP, Loset JR, Brun R (2015) Antiprotozoal activity profiling of approved drugs: a starting point toward drug repositioning. PLoS One 10:1–16Google Scholar
  18. Klionsky DJ, Abdelmohsen K, Abe A, Abedin MJ, Abeliovich H, Acevedo Arozena A, Adachi H, Adams CM, Adams PD, Adeli K, Adhihetty PJ, Adler SG, Agam G et al (2016) Guidelines for use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 12:1–222CrossRefGoogle Scholar
  19. Lazarin-Bidóia D, Desoti VC, Ueda-Nakamura T, Dias Filho BP, Nakamura CV, Silva SO (2013) Further evidence of the trypanocidal action of eupomatenoid-5: confirmation of involvement of reactive oxygen species and mitochondria owing to a reduction in trypanothione reductase activity. Free Radic Biol Med 60:17–28CrossRefGoogle Scholar
  20. Lorente SO, Rodrigues JCF, Jime C, Joyce-menekse M, Rodrigues C, Croft SL et al (2004) Novel azasterols as potential agents for treatment of leishmaniasis and trypanosomiasis. Antimicrob Agents Chemother 48:2937–2950CrossRefGoogle Scholar
  21. Maag RS, Hicks SW, Machamer CE (2003) Death from within: apoptosis and the secretory pathway. Curr Opin Cell Biol 15:456–461CrossRefGoogle Scholar
  22. McConville MJ, Saunders EC, Kloehn J, Dagley MJ (2015) Leishmania carbon metabolism in the macrophage phagolysosome-feast or famine? F1000Research 4:1–11CrossRefGoogle Scholar
  23. Menna-Barreto RFS, de Castro SL (2014) The double-edged sword in pathogenic trypanosomatids: the pivotal role of mitochondria in oxidative stress and bioenergetics. Biomed Res Int 2014:1–14CrossRefGoogle Scholar
  24. Menna-Barreto RFS, Corrêa JR, Cascabulho CM, Fernandes MC, Pinto V, Soares MJ et al (2009) Naphthoimidazoles promote different death phenotypes in Trypanosoma cruzi. Parasitology 136:499–510CrossRefGoogle Scholar
  25. Moradin N, Descoteaux A, Beverley SM (2012) Leishmania promastigotes: building a safe niche within macrophages. Front Cell Infect Microbiol 2:1–7CrossRefGoogle Scholar
  26. Mukherjee S, Mukherjee B, Mukhopadhyay R, Naskar K, Sundar S, Dujardin JC et al (2012) Imipramine is an orally active drug against both antimony sensitive and resistant Leishmania donovani clinical isolates in experimental infection. PLoS Negl Trop Dis 6:1–15CrossRefGoogle Scholar
  27. Munos B (2009) Lessons from 60 years of pharmaceutical innovation. Nat Rev Drug Discov 8:959–968CrossRefGoogle Scholar
  28. Okuno D, Lino R, Noji H (2011) Rotation and structure of FoF1-ATP synthase. J Biochem 149:655–664CrossRefGoogle Scholar
  29. Pace D (2014) Leishmaniasis. J Inf Secur 69:10–18Google Scholar
  30. Padhy B, Gupta Y (2011) Drug repositioning: re-investigating existing drugs for new therapeutic indications. J Postgrad Med 57:153–160CrossRefGoogle Scholar
  31. Proto WR, Coombs GH, Mottram JC (2012) Cell death in parasitic protozoa: regulated or incidental? Nat Rev Microbiol 11:58–66CrossRefGoogle Scholar
  32. Rivarola HW, Bustamante JM, Presti SL, Fernández AR, Enders JE, Gea S et al (2005) Trypanosoma cruzi: chemotherapeutic effects of clomipramine in mice infected with an isolate obtained from an endemic area. Exp Parasitol 111:80–86CrossRefGoogle Scholar
  33. Rodrigues JHS, Stein J, Strauss M, Rivarola HW, Ueda-Nakamura T, Nakamura CV, Duszenko M (2016) Clomipramine kills Trypanosoma brucei by apoptosis. Int J Med Microbiol 306:196–205CrossRefGoogle Scholar
  34. Rosenzweig D, Smith D, Opperdoes F, Stern S, Olafson RW, Zilberstein D (2007) Retooling Leishmania metabolism: from sand fly gut to human macrophage. FASEB J 22:590–602CrossRefGoogle Scholar
  35. `Safiulina D, Veksler V, Zharkovsky A, Kaasik A (2006) Loss of mitochondrial membrane potential is associated with increase in mitochondrial volume: physiological role in neurones. J Cell Physiol 206:347–353CrossRefGoogle Scholar
  36. Smirlis D, Duszenko M, Ruiz A, Scoulica E, Bastien P, Fasel N, Soteriadou K (2010) Targeting essential pathways in trypanosomatids gives insights into protozoan mechanisms of cell death. Parasit Vectors 3:107–132CrossRefGoogle Scholar
  37. Takahashi M, Shibata M, Niki E (2001) Estimation of lipid peroxidation of live cells using a fluorescent probe, diphenyl-1-pyrenylphosphine. Free Radic Biol Med 31(2):164–174CrossRefGoogle Scholar
  38. Walter P, Ron D (2011) The unfolded protein response: from stress pathway to homeostatic regulation. Science 334:1081–1086CrossRefGoogle Scholar
  39. WHO (2018) Leishmaniasis. World Health Organization. Accessed 06 July 2018
  40. Zilberstein D, Dwyer DM (1984) Antidepressants cause lethal disruption of membrane function in the human protozoan parasite Leishmania. Science 226:977–979CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Programa de Pós-Graduação em Ciências Biológicas, Área de Concentração Biologia Celular e MolecularUniversidade Estadual de MaringáMaringáBrazil
  2. 2.Programa de Pós-Graduação em Ciências FarmacêuticasUniversidade Estadual de MaringáMaringáBrazil
  3. 3.Laboratório de Inovação Tecnológica no Desenvolvimento de Fármacos e Cosméticos, Bloco B-08Universidade Estadual de MaringáMaringáBrazil

Personalised recommendations