Leishmaniasis is an infectious disease caused by protozoan parasites of the genus Leishmania. There is no vaccine against human leishmaniasis and the treatment of the disease would benefit from a broader spectrum and a higher efficacy of leishmanicidal compounds. We analyzed the leishmanicidal activity and the mechanism of action of the calcium ionophore, calcimycin. L. major promastigotes were coincubated with calcimycin and the viability of the cells was assessed using resazurin assay. Calcimycin displayed dose-dependent effect with IC50 = 0.16 μM. Analysis of propidium iodide/LDS-751 stained promastigotes revealed that lower concentrations of calcimycin had cytostatic effect and higher concentrations had cytotoxic effect. To establish the mechanism of action of calcimycin, which is known to stimulate activity of mammalian constitutive nitric oxide synthase (NOS), we coincubated L. major promastigotes with calcimycin and selective NOS inhibitors ARL-17477 or L-NNA. Addition of these inhibitors substantially decreased the toxicity of calcimycin to Leishmania promastigotes. In doing so, we demonstrated for the first time that calcimycin has a direct leishmanicidal effect on L. major promastigotes. Also, we showed that Leishmania constitutive Ca2+/calmodulin-dependent nitric oxide synthase is involved in the parasite cell death. These data suggest activation of Leishmania nitric oxide synthase as a new therapeutic approach.

Institute of Molecular Genetics of the ASCR v v i Prague Czech Republic

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Lipoldová M., Demant P. Genetic susceptibility to infectious disease: Lessons from mouse models of leishmaniasis. Nature Reviews Genetics. 2006;7(4):294–305. doi: 10.1038/nrg1832. PubMed DOI

Cruz I., Morales M. A., Noguer I., Rodríguez A., Alvar J. Leishmania in discarded syringes from intravenous drug users. The Lancet. 2002;359(9312):1124–1125. doi: 10.1016/S0140-6736(02)08160-6. PubMed DOI

Martín-Dávila P., Fortún J., López-Vélez R., et al. Transmission of tropical and geographically restricted infections during solid-organ transplantation. Clinical Microbiology Reviews. 2008;21(1):60–96. doi: 10.1128/CMR.00021-07. PubMed DOI PMC

Meinecke C. K., Schottelius J., Oskam L., Fleischer B. Congenital transmission of visceral leishmaniasis (Kala Azar) from an asymptomatic mother to her child. Pediatrics. 1999;104(5):p. e65. doi: 10.1542/peds.104.5.e65. PubMed DOI

Alvar J., Vélez I. D., Bern C., et al. Leishmaniasis worldwide and global estimates of its incidence. PLoS ONE. 2012;7(5) doi: 10.1371/journal.pone.0035671.e35671 PubMed DOI PMC

Du R., Hotez P. J., Al-Salem W. S., Acosta-Serrano A. Old World Cutaneous Leishmaniasis and Refugee Crises in the Middle East and North Africa. PLOS Neglected Tropical Diseases. 2016;10(5) doi: 10.1371/journal.pntd.0004545.e0004545 PubMed DOI PMC

Hotez P. J. Southern Europe’s Coming Plagues: Vector-Borne Neglected Tropical Diseases. PLOS Neglected Tropical Diseases. 2016;10(6) doi: 10.1371/journal.pntd.0004243.e0004243 PubMed DOI PMC

Lindgren E., Naucke T., Menne B. Europe. Climate variability and visceral leishmaniasis in Europe. 2004.

Naucke T. J., Menn B., Massberg D., Lorentz S. Sandflies and leishmaniasis in Germany. Parasitology Research. 2008;103(1):S65–S68. doi: 10.1007/s00436-008-1052-y. PubMed DOI

González C., Wang O., Strutz S. E., González-Salazar C., Sánchez-Cordero V., Sarkar S. Climate change and risk of leishmaniasis in North America: predictions from ecological niche models of vector and reservoir species. PLOS Neglected Tropical Diseases. 2010;4(1, article e585) doi: 10.1371/journal.pntd.0000585. PubMed DOI PMC

Srivastava S., Shankar P., Mishra J., Singh S. Possibilities and challenges for developing a successful vaccine for leishmaniasis. Parasites & Vectors. 2016;9(1, article no. 277) doi: 10.1186/s13071-016-1553-y. PubMed DOI PMC

Thomaz-Soccol V., Ferreira da E. S., Costa S. G., Letti L. A. J., Soccol C. R., Thomaz F. Recent advances in vaccines against Leishmania based on patent applications. Recent Pat Biotechnol. 2017 PubMed

Kobets T., Grekov I., Lipoldová M. Leishmaniasis: prevention, parasite detection and treatment. Current Medicinal Chemistry. 2012;19(10):1443–1474. doi: 10.2174/092986712799828300. PubMed DOI

Reed P. W., Lardy H. A. A23187: a divalent cation ionophore. The Journal of Biological Chemistry. 1972;247(21):6970–6977. PubMed

Pressman B. C. Biological applications of ionophores. Annual Review of Biochemistry. 1976;45:501–530. doi: 10.1146/annurev.bi.45.070176.002441. PubMed DOI

Reed P. W., Lardy H. W. Antibiotic A23187 as a probe for the study of calcium and magnesium function in biological systems. In: Mehlman M. H. H. R. W., editor. The Role of Membranes in Metabolic Regulation. New York, NY, USA: Academic Press; 1972. pp. 111–131.

Drenning J. A., Lira V. A., Simmons C. G., Soltow Q. A., Sellman J. E., Criswell D. S. Nitric oxide facilitates NFAT-dependent transcription in mouse myotubes. American Journal of Physiology-Cell Physiology. 2008;294(4):C1088–C1095. doi: 10.1152/ajpcell.00523.2007. PubMed DOI

Werner-Felmayer G., Werner E. R., Fuchs D., et al. Erratum: Ca2+/calmodulin-dependent nitric oxide synthase activity in the human cervix carcinoma cell line ME-180. Biochemical Journal. 1993;289(3):357–361. PubMed PMC

Paveto C., Pereira C., Espinosa J., et al. The nitric oxide transduction pathway in Trypanosoma cruzi. The Journal of Biological Chemistry. 1995;270(28):16576–16579. doi: 10.1074/jbc.270.28.16576. PubMed DOI

Genestra M., Guedes-Silva D., Souza W. J. S., et al. Nitric oxide synthase (NOS) characterization in Leishmania amazonensis axenic amastigotes. Archives of Medical Research. 2006;37(3):328–333. doi: 10.1016/j.arcmed.2005.07.011. PubMed DOI

Buchmuller-Rouiller Y., Mauel J. Macrophage activation for intracellular killing as induced by calcium ionophore: Correlation with biologic and biochemical events. The Journal of Immunology. 1991;146(1):217–223. PubMed

Lanza H., Afonso-Cardoso S. R., Silva A. G., et al. Comparative effect of ion calcium and magnesium in the activation and infection of the murine macrophage by Leishmania major. Biol Res. 2004;37(3):385–393. PubMed

Grekov I., Svobodová M., Nohýnková E., Lipoldová M. Preparation of highly infective Leishmania promastigotes by cultivation on SNB-9 biphasic medium. Journal of Microbiological Methods. 2011;87(3):273–277. doi: 10.1016/j.mimet.2011.08.012. PubMed DOI

Sharlow E. R., Close D., Shun T., et al. Identification of potent chemotypes targeting Leishmania major using a high-throughput, low-stringency, computationally enhanced, small molecule screen. PLOS Neglected Tropical Diseases. 2009;3(11, article e540) doi: 10.1371/journal.pntd.0000540. PubMed DOI PMC

De la Monte S. M., Chiche J.-D., Von dem Bussche A., et al. Nitric oxide synthase-3 overexpression causes apoptosis and impairs neuronal mitochondrial function: Relevance to Alzheimer's-type neurodegeneration. Laboratory Investigation. 2003;83(2):287–298. doi: 10.1097/01.LAB.0000056995.07053.C0. PubMed DOI

Regli L., Held M. C., Anderson R. E., Meyer F. B. Nitric oxide synthase inhibition by L-NAME prevents brain acidosis during focal cerebral ischemia in rabbits. Journal of Cerebral Blood Flow & Metabolism. 1996;16(5):988–995. doi: 10.1097/00004647-199609000-00024. PubMed DOI

Grekov I. Experimental murine leishmaniasis and its application for drug discovery and study of host-pathogen interactions [Ph.D. thesis] Prague, Czech Republic: Third Faculty of Medicine, Charles University; 2011.