title: Potent In Vitro Antiproliferative Synergism of Combinations of Ergosterol Biosynthesis Inhibitors against Leishmania amazonensis.
abstract: Leishmaniases comprise a spectrum of diseases caused by protozoan parasites of the Leishmania genus. Treatments available have limited safety and efficacy, high costs, and difficult administration. Thus, there is an urgent need for safer and more-effective therapies. Most trypanosomatids have an essential requirement for ergosterol and other 24-alkyl sterols, which are absent in mammalian cells. In previous studies, we showed that Leishmania amazonensis is highly susceptible to aryl-quinuclidines, such as E5700, which inhibit squalene synthase, and to the azoles itraconazole (ITZ) and posaconazole (POSA), which inhibit C-14α-demethylase. Herein, we investigated the antiproliferative, ultrastructural, and biochemical effects of combinations of E5700 with ITZ and POSA against L. amazonensis. Potent synergistic antiproliferative effects were observed against promastigotes, with fractional inhibitory concentration (FIC) ratios of 0.0525 and 0.0162 for combinations of E5700 plus ITZ and of E5700 plus POSA, respectively. Against intracellular amastigotes, FIC values were 0.175 and 0.1125 for combinations of E5700 plus ITZ and E5700 plus POSA, respectively. Marked alterations of the ultrastructure of promastigotes treated with the combinations were observed, in particular mitochondrial swelling, which was consistent with a reduction of the mitochondrial transmembrane potential, and an increase in the production of reactive oxygen species. We also observed the presence of vacuoles similar to autophagosomes in close association with mitochondria and an increase in the number of lipid bodies. Both growth arrest and ultrastructural/biochemical alterations were strictly associated with the depletion of the 14-desmethyl endogenous sterol pool. These results suggest the possibility of a novel combination therapy for the treatment of leishmaniasis.
authors.affiliation: Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janerio, Brazil Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Rio de Janeiro, Brazil.
authors.forename: S T
authors.initials: ST
authors.lastname: de Macedo-Silva
authors.affiliation: Instituto Nacional de Metrologia, Qualidade e Tecnologia, Rio de Janeiro, Brazil.
authors.forename: G
authors.initials: G
authors.lastname: Visbal
authors.affiliation: Instituto Venezolano de Investigaciones Científicas, Caracas, Venezuela.
authors.forename: J A
authors.initials: JA
authors.lastname: Urbina
authors.affiliation: Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janerio, Brazil Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Rio de Janeiro, Brazil Instituto Nacional de Metrologia, Qualidade e Tecnologia, Rio de Janeiro, Brazil.
authors.forename: W
authors.initials: W
authors.lastname: de Souza
authors.affiliation: Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janerio, Brazil Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Rio de Janeiro, Brazil Instituto Nacional de Metrologia, Qualidade e Tecnologia, Rio de Janeiro, Brazil Núcleo Multidisciplinar de Pesquisa UFRJ-Xerém, Divisão Biologia, Campus UFRJ-Xerém, Universidade Federal do Rio de Janeiro, Duque de Caxias, Rio de Janeiro, Brazil juliany.rodrigues@xerem.ufrj.br.
authors.forename: J C F
authors.initials: JC
authors.lastname: Rodrigues
mesh_terms: D058888:14-alpha Demethylase Inhibitors / Q000494:pharmacology*; D000818:Animals; D003470:Culture Media / Q000737:chemistry; D004357:Drug Synergism; D004359:Drug Therapy, Combination; D004875:Ergosterol / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D013186:Farnesyl-Diphosphate Farnesyltransferase / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D017964:Itraconazole / Q000494:pharmacology*; D007894:Leishmania mexicana / Q000187:drug effects* / Q000302:isolation & purification / Q000378:metabolism / Q000648:ultrastructure; D016774:Leishmaniasis, Diffuse Cutaneous / Q000469:parasitology; D066292:Lipid Droplets / Q000187:drug effects / Q000648:ultrastructure; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D021261:Parasitic Sensitivity Tests; D011725:Pyridines / Q000494:pharmacology*; D011812:Quinuclidines / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000819:agonists / Q000378:metabolism; D058886:Sterol 14-Demethylase / Q000378:metabolism; D014230:Triazoles / Q000494:pharmacology*; D014344:Trypanocidal Agents / Q000494:pharmacology*
chemical_list: D058888:14-alpha Demethylase Inhibitors; C489151:3-((2-benzyl-6-(3-hydroxy-4-methoxypyrrolidin-1-yl)pyridin-3-yl)ethynyl)quinuclidin-3-ol; D003470:Culture Media; D011725:Pyridines; D011812:Quinuclidines; D017382:Reactive Oxygen Species; D014230:Triazoles; D014344:Trypanocidal Agents; D017964:Itraconazole; C101425:posaconazole; D058886:Sterol 14-Demethylase; D013186:Farnesyl-Diphosphate Farnesyltransferase; D004875:Ergosterol