title: The cytotoxic activity of miltefosine against Leishmania and macrophages is associated with dynamic changes in plasma membrane proteins.
abstract: In this study, we combined electron paramagnetic resonance (EPR) spectroscopy with an analysis of biophysical cellular parameters to study the mechanisms underlying the in vitro anti-leishmanial activity of miltefosine (MT). A thiol-specific spin label attached to membrane-bound proteins of Leishmania amazonensis and peritoneal macrophages indicated that MT may bind to plasma membrane proteins in large quantities via a detergent-like action and cause structural changes associated with a marked increase in dynamics and exposure to an aqueous environment. EPR spectra of a spin-labeled stearic acid indicated strong interactions between the probe and membrane proteins and a marked increase in the membrane fluidity of MT-treated cells. The cytotoxicity of MT was found to depend on the cell concentration used in the assay. This dependence was described by an equation involving the 50% inhibitory concentrations of MT in the aqueous medium (cw50) and the cell membrane (cm50) and the membrane-aqueous medium partition coefficient of MT (K). With a cw50of 8.7μM, macrophages were less sensitive to MT than amastigotes and promastigotes of Leishmania, which had cw50values of 2.4-3.1μM. The estimated cm50of MT for Leishmania was 1.8M, which appears sufficient to cause ruptures or formation of pores in the plasma membrane. Additionally, we demonstrated that the changes in the plasma membrane detected by EPR spectroscopy occurred at cytotoxic concentrations of MT, as assessed through in vitro assays.
authors: Instituto de Física, Universidade Federal de Goiás, Goiânia, GO, Brazil; Instituto Federal Goiano, Urutaí, GO, Brazil.
authors: Kelly Souza
authors: KS
authors: Fernandes
authors: Instituto de Física, Universidade de Brasília, Brasília, DF, Brazil.
authors: Paulo Eduardo Narcizo
authors: PE
authors: de Souza
authors: Instituto de Patologia Tropical e Saúde Publica, Departamento de Imunologia e Patologia Geral, Universidade Federal de Goiás, Goiânia, GO, Brazil.
authors: Miriam Leandro
authors: ML
authors: Dorta
authors: Instituto de Física, Universidade Federal de Goiás, Goiânia, GO, Brazil. Electronic address: alonso@ufg.br.
authors: Antonio
authors: A
authors: Alonso
mesh_terms: D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology*; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D004578:Electron Spin Resonance Spectroscopy; D020128:Inhibitory Concentration 50; D007894:Leishmania mexicana / Q000187:drug effects* / Q000254:growth & development / Q000378:metabolism; D008018:Life Cycle Stages / Q000187:drug effects* / Q000502:physiology; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000469:parasitology; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000494:pharmacology; D061251:Primary Cell Culture; D015800:Protozoan Proteins / Q000737:chemistry* / Q000378:metabolism; D013113:Spin Labels
chemical_list: D000981:Antiprotozoal Agents; D008565:Membrane Proteins; D015800:Protozoan Proteins; D013113:Spin Labels; D010767:Phosphorylcholine; C039128:miltefosine
keywords: Leishmania; Macrophages; Membrane fluidity; Miltefosine; Protein dynamics