title: Miltefosine increases lipid and protein dynamics in Leishmania amazonensis membranes at concentrations similar to those needed for cytotoxicity activity.
abstract: Miltefosine (MT) is a membrane-active alkylphospholipid licensed for the topical treatment of breast cancer skin metastases and the oral treatment of leishmaniasis, although its mechanism of action remains unclear. Electron paramagnetic resonance (EPR) spectroscopy of a spin-labeled lipid and a thiol-specific spin label in the plasma membrane of Leishmania promastigotes showed that MT causes dramatic increases in membrane dynamics. Although these alterations can be detected using a spin-labeled lipid, our experimental results indicated that MT interacts predominantly with the protein component of the membrane. Cell lysis was also detected by analyzing the supernatants of centrifuged samples for the presence of spin-labeled membrane fragments and cytoplasmic proteins. Using a method for the rapid incorporation of MT into the membrane, these effects were measured immediately after treatment under the same range of MT concentrations that cause cell growth inhibition. Cytotoxicity, estimated via microscopic counting of living and dead cells, indicated ∼70% cell death at the concentration of MT at which EPR spectroscopy detected a significant change in membrane dynamics. After this initial impact on the number of viable parasites, the processes of cell death and growth continued during the first 4 h of incubation. The EPR spectra of spin-labeled membrane-bound proteins were consistent with more expanded and solvent-exposed protein conformations, suggesting a detergent-like action. Thus, MT may form micelle-like structures around polypeptide chains, and proteins with a higher hydrophobicity may induce the penetration of hydrophilic groups of MT into the membrane, causing its rupture.
authors.affiliation: Instituto de Física, Universidade Federal de Goiás, Goiânia, GO, Brazil.
authors.forename: Rodrigo Alves
authors.initials: RA
authors.lastname: Moreira
authors.affiliation: Instituto de Física, Universidade Federal de Goiás, Goiânia, GO, Brazil.
authors.forename: Sebastião Antonio
authors.initials: SA
authors.lastname: Mendanha
authors.affiliation: Instituto de Física, Universidade Federal de Goiás, Goiânia, GO, Brazil.
authors.forename: Kelly Souza
authors.initials: KS
authors.lastname: Fernandes
authors.affiliation: Instituto de Patologia Tropical e Saúde Publica, Departamento de Imunologia e Patologia Geral, Universidade Federal de Goiás, Goiânia, GO, Brazil.
authors.forename: Grazzielle Guimaraes
authors.initials: GG
authors.lastname: Matos
authors.affiliation: Instituto de Física, Universidade Federal de Goiás, Goiânia, GO, Brazil.
authors.forename: Lais
authors.initials: L
authors.lastname: Alonso
authors.affiliation: Instituto de Patologia Tropical e Saúde Publica, Departamento de Imunologia e Patologia Geral, Universidade Federal de Goiás, Goiânia, GO, Brazil.
authors.forename: Miriam Leandro
authors.initials: ML
authors.lastname: Dorta
authors.affiliation: Instituto de Física, Universidade Federal de Goiás, Goiânia, GO, Brazil alonso@if.ufg.br.
authors.forename: Antonio
authors.initials: A
authors.lastname: Alonso
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007894:Leishmania mexicana / Q000187:drug effects* / Q000378:metabolism; D008563:Membrane Lipids / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008823:Micelles; D056004:Molecular Dynamics Simulation; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D011487:Protein Conformation; D013113:Spin Labels
chemical_list: D000970:Antineoplastic Agents; D008563:Membrane Lipids; D008565:Membrane Proteins; D008823:Micelles; D013113:Spin Labels; D010767:Phosphorylcholine; C039128:miltefosine