Miltefosine Increases Lipid and Protein Dynamics in Leishmania amazonensis Membranes at Concentrations Similar to Those Needed for Cytotoxicity Activity

• Published in: Antimicrobial agents and chemotherapy Vol. 58; no. 6; pp. 3021 - 3028
• Main Authors: ALVES MOREIRA, Rodrigo, MENDANHA, Sebastião Antonio, FERNANDES, Kelly Souza, GUIMARES MATOS, Grazzielle, ALONSO, Lais, DORTA, Miriam Leandro, ALONSO, Antonio
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01-06-2014
• Subjects: Antibiotics. Antiinfectious agents. Antiparasitic agents; Antineoplastic Agents; Antineoplastic Agents - pharmacology; Biological and medical sciences; Cell Membrane - metabolism; Cell Survival - drug effects; Electron Spin Resonance Spectroscopy; Humans; Hydrophobic and Hydrophilic Interactions; Leishmania amazonensis; Leishmania mexicana; Leishmania mexicana - drug effects; Leishmania mexicana - metabolism; Mechanisms of Action: Physiological Effects; Medical sciences; Membrane Lipids; Membrane Lipids - metabolism; Membrane Proteins; Membrane Proteins - metabolism; Micelles; Molecular Dynamics Simulation; Pharmacology. Drug treatments; Phosphorylcholine; Phosphorylcholine - analogs & derivatives; Phosphorylcholine - chemistry; Phosphorylcholine - pharmacology; Protein Conformation; Spin Labels; Antineoplastic agent; Kinetoplastida; Protozoa; Phospholipid; Cytotoxicity; Lipids; Concentration; Biological activity; Protein; Miltefosine; Dynamics; Leishmania; Lysophospholipid
• ISSN: 0066-4804; 1098-6596
• DOI: 10.1128/AAC.01332-13

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.