Interação da miltefosina com os componentes lipídicos e proteicos das membranas de eritrócito e Leishmania estudada por ressonância paramagnética eletrônica
Carregando...
Data
2014-06-04
Autores
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Federal de Goiás
Resumo
Cutaneous leishmaniasis is a neglected tropical disease that infects millions of
people worldwide, representing a serious public health problem. The miltefosine (MT) is an
alkylphospholipid that has been approved for the treatment of breast cancer metastasis and
visceral leishmaniasis, although the mechanism of action at the molecular level is poorly
understood. Electron paramagnetic resonance (EPR) spectroscopy of the lipid spin lebel
analog of stearic acid (5-DSA) and the maleimide derivative spin label (6-MSL) covalently
bound to membrane proteins showed that the MT causes a large increase in the molecular
dynamics of erythrocyte membranes (ghosts) and detergent resistant membranes (DRMs)
prepared from erythrocyte membranes. In the vesicles of lipid raft constituents, it was shown
that 20 mol% sphingomyelin could be replaced by 20 mol% MT with no change in the molecular
dynamics. Furthermore, the effect of MT on DRMs was more pronounced than in
erythrocyte ghosts, supporting the hypothesis that MT is a lipid raft modulator. At the reported
MT-plasma concentrations found during the treatment of leishmaniasis (31-52μg/mL),
our measurements in blood plasma indicated a hemolytic level of 2-5% and also showed
that the MT concentration that changes the erythrocyte membrane fluidity to an extent that
is detectable by EPR spectroscopy causes about 46% hemolysis. Subsequently, EPR studies
performed with the same spin labels in the membrane of Leishmania (L.) amazonensis
(promastigote) showed changes similar to those found in erythrocyte membranes. Cytotoxic
effects on the parasites were also evaluated to investigate the relationships between the
cytotoxic potential of MT and its ability to alter membrane fluidity. The EPR data showed
that the minimum concentration of MT required to cause a change in the parasite membrane
occurred near the values of MT concentration which inhibits 50 % of cell growth (IC50); thus,
there is a correlation between the cytotoxicity and changes in the membrane. Although these
III
membrane 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.
Descrição
Palavras-chave
Citação
MOREIRA, Rodrigo Alves. Interação da miltefosina com os componentes lipídicos e proteicos das membranas de eritrócito e Leishmania estudada por ressonância paramagnética eletrônica. 2014. 139 f. Tese (Doutorado em Fisica) - Universidade Federal de Goiás, Goiânia, 2014.