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Previous issue date: 2015-04-14 === Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil === As diversas espécies de Leishmania são parasitas de considerável importância médica e econômica. As drogas atualmente utilizadas no tratamento da leishmaniose apresentam efeitos adversos, alta toxicidade, elevado custo e surgimento de cepas resistentes. Nesse contexto, inibidores proteolíticos é uma alternativa para o tratamento desta doença. Este estudo está focado nas calpaínas, que compreendem uma família de cisteína peptidases neutras dependentes de cálcio, envolvidas numa ampla variedade de funções fisiológicas. As calpaínas estão envolvidas em importantes doenças humanas, portanto inibidores de calpaínas já vêm sendo desenvolvidos e testados para o tratamento dessas doenças, alguns encontra-se em estágios avançados de triagem clínica. Dessa forma, o presente trabalho avalia a expressão e identificação de homólogos das calpaínas em Leishmania braziliensis e o efeito do inibidor de calpaínas MDL28170 em ensaios in vitro. Através da busca por sequências no GenBank, alinhamentos múltiplos, filogenia e análise de domínios conservados nas sequências obtidas, selecionamos como alvo inicial sequências de calpaínas que possuíam o maior número de domínios conservados. A análise da expressão gênica relativa indica que 13 das 20 calpaínas estudadas apresentam níveis constitutivos de mRNA nas formas procíclica e metacíclica de L. braziliensis. A expressão de cinco alvos é maior na forma procíclica, e a de um alvo é maior na forma metacíclica e há expressão estágio específica de apenas um alvo na forma procíclica. A partir de análises in silico, das sequências proteicas dessas calpaínas, selecionamos uma região consenso e um peptídeo correspondente a essa região foi sintetizado e empregado na imunização de coelhos
A reatividade do anticorpo gerado (Anti-TryTrip CALPAIN) foi avaliada por Western blotting, citometria de fluxo e imunocitoquímica. Em ensaios de Western blotting,verificamos que o anticorpo foi capaz de reagir contra uma proteína de 50 kDa. Já na imunolocalização foi possível observar calpaínas dispersas no citoplasma, membrana e núcleo. Além disso, através de citometria de fluxo, as moléculas homólogas às calpaínas foram identificadas em maior abundância no interior das células. Nos ensaios de inibição com o MDL28170, inibidor de calpaínas, foi possível observar a redução da proliferação nas formas promastigotas recém isoladas e múltiplas passagens de forma dose-dependente nas diferentes concentrações do inibidor ao longo de quatro dias. O efeito reversíveldo inibidor também foi avaliado nas formas promastigotas, com taxas menores comparado com o controle. O efeito do inibidor, também foi capaz de diminuir de maneira dose-dependente o índice de associação e aumentou o percentual de células com parasitos aderidos durante o processo de interação com macrófagos peritoneais. O parasito aumentou a expressão de uma peptidse clássica (gp63) quando tratado com o MDL28170, já a expressão de calpaínas e cpb não foi alterada pelo estresse induzido pelo composto. Estes dados sugerem mais estudos para melhor caracterizar as calpaínas em L. braziliensis e sugerem uma maior avaliação para uma possível associação de moléculas similares as calpaínas com a virulência ou não do parasito. Assim este trabalho acrescenta novas possibilidades para a utilização de inibidores de calpaínas como um potencial alvo de desenvolvimento para o tratamento da leishmaniose === The various species of Leishmania are parasites of considerable medical and economic
importance. The drugs used in the treatment of leishmaniasis have adverse effects, high toxicity,
high cost and emergence of resistant strains. In this context, proteolytic inhibitors could be an
alternative for the treatment of this disease. This study is focused on calpains, which comprise a
family of neutral cysteine peptidases, which are strictly dependent of calcium, and are there of
known as Calcium Dependent Peptidases.These enzymes play a variety of physiological
functions, and are involved in human diseases, therefore calpains inhibitors are under trial to
treat these diseases. Thus, this study aimed to assess calpain expression levels in Leishmania
braziliensis, as well as the effect of the calpain inhibitor MDL28170 in vitro. Through the
searching for sequencesin GenBank, multiple alignments, and phylogenetic analysis of the
obtained sequences conserved domains, selected as an initial target sequences of calpain which
possessed the greatest number of conserved domains. In this sense, we assessed the expression
levels of mRNA from a group of twenty sequences containing archetypal calpain domain. The
analysis indicated that 13 out of the 20 studied calpains have constitutive levels of mRNA
between the procyclic and metacyclic forms of L. braziliensis, while five calpains presented
higher expression levels at the procyclic stage, and only one sequence is augmented at the
metacyclic stage. One calpain molecule was found to be procyclic-specific. After that, we
selected a consensus region and a peptide was synthesized and used to immunize rabbits. The
reactivity of the antibody (anti-calpainTryTrip) was evaluated by Western blotting, flow
cytometry and immunocytochemistry. In Western blotting assays, we found that the anti-calpain
TriTryp antibody was able to recognize a 50 kDa protein. The immunolocalization assays
revealed calpain molecules present at the membrane, nucleus and dispersed throughout the
cytoplasm. Also, by flow cytometry, molecules homologous to calpains have been identified in
abundance within cells. In inhibition assays employing MDL28170, a potent and specific
calpain inhibitor, it was possible to observe a dose-dependent reduction in the proliferation rate,
either in freshly isolated promastigotes or multiple passages parasites. MDL28170 presents a
reversible inhibitory effect. The inhibitor was also able to decrease in a dose-dependent manner
the association index and the percentage of host cells with attached parasites during the process
of interaction with peritoneal macrophages. Finally, MDL28170 enhanced the expression of
gp63 molecules, while cpb and calpains expression were not affect. Further studies to better
characterize the calpain in L. braziliensis should be performed, aiming to add new possibilities
for the exploitation of calpain inhibitors as a potential for the treatment of leishmaniasis. === The various species of Leishmania are parasites of considerable medical and economic
importance. The drugs used in the treatment of leishmaniasis have adverse effects, high toxicity,
high cost and emergence of resistant strains. In this context, proteolytic inhibitors could be an
alternative for the treatment of this disease. This study is focused on calpains, which comprise a
family of neutral cysteine peptidases, which are strictly dependent of calcium, and are there of
known as Calcium Dependent Peptidases.These enzymes play a variety of physiological
functions, and are involved in human diseases, therefore calpains inhibitors are under trial to
treat these diseases. Thus, this study aimed to assess calpain expression levels in Leishmania
braziliensis, as well as the effect of the calpain inhibitor MDL28170 in vitro. Through the
searching for sequencesin GenBank, multiple alignments, and phylogenetic analysis of the
obtained sequences conserved domains, selected as an initial target sequences of calpain which
possessed the greatest number of conserved domains. In this sense, we assessed the expression
levels of mRNA from a group of twenty sequences containing archetypal calpain domain. The
analysis indicated that 13 out of the 20 studied calpains have constitutive levels of mRNA
between the procyclic and metacyclic forms of L. braziliensis, while five calpains presented
higher expression levels at the procyclic stage, and only one sequence is augmented at the
metacyclic stage. One calpain molecule was found to be procyclic-specific. After that, we
selected a consensus region and a peptide was synthesized and used to immunize rabbits. The
reactivity of the antibody (anti-calpainTryTrip) was evaluated by Western blotting, flow
cytometry and immunocytochemistry. In Western blotting assays, we found that the anti-calpain
TriTryp antibody was able to recognize a 50 kDa protein. The immunolocalization assays
revealed calpain molecules present at the membrane, nucleus and dispersed throughout the
cytoplasm. Also, by flow cytometry, molecules homologous to calpains have been identified in
abundance within cells. In inhibition assays employing MDL28170, a potent and specific
calpain inhibitor, it was possible to observe a dose-dependent reduction in the proliferation rate,
either in freshly isolated promastigotes or multiple passages parasites. MDL28170 presents a
reversible inhibitory effect. The inhibitor was also able to decrease in a dose-dependent manner
the association index and the percentage of host cells with attached parasites during the process
of interaction with peritoneal macrophages. Finally, MDL28170 enhanced the expression of
gp63 molecules, while cpb and calpains expression were not affect. Further studies to better
characterize the calpain in L. braziliensis should be performed, aiming to add new possibilities
for the exploitation of calpain inhibitors as a potential for the treatment of leishmaniasis.
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