Dados do Trabalho

Título

Serine protease of Leishmania amazonensis as potential druggable target using N-p-Tosyl-L-phenylalanine Chloromethyl Ketone(TPCK)

Introdução

Serine proteases (SP) have been classified into evolutionarily unrelated clans, and can be subdivided into families, based on their catalytic mechanism. A catalytic triad of SP is typically composed of histidine, serine, and aspartic residues in the active site. Despite this highly evolutionarily conserved triad in the active site, studies have shown unusual folds unrelated to any other serine protease, with an active site consisting instead of histidine, an aspartic acid, or even a tetrad consisting of serine, two histidine, and aspartic acid.In Leishmania spp. the SP are distributed in 6 clans among 8 families, and they represent one of three protease groups within the genome. In addition, the importance of SP in other protozoans has already been described.

Objetivo (s)

We evaluated the antileishmanial effect of TPCK at the enzymatic level, in intracellular amastigotes, and in a murine model. Our group has already described that TPCK inhibited L.amazonensis promastigotes close to 20 uM. Furthermore, we saw that in intracellular amastigotes this inhibition was 16.6 uM, showing the potential of this drug. Concerning L. amazonensis intracellular amastigotes, treatment with TPCK also caused ultrastructural changes. Macrophages infected with L. amazonensis and untreated presented amastigotes inside parasitophorous vacuoles, with parasite organelles exhibiting regular morphology.

Material e Métodos

After treatment with TPCK at 15 μM, membrane material was observed within the parasitophorous vacuole. Furthermore, large vacuoles in the parasite cytoplasm containing granular material and the presence of electron-dense material were also observed.Treatment with 30 μM caused more damage to amastigotes than the lower concentration, and cytoplasmic vacuoles containing granular material were also observed within these treated amastigotes.

Resultados e Conclusão

Concentric membrane profiles forming myelinic figures were noted. Some figures indicates parasites with ruptured plasma membranes.Vacuoles were also observed in the cytoplasm of macrophages infected and treated with TPCK. To test the enzymatic action of the compound against the serine protection substrate,TPCK was able to inhibit the enzymatic degradation, demonstrating that its action is directed to the specific substrate. In addition, in vivo effect of L. amazonensis animals treated with TPCK displayed reduced injury in the chronic phase of the disease, and not was toxic for hepatic or kidney.

Palavras-chave

Protease,microscopy, Leishmania,TPCK

Agradecimentos

FAPERJ,CNPq