Dados do Trabalho

Título

EFFECTS OF SERINE PROTEASE INHIBITOR N-TOSYL-L-PHENYLALANINE CHLOROMETHYL KETONE (TPCK) ON LEISHMANIA AMAZONENSIS

Introdução

Leishmaniasis chemotherapy exhibit different limitations and to search new compounds with leishmanicidal activity is very important. Serine proteases are involved in the pathogenesis of different parasites, including Leishmania.

Objetivo(s)

Evaluate the in vitro and in vivo effects of a serine protease inhibitor (TPCK), against Leishmania amazonensis.

Material e Métodos

The anti-promastigote effect and the toxicity on macrophages were determined by MTT colorimetric method. The anti-amastigote activity was analyzed by counting intracellular parasites after Giemsa staining. Stains with H2DCFDA and Nile Red were performed on L. amazonensis promastigotes treated with TPCK to determine the occurrence of oxidative stress and to evaluate the lipid content of the parasites, respectively. Transmission electron microscopy (TEM) was used to determine TPCK-induced ultracellular changes on intracellular amastigotes. The in vivo effect of TPCK was determined in BALB/c mice infected with L. amazonensis and treated for a total of 10 doses of 45 and 60 mg/kg intraperitoneally. In vivo toxicity was evaluated by determination of creatinine, AST (aspartate aminotransferase), and ALT (alanine aminotransferase) levels in the serum of L. amazonensi-infected BALB/c and treated with TPCK, using laboratory kits colorimetric kinetic test.

Resultados e Conclusão

TPCK exhibited low toxicity against mammalian cells (CC50 138.8 µM) and was active against L. amazonensis promastigotes (IC50 = 14.6 µM) and intracellular amastigotes (IC50 values = 14.2µM), displaying selectivity for parasite when compared to host cells (selectivity index close to 10). L. amazonensis promastigotes treated with TPCK presented oxidative stress and reduction of lipids content. Analyzes by TEM revealed the presence of large cytoplasmic vacuoles inside intracellular amastigotes treated with TPCK and some vacuoles containing a membranous profile or electron dense material. BALB/c mice infected with L. amazonensis and treated with TPCK had a reduction in lesion size and parasite loads in the footpad and spleen. TPCK did not altered creatinine, AST and ALT levels in the serum of infected animals. This work highlights effects TPCK against L. amazonensis and confirms the importance of serine proteases as drug targets in Leishmania spp.

Palavras-chave

SERINE PROTEASES; TPCK; LEISHMANIASIS; LEISHMANIA AMAZONENSIS.