RESUMO
Three proteinase inhibitors, one peptidyl acyloxymethyl ketone (AMK), Z-Phe-Lys-CH2-OCO-(2,4,6-Me3)Ph.HCl, and two diazomethyl ketones (DMKs), Z-Phe-Phe-DMK and Z-Phe-Ala-DMK, have been studied for their effects in vitro on the four developmental stages of Trypanosoma cruzi. The three inhibitors penetrated living parasites and inhibited the major cysteine proteinase, cruzipain. The AMK was the most potent inhibitor of cruzipain itself and at 20 microM caused lysis of epimastigotes and trypomastigotes. When at lower concentrations, however, it had little effect on epimastigote growth but reduced metacyclogenesis. The DMKs had no effect against epimastigotes but inhibited differentiation to metacyclics. All three inhibitors markedly reduced infection of Vero cells by the parasite and the multiplication of the intracellular amastigotes, whereas release of trypomastigotes was almost entirely prevented. The results confirm the importance of cysteine proteinases in the life cycle of T. cruzi, and suggest that the differentiation steps are the most susceptible to cysteine proteinase inhibitors.
Assuntos
Inibidores de Cisteína Proteinase/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Chlorocebus aethiops , Cisteína Endopeptidases/metabolismo , Diazometano/análogos & derivados , Diazometano/farmacologia , Dipeptídeos/farmacologia , Cetonas/farmacologia , Proteínas de Protozoários , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/patogenicidade , Células Vero/parasitologiaRESUMO
The proteinases of Leishmania mexicana mexicana amastigotes and promastigotes have been analysed by electrophoresis on polyacrylamide gels containing denatured haemoglobin. Eleven bands of activity were detected indicating multiple proteinases. These were significant quantitative and qualitative differences between the proteinases of the two developmental forms. Four, B-E, were present in both forms but were of much higher activity in the amastigote. There were two major activities in promastigotes, A and D. The other proteinases, F-K, were of lower activity; I and K were not detected in promastigotes. All proteinases were active optimally at pH 4.0. Most of them, including the major proteinases A-E, were thiol proteinases since they were stimulated by 1 mM dithiothreitol and were sensitive to inhibitors such as HgCl2, leupeptin, antipain and iodoacetic acid.