Proteinase inhibitors protect Leishmania amazonensis amastigotes from destruction by amino acid esters

Abstract

Lysosomotropic animo acid esters and amides kill Leishmania amazonensis amastigotes by a mechanism which probably involves enzymatic hydrolysis of the compounds and rapid accumulation of less permeant amino acid within the parasites. We show here that, in agreement with this model, the proteinase inhibitors antipain and chymostatin prevented the killing of intracellular and isolated parasites by l-leucine methyl ester (Leu-OMe). Survival of Leishmania within macrophages was assessed microscopically, and that of isolated amastigotes was measured by tetrazolium (MTT) reduction. Near maximal protection of intracellular parasites was obtained after 24 h incubation of macrophage cultures with 50 μg ml −1 of antipain or chymostatin. In contrast, isolated amastigotes were significantly protected after 15–60 min incubation with as little as 0.25 μg ml −1 antipain or chymostatin. Incubation for > 1 h with chymostatin or > 4 h with antipain alone resulted in loss of viability of the parasites. Protective activity was only slightly diminished by 20 h chase of isolated parasites in inhibitor-free medium. Two synthetic chymostatin analogues, Z-Val-Phe-Se and Z-Ile-Phe-Sc, protected isolated amastigotes at 4 or 10 μg ml −1. With the exception of Trp-NH 2, the toxicity of which was only minimally inhibited, antipain and chymostatin also prevented parasite destruction by other amino acid derivatives. Finally, in concentration-dependent fashion, the inhibitors reduced the accumulation of [ 3H]leucine in isolated amastigotes incubated with [ 3H]Leu-OMe. Since uptake of labelled ester was unaffected, we postulate that protection involves inhibition of the parasite enzymes which hydrolyse the amino acid derivatives.