Uptake of Z-Tyr{125I}-AlaCHN2, an irreversible cysteine proteinase inhibitor, by lesion amastigotes, axenic amastigotes and promastigotes of Leishmania mexicana

Radioiodinated N-benzyloxycarbonyl-tyrosyl-alanyl diazomethane (Z-Tyr[l25I]-AlaCHN2) was previously shown to selectively label two (28 and 31 kDa) Leishmania mexicana cysteine proteinases common to both the promastigote and the amastigote stages. Here we have confirmed the specificity of the compound towards two similar enzymes of axenic L. mexicana amastigotes and demonstrated that lesion amastigotes, axenic amastigotes and stationary promastigotes internalized the l25I-labeled inhibitor at different rates. Uptake of Z-Tyr[l25I]-AlaCHN2 by the parasites, which was not significantly modified by changing the medium pH, was clearly correlated with the binding of the compound to the 28- and 3l-kDa cysteine proteinases, as judged by the specificity of enzyme labeling in gelatin gels and the recovery of 75 per cent or more parasite-associated radioactivity in TCA-insoluble fractions. For all three developmental stages, uptake markedly increased with time and linearly up to 60 min, but throughout the period examined, radiolabel accumulation occurred more efficiently in amastigotes. By 5 h, when values were near or at saturation, radioactivity (in cpm/mug of total protein) associated with lesion amastigotes was 1.8- and 2.9-times that recovered from axenic amastigotes and stationary promastigotes, respectively. Pulse-chase experiments, in which cysteine proteinases were fully blocked with Z-Phe-AlaCHN2 prior to the pulse with Z-Tyr[l25I]-AlaCHN2, showed that labeling of the amastigote enzymes could be partially restored, whereas labeling of promastigote proteinases could not, after a 5-h chase period in inhibitor-free medium.

From lysosomes to cells, from cells to Leishmania: amino acid esters as potential chemotherapeutic agents

Amino acid esters can disrupt lysosomes and damage monocytes and certain lymphocyte populations. Lysosomal disruption involves pH trapping of the esters, followed by their hydrolyssis by as yet unidentified enzymes. Accumulation of the more polar amino acids is assumed to cause osmotic lysis of the organelles. we have discovered that certain amino acid esters and amides destroy Leishmania mexicana amazonensis amastigores lodged within macrophages in culture, as well as parasites isolated from mouse lesions. This paper reviews the amino acid specificity of parasite killing, the resistance of amastigotes derived from infection of macrophages with promastigotes, the involvement of an acidified compartment within the parasites, and the protection conferred by other amino acid esters, and the protease inhibitors antipain and chymostatin, aginst the destruction of amastigotes by Leucine-methyl ester. Studies with tritiated esters confirm the critical role of ester hydrolysis for leishmanicidal activity and strengthen the view that similar mechanisms underlie disruption of lysosomes and destruction of Leismania. Characterization of the parasite organelles and of the enzymes involved in the leishmanicidal activity as well as structure-activity studies may permit the design of compounds mor selective for the parasites