Aziridine-2,3-Dicarboxylate-Based Cysteine Cathepsin Inhibitors Induce Cell Death inLeishmania majorAssociated with Accumulation of Debris in Autophagy-Related Lysosome-Like Vacuoles

The papain-like cysteine cathepsins expressed byLeishmania play a key role in the life cycle of these parasites, turning them into attractive targets for the development of new drugs. We previously demonstrated that two compounds of a series of peptidomimetic aziridine-2,3-dicarboxylate [Azi(OBn)2 ]-based inhibitors, Boc-(S )-Leu-(R )-Pro-(S ,S )-Azi(OBn)2 (compound 13b) and Boc-(R )-Leu-(S )-Pro-(S ,S )-Azi(OBn)2 (compound 13e), reduced the growth and viability ofLeishmania major and the infection rate of macrophages while not showing cytotoxicity against host cells. In the present study, we characterized the mode of action of inhibitors 13b and 13e inL. major . Both compounds targeted leishmanial cathepsin B-like cysteine cathepsin cysteine proteinase C, as shown by fluorescence proteinase activity assays and active-site labeling with biotin-tagged inhibitors. Furthermore, compounds 13b and 13e were potent inducers of cell death in promastigotes, characterized by cell shrinkage, reduction of mitochondrial transmembrane potential, and increased DNA fragmentation. Transmission electron microscopic studies revealed the enrichment of undigested debris in lysosome-like organelles participating in micro- and macroautophagy-like processes. The release of digestive enzymes into the cytoplasm after rupture of membranes of lysosome-like vacuoles resulted in the significant digestion of intracellular compartments. However, the plasma membrane integrity of compound-treated promastigotes was maintained for several hours. Taken together, our results suggest that the induction of cell death inLeishmania by cysteine cathepsin inhibitors 13b and 13e is different from mammalian apoptosis and is caused by incomplete digestion in autophagy-related lysosome-like vacuoles.