The therapeutic potential of synthetic inhibitors to the major
cysteine-proteinase from
Trypanosoma cruzi (cruzain or
cruzipain) was recently demonstrated in animal models of
Chagas' disease. A possible limitation of this strategy would be the emergence of parasite populations developing resistance to
cysteine-proteinase inhibitors. Here, we describe the properties of a phenotypically stable T. cruzi cell line (R-Dm28) that displays increased resistance to Z-(SBz)Cys-Phe-CHN2, an irreversible
cysteine-proteinase inhibitor which preferentially inactivates
cathepsin L-like
enzymes. Isolated from axenic cultures of the parental cells (IC50 1.5 microM), R-Dm28 epimastigotes exhibited 13-fold (IC50) 20 microM) higher resistance to this inhibitor and did not display cross-resistance to unrelated
trypanocidal drugs, such as benznidazol and
nifurtimox. Western blotting (with mAb), affinity labeling (with
biotin-LVG-CHN2) and FACS analysis of R-Dm28 log-phase epimastigotes revealed that the
cruzipain target was expressed at lower levels, as compared with Dm28c. Interestingly, this deficit was paralleled by increased expression of an unrelated Mr 30 000
cysteine-proteinase whose activity was somewhat refractory to inhibition by Z-(SBz)Cys-Phe-CHN,. N-terminal sequencing of the affinity-purified
biotin-LVG-
proteinase complex allowed its identification as a
cathepsin B-like
enzyme. Increased antigenic deposits of this
proteinase were found in the grossly enlarged and electron dense reservosomes from R-Dm28 epimastigotes. Our data suggest that R-Dm28 resistance to toxic effects induced by the synthetic inhibitor may result from decreased availability of the most sensitive
cysteine-proteinase target,
cruzipain. The deficit in metabolic functions otherwise mediated by this
cathepsin L-like
proteinase is likely compensated by increased expression/accumulation of a
cathepsin B-like target.