Cysteine proteinase inhibitors kill cultured bloodstream forms of Trypanosoma brucei brucei

Trypanosoma brucei brucei is a causative agent of bovine trypanosomiasis (n agana), a disease of considerable economic significance in much of Africa. Here we report investigations on the effects of various irreversible cystei ne proteinase inhibitors, including vinyl sulfones (VS), peptidyl chloromet hylketones (CMK), diazomethylketones, and fluoromethylketones, on the major lysosomal cysteine proteinase (trypanopain-Tb) of T. b. brucei and on in v itro-cultured bloodstream forms of the parasite. Many of the rested inhibit ors were trypanocidal at low micromolar concentrations. Methylpiperazine ur ea-Phe-homoPhe-VS was the most effective trypanocidal agent, killing 50% of test populations at a working concentration of 0.11 mu M, while carbobenzo xy-Phe-Phe-CMK was the most trypanocidal of the methylketones with an IC50 of 3.6 mu M. Labelling of live and lysed T. b. brucei with biotinylated inh ibitor derivatives suggests that trypanopain-Tb is the likely intracellular target for these inhibitors, Kinetic analysis of the inhibition of purifie d trypanopain-Tb by the inhibitors showed that most had k(ass) values in th e 10(6) M-1 s(-1) range. We conclude that cysteine proteinase inhibitors ha ve potential as trypanocidal agents and that a major target of these compou nds is the lysosomal enzyme trypanopain-Tb. (C) 1999 Academic Press.