The cysteine proteinase inhibitor Z-Phe-Ala-CHN2 alters cell morphology and cell division activity of Trypanosoma brucei bloodstream forms in vivo

Abstract

BackgroundCurrent chemotherapy of human African trypanosomiasis or sleeping sickness relies on drugs developed decades ago, some of which show toxic side effects. One promising line of research towards the development of novel anti-trypanosomal drugs are small-molecule inhibitors of Trypanosoma brucei cysteine proteinases.ResultsIn this study, we demonstrate that treatment of T. brucei-infected mice with the inhibitor, carbobenzoxy-phenylalanyl-alanine-diazomethyl ketone (Z-Phe-Ala-CHN2), alters parasite morphology and inhibits cell division. Following daily intra-peritoneal administration of 250 mg kg-1 of Z-Phe-Ala-CHN2 on days three and four post infection (p.i.), stumpy-like forms with enlarged lysosomes were evident by day five p.i. In addition, trypanosomes exposed to the inhibitor had a 65% greater protein content than those from control mice. Also, in contrast to the normal 16% of parasites containing two kinetoplasts – a hallmark of active mitosis, only 4% of trypanosomes exposed to the inhibitor were actively dividing, indicating cell cycle-arrest.ConclusionWe suggest that inhibition of endogenous cysteine proteinases by Z-Phe-Ala-CHN2 depletes the parasite of essential nutrients necessary for DNA synthesis, which in turn, prevents progression of the cell cycle. This arrest then triggers differentiation of the long-slender into short-stumpy forms.

Cytosolic Peroxidases Protect the Lysosome of Bloodstream African Trypanosomes from Iron-Mediated Membrane Damage

“…The enlarged but still confined fluorescence observed in 5–10% of the cells (2 in Figure 2A) suggests that swelling of the organelle can occur as an intermediate step. This has been observed in parasites that were treated with protease inhibitors or human serum or upon ablation of p67, a lysosomal transmembrane glycoprotein [11], [25], [26]. Trypanolysis caused by apoL-I, crucial component of the trypanolytic factor present in human serum, involves the formation of anion-selective pores in the lysosomal membrane of the parasite, a process which is abolished by addition of 1 mM 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) to the culture medium [26], [27].…”

The cell biology of Trypanosoma brucei differentiation

“…Treatment with troglitazone, a thiazolidinedione that acts on the peroxisome proliferating-activated receptor gamma in higher eukaryotes, also promotes morphological transformation of pleomorphic lines and causes cell growth arrest in monomorphic cells (although not at a specific cell-cycle position) [13]. Cell-cycle arrest and development to a stumpy morphology has also been observed with several other compounds or genetic perturbations that inhibit cell growth in bloodstream forms [14,15]. However, the precise relationship between cells described as ‘stumpy-like’ and the stumpy cells that arise in response to SIF in vivo or in vitro is not clear.…”

Crystal Structures of TbCatB and Rhodesain, Potential Chemotherapeutic Targets and Major Cysteine Proteases of Trypanosoma brucei

“…TbCatB may therefore represent the more promising target for novel cysteine protease inhibitors targeting T. brucei infection. A cysteine protease inhibitor, Z-Phe-Ala-CHN2, has been shown to be lethal to T. brucei both in vitro and in vivo [18] , [19] and our efforts are focused on elucidating key features of inhibitors that will optimize both specificity and potency.…”