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, 188 (4), 725-34

Cysteine Protease Inhibitors Cure an Experimental Trypanosoma Cruzi Infection

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Cysteine Protease Inhibitors Cure an Experimental Trypanosoma Cruzi Infection

J C Engel et al. J Exp Med.

Abstract

Trypanosoma cruzi is the causative agent of Chagas' disease. The major protease, cruzain, is a target for the development of new chemotherapy. We report the first successful treatment of an animal model of Chagas' disease with inhibitors designed to inactivate cruzain. Treatment with fluoromethyl ketone-derivatized pseudopeptides rescued mice from lethal infection. The optimal pseudopeptide scaffold was phenylalanine-homophenylalanine. To achieve cure of infection, this pseudopeptide scaffold was incorporated in a less toxic vinyl sulfone derivative. N-methyl piperazine-Phe-homoPhe-vinyl sulfone phenyl also rescued mice from a lethal infection. Six of the treated mice survived over nine months, three without further treatment. Three mice that had entered the chronic stage of infection were retreated with a 20-d regimen. At the conclusion of the experiments, five of the six mice had repeated negative hemacultures, indicative of parasitological cure. Studies of the effect of inhibitors on the intracellular amastigote form suggest that the life cycle is interrupted because of inhibitor arrest of normal autoproteolytic cruzain processing at the level of the Golgi complex. Parasites recovered from the hearts of treated mice showed the same abnormalities as those treated in vitro. No abnormalities were noted in the Golgi complex of host cells. This study provides proof of concept that cysteine protease inhibitors can be given at therapeutic doses to animals to selectively arrest a parasitic infection.

Figures

Figure 1
Figure 1
Levels of parasitemia and survival of mice treated with peptidomimetic fluoromethyl ketones. CH3 mice were infected with T. cruzi and treated twice daily with 1 mg i.p. of Z-F-Ala-FMK (•); Mu-bsu-hF-FMK (○); Mu-F-hF-FMK (▵); and controls with and without DMSO i.p. (□, ▪). Parasitemias were determined every 48 h in each animal on alternating days. Results are mean of two to three animals per day.
Figure 2
Figure 2
Treatment of T. cruzi–infected macrophages with a cysteine protease inhibitor and a fluorescent probe specific for the Golgi complex. Phase contrast (A) and fluorescence (C) microphotograph of an untreated T. cruzi–infected macrophage. Several intracellular amastigotes are visible within the cytoplasm of the host cell. The Golgi complex (G) of the host cell is labeled. Bodipy FL does not induce visible fluorescence in the Golgi complex of untreated amastigotes. Phase contrast (B) and fluorescence microphotograph (D) of an infected macrophage treated with 20 μM Mu-F-hF-VSφ for 48 h. Large, fluorescent Golgi vesicles (g) are evident in CPI-treated amastigotes indicative of vesicle dilation abnormality (21). a, amastigote; G, macrophage– Golgi complex; g, amastigote–Golgi complex; N, macrophage nucleus.
Figure 3
Figure 3
Electronmicroscopy of T. cruzi intracellular amastigotes. T. cruzi amastigotes observed within culture macrophages (A and B) or heart muscle of Mu-F-hF-VSφ–treated mice (C). The normal ultrastructure of untreated intracellular amastigotes within irradiated macrophages (A) contrasts with the altered morphology of intracellular amastigotes treated with 20 μM of Mu-F-hF-VSφ for 48 h (B). More dilated Golgi vesicles and perinuclear membrane similar to that reported in treated epimastigotes (21). Similar ultrastructural alterations were observed in amastigotes isolated from heart muscle (C) of experimentally infected mice treated with 2 mg/d i.p. Mu-F-hF-VSφ for 4 d before necropsy and isolation of heart muscle. A, Untreated amastigotes; B, CPI-treated cell culture amastigote; C, amastigote infecting the heart muscle of a CPI-treated animal. Nuclear membrane (large arrows); Golgi complex (small arrow); vesicle (▴). No abnormalities were noted in Golgi complex or other organelles of host cells. Bar, 1 μm.
Figure 4
Figure 4
Immunoelectronmicroscopy of cell surface membranes of T. cruzi amastigotes. Cell surface membranes of untreated (A) and Mu-F-hF-VSφ–treated (B) amastigotes were immunocytochemically labeled with a specific anti-cruzain antibody. Note markedly diminished gold label on surface of treated parasite consistent with retention of unprocessed cruzain in Golgi (21). PM, parasite cell surface membrane; IGL, immunogold label. Bar, 0.2 μm.
Figure 5
Figure 5
Autoradiogram of extract from radiolabeled [14C]Mu-F-hF-VSφ intracellular amastigotes. Binding of the radiolabeled inhibitor to amastigote cruzain was abolished by preincubation with unlabeled Mu-F-hF-VSφ followed by [14C]CPI. Epimastigote cruzain and recombinant cruzain controls are shown as standards. Note two species labeled in amastigotes that comigrate with either epimastigote cruzain (50 kD) which has both catalytic and COOH-terminal domains (13) or with recombinant cruzain (30 kD) that only has catalytic domain. Lane 1, T. cruzi intracellular amastigotes labeled with [14C]CPI; lane 2, T. cruzi intracellular amastigotes preincubated with unlabeled inhibitor; lane 3, sample buffer; lane 4, recombinant cruzain labeled with [14C]CPI; lane 5, epimastigotes radiolabeled with [14C]Mu-F-hF-VSφ. Note 57/51-kD doublet characteristic of native cruzain which retains COOH-terminal domain (13). Recombinant cruzain lacks the COOH-terminal domain (14).

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