Multiple effects of pepstatin A on Trypanosoma cruzi epimastigote forms

Abstract

Herein, we have aimed to explore the effects of pepstatin A, a powerful aspartic protease inhibitor, on Trypanosoma cruzi, the etiologic agent of Chagas' disease. Pepstatin A arrested the proliferation of epimastigotes of T. cruzi (clone Dm28c, TcI lineage), in both dose- and time-dependent manner. The IC(50) value was calculated to be 36.2 μM after 96 h of parasite-drug contact. The parasite treatment with pepstatin A resulted in significant morphological alterations, including parasites becoming round in shape, reduction (≈25%) of the parasite size, and parasites presenting parts or the whole flagellum detached from the cell body. Cell lysis was not observed, resulting in a trypanostatic effect. The treatment of different T. cruzi strains, belonging to distinct phylogenetic lineages, with pepstatin A at 36.2 μM resulted in growth inhibition as follows: 28% to Y (TcII), 45% to CL Brener (TcII), 45.4% to 4167 (Z3), and 26.4% to 3663 (Z3) strains. The hydrolysis of a cathepsin D fluorogenic substrate (7-methoxycoumarin-4-acetyl-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(DNP)-D: -Arg-amide) by T. cruzi epimastigote extract was inhibited (≈65%) by pepstatin A at 10 μM, suggesting that an aspartic protease may be the intracellular target of this inhibitor. Curiously, pepstatin A induced an increase of 54% and 98%, respectively, in the surface expression of gp63- and calpain-related molecules in epimastigotes, but not in the cruzipain level, as well as stimulated the epimastigote-to-trypomastigote differentiation in a dose-dependent manner. However, approximately 45% of the trypomastigotes had their flagellum detached from the cell body. These results contribute to understand the possible role of aspartic proteases in the physiology of T. cruzi cells, adding new in vitro insights into the possibility of exploiting aspartic protease as promising targets to treat Chagas' disease.

Decoding the Anti-Trypanosoma cruzi Action of HIV Peptidase Inhibitors Using Epimastigotes as a Model

“…The HIV PIs induced several alterations on epimastigote forms similar to those caused by pepstatin A, a prototypal aspartic peptidase inhibitor [12] . Due the morphological alterations observed after treatment with the HIV PIs by light microscopy, we aimed to analyze the T. cruzi clone Dm28c epimastigotes alterations more deeply by means of transmission electron microscopy.…”

“…In trypanosomatids, it has been observed that the aspartic peptidase inhibitors commonly used in HAART were capable of suppressing the proliferation of Leishmania species [26] - [28] . In an initial approach to study the effects of aspartic peptidase inhibitors against T. cruzi , we have previously demonstrated that the classic aspartic peptidase inhibitor pepstatin A was able to inhibit T. cruzi clone Dm28c epimastigote growth in a typical dose-dependent manner, with an IC 50 value of 36.2 µM after 96 h [12] . In the present work, we showed that HIV PIs inhibited the proliferation of T. cruzi clone Dm28c epimastigotes at much lower concentrations than pepstatin A, being ritonavir, lopinavir and nelfinavir the most effective compounds tested.…”

“…The effects of six distinct HIV PIs (amprenavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir) on T. cruzi clone Dm28c epimastigote forms were assessed by a method similar to that previously described elsewhere [12] . Briefly, epimastigotes were counted using a Neubauer chamber and resuspended in fresh medium to a final concentration of 5×10 6 viable epimastigotes per milliliter.…”

“…The reaction was started by the addition of the substrate (2 µM) to the parasite extract (40 µg protein) in a total volume of 60 µl of a buffer containing 0.2 M sodium phosphate, 0.1 M citric acid, 1 mM EDTA, 10 µM E-64, pH 4.0, in the presence or the absence of each HIV PI at 10, 50 or 100 µM. The reaction mixture was incubated at 37°C for 2 h. The assays were controlled for self-liberation of the fluorophore over the same time interval [12] .…”

“…Pepstatin A arrested the proliferation of epimastigotes, in both dose- and time-dependent manner. The treatment of parasite with pepstatin A resulted in significant morphological alterations, as detected by light microscopy analysis [12] . Nevertheless, pepstatin-like drugs are not used clinically because of their metabolism in the liver and rapid clearance from blood [13] .…”