Multiple effects of pepstatin A on Trypanosoma cruzi epimastigote forms.
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.
Laboratório de Bioquímica de Proteases, Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de Góes (IMPG), Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil.
Leandro S
LS
Sangenito
Keyla C
KC
Gonçalves
Erika A
EA
Abi-Chacra
Cátia L
CL
Sodré
Claudia M
CM
d'Avila-Levy
Marta H
MH
Branquinha
André L S
AL
Santos
D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology*; D005407:Flagella / Q000187:drug effects; D006801:Humans; D020128:Inhibitory Concentration 50; D008853:Microscopy; D021261:Parasitic Sensitivity Tests; D010436:Pepstatins / Q000494:pharmacology*; D014349:Trypanosoma cruzi / Q000166:cytology / Q000187:drug effects* / Q000254:growth & development
D000981:Antiprotozoal Agents; D010436:Pepstatins; C031375:pepstatin