Abstract:The treatment for both leishmaniasis and trypanosomiasis, which are severe human infections caused by trypanosomatids belonging to Leishmania and Trypanosoma genera, respectively, is extremely limited because of concerns of toxicity and efficacy with the available anti-protozoan drugs, as well as the emergence of drug resistance. Consequently, the urgency for the discovery of new trypanosomatid targets and novel bioactive compounds is particularly necessary. In this context, the investigation of changes in parasite gene expression between drug resistant/sensitive strains and in the up-regulation of virulence-related genes in infective forms has brought to the fore the involvement of calpain-like proteins in several crucial pathophysiological processes performed by trypanosomatids. These studies were encouraged by the publication of the complete genome sequences of three human pathogenic trypanosomatids, Trypanosoma brucei, Trypanosoma cruzi and Leishmania major, which allowed in silico analyses that in turn directed the identification of numerous genes with interesting chemotherapeutic characteristics, including a large family of calpain-related proteins, in which to date 23 genes were assigned as calpains in T. brucei, 40 in T. cruzi and 33 in L. braziliensis. In the present review, we intend to add to these biochemical/biological reports the investigations performed upon the inhibitory capability of calpain inhibitors against human pathogenic trypanosomatids.

BackgroundHuman cutaneous leishmaniasis is caused by distinct species, including Leishmania amazonensis. Treatment of cutaneous leishmaniasis is far from satisfactory due to increases in drug resistance and relapses, and toxicity of compounds to the host. As a consequence for this situation, the development of new leishmanicidal drugs and the search of new targets in the parasite biology are important goals.Methodology/Principal FindingsIn this study, we investigated the mechanism of death pathway induced by the calpain inhibitor MDL28170 on Leishmania amazonensis promastigote forms. The combined use of different techniques was applied to contemplate this goal. MDL28170 treatment with IC50 (15 µM) and two times the IC50 doses induced loss of parasite viability, as verified by resazurin assay, as well as depolarization of the mitochondrial membrane, which was quantified by JC-1 staining. Scanning and transmission electron microscopic images revealed drastic alterations on the parasite morphology, some of them resembling apoptotic-like death, including cell shrinking, surface membrane blebs and altered chromatin condensation pattern. The lipid rearrangement of the plasma membrane was detected by Annexin-V labeling. The inhibitor also induced a significant increase in the proportion of cells in the sub-G0/G1 phase, as quantified by propidium iodide staining, as well as genomic DNA fragmentation, detected by TUNEL assay. In cells treated with MDL28170 at two times the IC50 dose, it was also possible to observe an oligonucleossomal DNA fragmentation by agarose gel electrophoresis.Conclusions/SignificanceThe data presented in the current study suggest that MDL28170 induces apoptotic marker expression in promastigotes of L. amazonensis. Altogether, the results described in the present work not only provide a rationale for further exploration of the mechanism of action of calpain inhibitors against trypanosomatids, but may also widen the investigation of the potential clinical utility of calpain inhibitors in the chemotherapy of leishmaniases.

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.

ObjectiveThe low investment in research, diagnosis and treatment are factors that contribute to the continuity of Chagas’ disease as a neglected tropical diseases (NTDs). In this context, the repositioning of drugs represents a useful strategy, in the search for new chemotherapeutic approaches for NTDs. HIV aspartic peptidase inhibitors (HIV IPs) are good candidates for drug repurposing. Here, we modeled the three dimensional structure of an aspartyl peptidase of Trypanosoma cruzi, the causative agent of Chagas’ disease, aligned it to the HIV aspartyl peptidase and performed docking binding assays with the HIV PIs.ResultsThe 3D structure confirmed the presence of acid aspartic residues, which are critical to enzyme activity. The docking experiment revealed that HIV IPs bind to the active site of the enzyme, being ritonavir and lopinavir the ones with greater affinity. Benznidazole presented the worst binding affinity, this drug is currently used in Chagas’ disease treatment and was included as negative control. These results together with previous data on the trypanocidal effect of the HIV PIs support the hypothesis that a T. cruzi aspartyl peptidase can be the intracellular target of these inhibitors. However, the direct demonstration of the inhibition of T. cruzi aspartyl peptidase activity by HIV PIs is still a goal to be persuaded.Electronic supplementary materialThe online version of this article (10.1186/s13104-018-3927-z) contains supplementary material, which is available to authorized users.