The increase of leishmaniasis cases worldwide and the emergence of Leishmania strains resistant to current treatments make necessary to find new therapeutic targets. Proteases are appealing drug targets because they play pivotal roles in facilitating parasite survival and promoting pathogenesis. Enzymes belonging to the dipeptidyl peptidase 3 (DPP3) group have been described in different organisms such as mammals, insects and yeast, in which these enzymes have been involved in both protein turnover and protection against oxidative damage. The aim of this work was to characterize the structure and function of the Leishmania braziliensis DPP3 (LbDPP3) protein as the first step to elucidate its suitability as a potential drug target. Sequence alignment showed 43% of identity between LbDPP3 and its human orthologous (hDPP3) enzyme. Although the modeled protein adopted a globally conserved three-dimensional (3D) structure, structural differences were found in the vicinity of the active site and the substrate binding-cleft. In addition, the Leishmania protein was expressed as a soluble recombinant protein and its kinetics parameters were determined using the z-Arginine-Arginine-AMC substrate. The LbDPP3 activity was maximal at pH values between 8.0–8.5. Interestingly, classical enzyme inhibitors such as the tynorphin and its derivative peptide IVYPW were found to actively inhibit the LbDPP3 activity. Moreover, these DPP3 inhibitors showed a detrimental effect upon parasite survival, decreasing the viability of promastigotes by up to 29%. Finally, it was observed that LbDPP3 was equally expressed along the in vitro differentiation from promastigotes to axenic amastigotes. In conclusion, these findings suggest that the L. brazileinsis DPP3 could be a promising drug target.

Trypanosomatid type I nitroreductases (NTRs), i.e., mitochondrial enzymes that metabolise nitroaromatic pro-drugs, are essential for parasite growth, infection, and survival. Here, a type I NTR of non-virulent protozoan Trypanosoma rangeli is described and compared to those of other trypanosomatids. The NTR gene was isolated from KP1(+) and KP1(-) strains, and its corresponding transcript and 5’ untranslated region (5’UTR) were determined. Bioinformatics analyses and nitro-drug activation assays were also performed. The results indicated that the type I NTR gene is present in both KP1(-) and KP1(+) strains, with 98% identity. However, the predicted subcellular localisation of the protein differed among the strains (predicted as mitochondrial in the KP1(+) strain). Comparisons of the domains and 3D structures of the NTRs with those of orthologs demonstrated that the nitroreductase domain of T. rangeli NTR is conserved across all the strains, including the residues involved in the interaction with the FMN cofactor and in the tertiary structure characteristics of this oxidoreductase protein family. mRNA processing and expression were also observed. In addition, T. rangeli was shown to be sensitive to benznidazole and nifurtimox in a concentration-dependent manner. In summary, T. rangeli appears to have a newly discovered functional type I NTR.