The cysteine protease cruzipain is considered to be a validated target for therapeutic intervention in the treatment of Chagas disease. Anti-trypanosomal activity against the CL Brener strain of T. cruzi was observed in the 0.1 μM to 1 μM range for three nitrile-based cysteine protease inhibitors based on two scaffolds known to be associated with cathepsin K inhibition. The two compounds showing the greatest potency against the trypanosome were characterized by EC₅₀ values (0.12 μM and 0.25 μM) that were an order of magnitude lower than the corresponding K_i values measured against cruzain, a recombinant form of cruzipain, in an enzyme inhibition assay. This implies that the anti-trypanosomal activity of these two compounds may not be explained only by the inhibition of the cruzain enzyme, thereby triggering a putative polypharmacological profile towards cysteine proteases. Chagas disease is still considered a neglected tropical disease (NTD). There are more than 5 million people infected worldwide of which 99% are located in the Americas. Chagas disease has huge social and economic consequences for countries with significant proportions of their populations living in poverty. Chagas disease causes approximately seven thousand deaths per year and half a million people live with disabilities caused by the disease. Based on disability-adjusted life-years (DALYs), the disease burden of Chagas disease is five times greater than malaria and is approximately one-fifth of HIV/AIDS in the Latin American and Caribbean region. Despite being characterized over a century ago by Carlos Chagas who identified Trypanosoma cruzi as the causative agent, treatment of the disease is restricted to just two drugs (benznidazole and nifurtimox) that are effective only in the acute phase of the disease. Failure to rapidly diagnose T. cruzi infections and a poor side effect profile that causes many patients to abandon treatment both limit the effectiveness of treatment in the acute phase Chagas disease and many patients ultimately progress to the chronic phase. In this study, we have identified three compounds with anti-trypanosomal effects on the infective T. cruzi CL Brener form prevalent in various regions of the Americas, with potency in the 0.1 μM to 1 μM range and minimal cytotoxicity, even at 128 μM. Additionally, two of these compounds are significantly more potent against the parasite than against the recombinant form of the cysteine protease cruzipain which is generally considered to be a valid target for therapeutic intervention in the treatment of Chagas disease. These observations raise questions about the relevance of cruzain inhibition as a predictor of anti-trypanosomal activity and reinforce the case for using phenotypic assays in the search for new antichagasic agents. Correlation between enzyme inhibition and activity in cell-based assays is a general issue in drug discovery and we discuss the importance of intracellular unbound concentration in this context. We believe that this study is of significant interest both on account of the potent anti-trypanosomal activity observed for three of the compounds studied and the weak link between this activity and cruzain inhibition.

In the protozoan parasite Leishmania, most genes encoding for ribosomal proteins (RPs) are present as two or more copies in the genome. However, their untranslated regions (UTRs) are predominantly divergent and might be associated with a distinct regulation of the expression of paralogous genes. Herein, we investigated the expression profiles of two RPs (S16 and L13a) encoded by duplicated genes in Leishmania major. The genes encoding for the S16 protein possess identical coding sequences (CDSs) and divergent UTRs, whereas the CDSs of L13a diverge by two amino acids and by their UTRs. Using CRISPR/Cas9 genome editing, we generated knockout (Δ) and endogenously tagged transfectants for each paralog of L13a and S16 genes. Combining tagged and Δ cell lines we found evidence of differential expression of both RPS16 and RPL13a isoforms throughout parasite development, with one isoform consistently more abundant than its respective copy. In addition, compensatory expression was observed for each paralog upon deletion of the corresponding isoform, suggesting functional conservation between these proteins. This differential expression pattern relates to post-translational processes, given compensation occurs at the level of the protein, with no alterations detected at transcript level. Ribosomal profiles for RPL13a indicate a standard behavior for these paralogues suggestive of interaction with heavy RNA-protein complexes, as already reported for other RPs in trypanosomatids. We identified paralog-specific bound to their 3’UTRs which may be influential in regulating paralog expression. In support, we identified conserved cis-elements within the 3’UTRs of RPS16 and RPL13a; cis-elements exclusive to the UTR of the more abundant paralog or to the less abundant ones were identified.