Leishmania Ribosomal Protein (RP) paralogous genes compensate each other's expression maintaining protein native levels.

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.

Functional Study of Leishmania braziliensis Protein Arginine Methyltransferases (PRMTs) Reveals That PRMT1 and PRMT5 Are Required for Macrophage Infection.

In trypanosomatids, regulation of gene expression occurs mainly at the posttranscriptional level, and RNA-binding proteins (RBPs) are key players in determining the fates of transcripts. RBPs are targets of protein arginine methyltransferases (PRMTs), which posttranslationally regulate the RNA-binding capacity and other RBP interactions by transferring methyl groups to arginine residues (R-methylation). Herein, we functionally characterized the five predicted PRMTs in Leishmania braziliensis by gene knockout and endogenous protein HA tagging using CRISPR/Cas9 gene editing. We report that R-methylation profiles vary among Leishmania species and across L. braziliensis lifecycle stages, with the peak PRMT expression occurring in promastigotes. A list of PRMT-interacting proteins was obtained in a single coimmunoprecipitation assay using HA-tagged PRMTs, suggesting a network of putative targets of PRMTs and cooperation between the R-methylation writers. Knockout of each L. braziliensis PRMT led to significant changes in global arginine methylation patterns without affecting cell viability. Deletion of either PRMT1 or PRMT3 disrupted most type I PRMT activity, resulting in a global increase in monomethyl arginine levels. Finally, we demonstrate that L. braziliensis PRMT1 and PRMT5 are required for efficient macrophage infection in vitro, and for axenic amastigote proliferation. The results indicate that R-methylation is modulated across lifecycle stages in L. braziliensis and show possible functional overlap and cooperation among the different PRMTs in targeting proteins. Overall, our data suggest important regulatory roles of these proteins throughout the L. braziliensis life cycle, showing that arginine methylation is important for parasite-host cell interactions.

Dipeptidyl nitrile derivatives suppress the Trypanosoma cruzi in vitro infection.

Chagas disease affects several countries around the world with health and sanitation problems. Cysteine proteases are essential for the virulence and replication of the Trypanosoma cruzi, being modulated by dipeptidyl nitriles and derivatives. Here, four dipeptidyl nitrile derivatives were assayed in three T. cruzi morphologies and two strains (Tulahuen and Y) using a set of assays: (i) analysis of the inhibitory activity against cysteine proteases; (ii) determination of the cytotoxic activity and selectivity index; (iii) verification of the inhibition of the trypomastigote invasion in the host cell. These compounds could inhibit the activity of cysteine proteases using the selective substrate Z-FR-MCA for the trypomastigote lysate and extracellular amastigotes. Interestingly, these compounds did not present relevant enzymatic inhibition for the epimastigote lysate. Most of the substances were also cytotoxic and selective against the trypomastigotes and intracellular amastigotes. The best compound of the series (Neq0662) could reduce the enzymatic activity of the cysteine proteases for the trypomastigotes and amastigotes. It was equipotent to the benznidazole drug in the cytotoxic studies using these two parasite forms. Neq0662 was also selective for the parasite, and it inhibited the invasion of the mammalian host cell in all conditions tested at 10 µM. The stereochemistry of the trifluoromethyl group was an important factor for the bioactivity when the two diastereomers (Neq0662 and Neq0663) were compared. All-in-all, these results indicate that these compounds could move further in the drug development stage because of its promising bioactive profile.

Dipeptidyl nitrile derivatives have cytostatic effects against Leishmania spp. promastigotes.

Cysteine proteases are involved in critical cell processes to the protozoa from Leishmania genus, and their inhibition is a therapeutic alternative to treat the disease. In this work, derivatives of dipeptidyl nitriles acting as reversible covalent inhibitors of cysteine proteases were studied as cytostatic agents. The proteolytic activity inside the living and lysed parasite cells was quantified using a selective substrate for cysteine proteases (Z-FR-MCA) from Leishmania amazonensis and L. infantum. The overall proteolytic activity of intact cells and even cell extracts was only marginally affected at high concentrations, with the observation of cytostatic activity and cell cycle arrest of promastigotes. However, the cytotoxic effects were only observed for infected J774 macrophages, which impaired further analysis of the amastigote infection. Therefore, the proteolytic inhibition in intact L. amazonensis and L. infantum promastigotes had no relationship to the cytostatic activity, which emphasizes that these dipeptidyl nitriles act through another mechanism of action.

Biological Activity and Physicochemical Properties of Dipeptidyl Nitrile Derivatives Against Pancreatic Ductal Adenocarcinoma Cells.

BACKGROUND: Pancreatic cancer is one of the most aggressive types with high mortality in patients. Therefore, studies to discover new drugs based on cellular targets have been developed to treat this disease. Due to the importance of Cysteine Protease (CP) to several cellular processes in cancer cells, CP inhibitors have been studied as novel alternative approaches for pancreatic cancer therapy. OBJECTIVE: The cytostatic potential of new CP inhibitors derived from dipeptidyl nitriles is analyzed in vitro using pancreatic cancer (MIA PaCa-2) cells. METHODS: The cytotoxic and cytostatic activities were studied using MTT colorimetric assay in 2D and 3D cultures. Colony formation, migration in Boyden chamber and cell cycle analysis were applied to further study the cytostatic activity. The inhibition of cysteine proteases was evaluated with Z-FR-MCA selective substrate, and ROS evaluation was performed with DCFH-DA fluorophore. Permeability was investigated using HPLC-MS to obtain log kw. Combination therapy was also evaluated using the best compound with gemcitabine. RESULTS: The inhibition of intracellular CP activity by the compounds was confirmed, and the cytostatic effect was established with cell cycle retention in the G1 phase. CP inhibitors were able to reduce cell proliferation by 50% in the clonogenic assay, and the same result was achieved for the migration assay, without any cytotoxic effect. The Neq0554 inhibitor was also efficient to increase the gemcitabine potency in the combination therapy. Physicochemical properties using an artificial membrane model quantified 1.14 ≥ log Kw ≥ 0.75 for all inhibitors (also confirmed using HPLC-MS analysis) along with the identification of intra and extracellular metabolites. Finally, these dipeptidyl nitrile derivatives did not trigger the formation of reactive oxygen species, which is linked to genotoxicity. CONCLUSION: Altogether, these results provide a clear and favorable picture to develop CP inhibitors in pre-clinical assays.