We examined strains of Trypanosoma cruzi isolated from patients with acute Chagas disease that had been acquired by oral transmission in the state of Santa Catarina, Brazil (2005) and two isolates that had been obtained from a marsupial (Didelphis aurita) and a vector (Triatoma tibiamaculata). These strains were characterised through their biological behaviour and isoenzymic profiles and genotyped according to the new Taxonomy Consensus (2009) based on the discrete typing unities, that is, T. cruzi genotypes I-VI. All strains exhibited the biological behaviour of biodeme type II. In six isolates, late peaks of parasitaemia, beyond the 20th day, suggested a double infection with biodemes II + III. Isoenzymes revealed Z2 or mixed Z1 and Z2 profiles. Genotyping was performed using three polymorphic genes (cytochrome oxidase II, spliced leader intergenic region and 24Sα rRNA) and the restriction fragment length polymorphism of the kDNA minicircles. Based on these markers, all but four isolates were characterised as T. cruzi II genotypes. Four mixed populations were identified: SC90, SC93 and SC97 (T. cruzi I + T. cruzi II) and SC95 (T. cruzi I + T. cruzi VI). Comparison of the results obtained by different methods was essential for the correct identification of the mixed populations and major lineages involved indicating that characterisation by different methods can provide new insights into the relationship between phenotypic and genotypic aspects of parasite behaviour.

The Serine/arginine-rich protein kinases (SRPK) are involved in pre-mRNA splicing control through the phosphorylation of the SR protein family of splicing factors. Over the last years, several studies have shown the relevance of SRPK for human cancers and their potential as promising drug targets. In this context, we have previously selected three trifluoromethyl arylamides (named here as SRVIC24, SRVIC30 and SRVIC36) with improved in vitro antileukemia effect and ability of impairing the cellular activity of SRPK. Given the increasing amount of reports on the implication of these kinases in metastatic cancers, in this study, we have evaluated the antimetastatic effect of these compounds and the known SRPK inhibitor (SRPIN340) on a murine model of metastatic melanoma. The compounds were able to impact the melanoma cell metastatic behavior by decreasing migration, invasion, adhesion, and colony formation in in vitro assays. Also, they presented antimetastatic in vivo activity, without apparent signs of systemic toxicity after treatments, as revealed by the histology of organs and analysis of key serum biochemical markers. Moreover, the effect of the treatments on SRPK1 nuclear translocation and SR protein phosphorylation was observed. Finally, molecular docking studies were carried out to gain structural information on the SRPK-compound complexes. Together, these data suggest that SRPK pharmacological inhibition should be considered as an interesting therapeutic strategy against metastatic cancers.

Cysteine proteases are essential hydrolytic enzymes present in the majority of organisms, including viruses and unicellular parasites. Despite the high sequence identity displayed among these proteins, specific structural features across different species grant distinct functions to these biomolecules, frequently related to pathological conditions. Consequently, their relevance as promising targets for potential specific inhibitors has been highlighted and occasionally validated in recent decades. In this review, we discuss the recent outcomes on structure-based campaigns aiming the discovery of new inhibitor prototypes against cruzain and falcipain, as alternative therapeutic tools for Chagas disease and malaria treatments, respectively. Computational and synthetic approaches have been combined on hit optimization strategies and are also discussed herein. These rationales are extended to additional tropical infectious and neglected pathologies, such as schistosomiasis, leishmaniasis and babesiosis, and also to Alzheimer's Disease, a widespread neurodegenerative disease poorly managed by current available drugs and recently linked to particular physiopathological roles of human cysteine proteases.

Cruzipains are the main papain-like cysteine proteases of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease. Encoded by a multigenic family, previous studies have estimated the presence of dozens of copies spread over multiple chromosomes in different parasite strains. Here, we describe the complete gene repertoire of cruzipain in three parasite strains, their genomic organization, and expression pattern throughout the parasite life cycle. Furthermore, we have analyzed primary sequence variations among distinct family members as well as structural differences between the main groups of cruzipains. Based on phylogenetic inferences and residue positions crucial for enzyme function and specificity, we propose the classification of cruzipains into two families (I and II), whose genes are distributed in two or three separate clusters in the parasite genome, according with the strain. Family I comprises nearly identical copies to the previously characterized cruzipain 1/cruzain, whereas Family II encompasses three structurally distinct sub-types, named cruzipain 2, cruzipain 3, and cruzipain 4. RNA-seq data derived from the CL Brener strain indicates that Family I genes are mainly expressed by epimastigotes, whereas trypomastigotes mainly express Family II genes. Significant differences in the active sites among the enzyme sub-types were also identified, which may play a role in their substrate selectivity and impact their inhibition by small molecules.

No abstract