Optimization of benzenesulfonyl derivatives as anti-Trypanosomatidae agents: Structural design, synthesis, and pharmacological assessment against Trypanosoma cruzi and Leishmania infantum.

Leishmaniasis and Chagas disease are neglected tropical diseases caused by Trypanosomatidae parasites. Given the numerous limitations associated with current treatments, such as extended treatment duration, variable efficacy, and severe side effects, there is an urgent imperative to explore novel therapeutic options. This study details the early stages of hit-to-lead optimization for a benzenesulfonyl derivative, denoted as initial hit, against Trypanossoma cruzi (T. cruzi), Leishmania infantum (L. infantum) and Leishmania braziliensis (L. braziliensis). We investigated structure - activity relationships using a series of 26 newly designed derivatives, ultimately yielding potential lead candidates with potent low-micromolar and sub-micromolar activities against T. cruzi and Leishmania spp, respectively, and low in vitro cytotoxicity against mammalian cells. These discoveries emphasize the significant promise of this chemical class in the fight against Chagas disease and leishmaniasis.

Evaluation and discovery of novel benzothiazole derivatives as promising hits against Leishmania infantum.

An early exploration of the benzothiazole class against two kinetoplastid parasites, Leishmania infantum and Trypanosoma cruzi, has been performed after the identification of a benzothiazole derivative as a suitable antileishmanial initial hit. The first series of derivatives focused on the acyl fragment of its class, evaluating diverse linear and cyclic, alkyl and aromatic substituents, and identified two other potent compounds, the phenyl and cyclohexyl derivatives. Subsequently, new compounds were designed to assess the impact of the presence of diverse substituents on the benzothiazole ring or the replacement of the endocyclic sulfur by other heteroatoms. All compounds showed relatively low cytotoxicity, resulting in decent selectivity indexes for the most active compounds. Ultimately, the in vitro ADME properties of these compounds were assessed, revealing a satisfying water solubility, gastrointestinal permeability, despite their low metabolic stability and high lipophilicity. Consequently, compounds 5 and 6 were identified as promising hits for further hit-to-lead exploration within this benzothiazole class against L. infantum, thus providing promising starting points for the development of antileishmanial candidates.

A specific Leishmania infantum polyepitope vaccine triggers Th1-type immune response and protects against experimental visceral leishmaniasis.

The development of an immunogenic, effective, and safe vaccine is essential as an alternative for disease control. The present study aimed to evaluate the immunogenicity and efficacy potential of a polyepitope T-cell antigen candidate against visceral leishmaniasis in a murine model. BALB/c mice were immunized with three doses subcutaneously with Poly-T Leish alone or adjuvanted with Saponin plus Monophosphoryl lipid A, with 15-day intervals between doses, and challenged with 107 stationary-phase Leishmania infantum promastigotes via tail vein. Immunogenicity and parasitism in spleen and liver of immunized mice were evaluated 45 days post-challenge. Our results revealed that the immunization with Poly-T Leish and Poly-T Leish/SM increases the percentage of specific T (CD4+ and CD8+) lymphocytes proliferation in vitro after antigen-specific stimulation. Also, Poly-T Leish and Poly-T Leish/SM groups showed a high percentage of IFN-γ and TNF-α-producing T cells, meanwhile, the Poly-T Leish/SM group also showed an increased percentage of multifunctional T cells producing double and triple-positive (IFN-γ+TNF-α+IL-2+) cytokines. The immunization with Poly-T Leish or Poly-T Leish/SM stimulated a decreased IL-4 and IL-10 compared to the Saline and adjuvant group. Poly-T Leish/SM immunized mice exhibit a noteworthy reduction in the parasite burden (spleen and liver) through real-time PCR (96%). Moreover, we observed higher nitrite secretion in 120-hour stimulated-culture supernatant using Griess method. We demonstrated that the Poly-T Leish/SM candidate was potentially immunogenic, providing enhancement of protective immune mechanisms, and conferred protection reducing parasitism. Our candidate was considered potential against visceral leishmaniasis, and eventually, could be tested in phase I and II clinical trials in dogs.

Synthesis, Design, and Structure-Activity Relationship of a Benzenesulfonylpiperazine Series against Trypanosoma cruzi.

Chagas disease is a neglected tropical disease, endemic in Latin America and caused by the protozoan parasite Trypanosoma cruzi. Available treatments show low cure efficacy during the chronic phase of the disease and cause a series of side effects, reinforcing the need to develop new drugs against Chagas disease. In this work, we describe the optimization of a trypanocidal hit compound recently reported in phenotypic high-throughput screening studies against Trypanosoma cruzi. A hit-to-lead process was initiated and a structure-activity relationship against Trypanosoma cruzi was obtained after the synthesis and biological evaluation of 22 new benzenesulfonylpiperazine derivatives. From this structure-activity relationship study, we identified three compounds with a promising predicted ADMET profile and potency comparable to the reference drug benznidazole, which are candidates for further development towards therapies for Chagas disease.

Expression Profile of Genes Related to the Th17 Pathway in Macrophages Infected by Leishmania major and Leishmania amazonensis: The Use of Gene Regulatory Networks in Modeling This Pathway.

Leishmania amazonensis and Leishmania major are the causative agents of cutaneous and mucocutaneous diseases. The infections' outcome depends on host-parasite interactions and Th1/Th2 response, and in cutaneous form, regulation of Th17 cytokines has been reported to maintain inflammation in lesions. Despite that, the Th17 regulatory scenario remains unclear. With the aim to gain a better understanding of the transcription factors (TFs) and genes involved in Th17 induction, in this study, the role of inducing factors of the Th17 pathway in Leishmania-macrophage infection was addressed through computational modeling of gene regulatory networks (GRNs). The Th17 GRN modeling integrated experimentally validated data available in the literature and gene expression data from a time-series RNA-seq experiment (4, 24, 48, and 72 h post-infection). The generated model comprises a total of 10 TFs, 22 coding genes, and 16 cytokines related to the Th17 immune modulation. Addressing the Th17 induction in infected and uninfected macrophages, an increase of 2- to 3-fold in 4-24 h was observed in the former. However, there was a decrease in basal levels at 48-72 h for both groups. In order to evaluate the possible outcomes triggered by GRN component modulation in the Th17 pathway. The generated GRN models promoted an integrative and dynamic view of Leishmania-macrophage interaction over time that extends beyond the analysis of single-gene expression.

Comparative transcriptomic analysis of antimony resistant and susceptible Leishmania infantum lines.

BACKGROUND: One of the major challenges to leishmaniasis treatment is the emergence of parasites resistant to antimony. To study differentially expressed genes associated with drug resistance, we performed a comparative transcriptomic analysis between wild-type and potassium antimonyl tartrate (SbIII)-resistant Leishmania infantum lines using high-throughput RNA sequencing. METHODS: All the cDNA libraries were constructed from promastigote forms of each line, sequenced and analyzed using STAR for mapping the reads against the reference genome (L. infantum JPCM5) and DESeq2 for differential expression statistical analyses. All the genes were functionally annotated using sequence similarity search. RESULTS: The analytical pipeline considering an adjusted p-value < 0.05 and fold change > 2.0 identified 933 transcripts differentially expressed (DE) between wild-type and SbIII-resistant L. infantum lines. Out of 933 DE transcripts, 504 presented functional annotation and 429 were assigned as hypothetical proteins. A total of 837 transcripts were upregulated and 96 were downregulated in the SbIII-resistant L. infantum line. Using this DE dataset, the proteins were further grouped in functional classes according to the gene ontology database. The functional enrichment analysis for biological processes showed that the upregulated transcripts in the SbIII-resistant line are associated with protein phosphorylation, microtubule-based movement, ubiquitination, host-parasite interaction, cellular process and other categories. The downregulated transcripts in the SbIII-resistant line are assigned in the GO categories: ribonucleoprotein complex, ribosome biogenesis, rRNA processing, nucleosome assembly and translation. CONCLUSIONS: The transcriptomic profile of L. infantum showed a robust set of genes from different metabolic pathways associated with the antimony resistance phenotype in this parasite. Our results address the complex and multifactorial antimony resistance mechanisms in Leishmania, identifying several candidate genes that may be further evaluated as molecular targets for chemotherapy of leishmaniasis.

Diversity and genome mapping assessment of disordered and functional domains in trypanosomatids.

The proteins that have structural disorder exemplify a class of proteins which is part of a new frontier in structural biology that demands a new understanding of the paradigm of structure/function correlations. In order to address the location, relative distances and the functional/structural correlation between disordered and conserved domains, consensus disordered predictions were mapped together with CDD domains in Leishmania braziliensis M2904, Leishmania infantum JPCM5, Trypanosoma cruzi CL-Brener Esmeraldo-like, Trypanosoma cruzi Dm28c, Trypanosoma cruzi Sylvio X10, Blechomonas ayalai B08-376 and Paratrypanosoma confusum CUL13 predicted proteomes. Our results depicts the role of protein disorder in key aspects of parasites biology highlighting: a) statistical significant association between genome structural location of protein disordered consensus stretches and functional domains; b) that disordered protein stretches appear in greater percentage at upstream or downstream position of the predicted domain; c) a possible role of structural disorder in several gene expression, control points that includes but are not limited to: i) protein folding; ii) protein transport and degradation; and iii) protein modification. In addition, for values of protein with disorder content greater than 40%, a small percentage of protein binding sites in IDPs/IDRs, a higher hypothetical protein annotation frequency was observed than expected by chance and trypanosomatid multigene families linked with virulence are rich in protein with disorder content. SIGNIFICANCE: T. cruzi and Leishmania spp are the etiological agents of Chagas disease and leishmaniasis, respectively. Currently, no vaccine or effective drug treatment is available against these neglected diseases and the knowledge about the post-transcriptional and post-translational mechanisms of these organisms, which are key for this scenario, remain scarce. This study depicts the potential impact of the proximity between protein structural disorder and functional domains in the post-transcriptional regulation of pathogenic versus human non-pathogenic trypanosomatids. Our results revealed a significant statistical relationship between the genome structural locations of these two variables and disordered regions appearing more frequently at upstream or downstream positions of the CDD locus domain. This flexibility feature would maintain structural accessibility of functional sites for post-translational modifications, shedding light into this important aspect of parasite biology. This hypothesis is corroborated by the functional enrichment analysis of disordered proteins subset that highlight the involvement of this class of proteins in protein folding, protein transport and degradation and protein modification. Furthermore, our results pointed out: a) the impact of protein disorder in the process of genome annotation (proteins tend to be annotated as hypothetical when the disorder content reaches ~40%); b) that trypanosomatid multigenic families linked with virulence have a key protein disorder content.

In silico analysis of putative dormancy genes in Plasmodium vivax.

Plasmodium vivax is the most widely spread species causing human malaria. The control of malaria caused by P. vivax has been largely hampered by its ability to develop a dormant liver stage that can generate a new blood infection at different periods of time. Unfortunately, the mechanisms of dormancy in P. vivax have not been thoroughly elucidated to date. In this study, the putative dormancy genes were analyzed to select genes with less genetic variability to maintain the function of relapsing. Expression data concerning these genes were searched to support the selection. Protein interactions among selected gene products were identified based on known and predicted protein-protein interaction using String database. Potentially interacting proteins (n = 15) were used to propose a mechanism involved in dormancy based on the differential vesicular transport due to the iron available in the hepatocyte.

Shotgun proteomics to unravel the complexity of the Leishmania infantum exoproteome and the relative abundance of its constituents.

The exoproteome of some Leishmania species has revealed important insights into host-parasite interaction, paving the way for the proposal of novel disease-oriented interventions. The focus of the present investigation constituted the molecular profile of the L. infantum exoproteome revealed by a shotgun proteomic approach. Promastigotes under logarithmic phase of growth were obtained and harvested by centrifugation at different time points. Cell integrity was evaluated through the counting of viable parasites using propidium iodide labeling, followed by flow cytometry analysis. The 6h culture supernatant, operationally defined here as exoproteome, was then conditioned to in solution digestion and the resulting peptides submitted to mass spectrometry. A total of 102 proteins were identified and categorized according to their cellular function. Their relative abundance index (emPAI) allowed inference that the L. infantum exoproteome is a complex mixture dominated by molecules particularly involved in nucleotide metabolism and antioxidant activity. Bioinformatic analyses support that approximately 60% of the identified proteins are secreted, of which, 85% possibly reach the extracellular milieu by means of non-classic pathways. At last, sera from naturally infected animals, carriers of differing clinical forms of Canine Visceral Leishmaniasis (CVL), were used to test the immunogenicity associated to the L. infantum exoproteome. Western blotting experiments revealed that this sub-proteome was useful at discriminating symptomatic animals from those exhibiting other clinical forms of the disease. Collectively, the molecular characterization of the L. infantum exoproteome and the preliminary immunoproteomic assays opened up new research avenues related to treatment, prognosis and diagnosis of CVL.