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Leishmaniasis remains an important neglected tropical disease, and current treatments are limited by toxicity, resistance, and low bioavailability. In this study, novel guanidine derivatives were evaluated through an integrated approach, combining in silico physicochemical profiling with in vitro biological assays using Leishmania (Viannia) braziliensis, the etiological agent of American Tegumentary Leishmaniasis (ATL). Most compounds exhibited favorable drug-like properties, though variations in lipophilicity and solubility influenced biological performance. Among the tested molecules, FURL-G5 emerged as the most promising candidate, showing potent activity against promastigote forms and low cytotoxicity in murine macrophages, resulting in high selectivity indices (SI > 10), comparable to those of LQOF-G1, a compound with previously established leishmanicidal effects. These compounds were also tested on intracellular amastigotes, drastically reducing the infection rate of macrophages. The integration of an in silico approach and biological validation enabled rational compound prioritization and supports the early-stage development of these scaffolds. Overall, this study reinforces the potential of guanidine-based compounds as leads for innovative ATL drug discovery and demonstrates the value of multidisciplinary strategies for identifying selective and safe therapeutic candidates.

Glucantime (Sb V ) is the first-line treatment against American Tegumentary Leishmaniasis. Resistance cases to this drug have been reported and related to host characteristics and parasite phenotypes. In this study, 12 Leishmania ( Viannia ) braziliensis isolates from patients that presented clinical cure (Responders—R) and relapse or therapeutic failure (Non-responders—NR) after treatment with antimony, were analyzed. These parasites were assessed by in vitro susceptibility to Sb III and Sb V , serine proteases activity measured with substrate (z-FR-AMC) and specific inhibitors (TLCK, AEBSF and PMSF). In vitro susceptibility of axenic amastigotes to Sb III showed a significant difference between R and NR groups. The protease assays showed that TLCK inhibited almost 100% of activity in both axenic amastigotes and promastigotes while AEBSF inhibited around 70%, and PMSF showed lower inhibition of some isolates. Principal component and clustering analysis performed with these data yielded one homogeneous cluster with only NR isolates and three heterogeneous clusters with R and NR isolates. Additionally, differential expression of subtilisins (LbrM.13.0860 and LbrM.28.2570) and TXNPx (LbrM.15.1080) was evaluated in promastigotes and axenic amastigotes from both groups. The results showed a higher expression of LbrM.13.0860 and LbrM.15.1080 genes in axenic amastigotes, while LbrM.28.2570 gene had the lowest expression in all isolates, regardless of the parasite form. The data presented here show a phenotypic heterogeneity among the parasites, suggesting that exploration of in vitro phenotypes based on Sb III and serine proteases profiles can aid in the characterization of L. ( V. ) braziliensis clinical isolates.

Background/Objectives: Cutaneous leishmaniasis (CL) remains a global health challenge, with treatment options often limited by drug resistance and systemic toxicity. Amphotericin B (AmB) represents a promising alternative. but intravenous administration causes severe systemic adverse effects. Despite growing interest in topical therapies, knowledge gaps remain regarding the comparative efficacy of delivery systems, including the influence of treatment timing and potential intrinsic effects. This study aimed to develop and characterize different topical AmB formulations (polymeric nanoparticles (PCL-AmB), a lipid-based (Oil_AmB) formulation, and a gel emulsion) to evaluate their in vivo efficacy against CL in a murine model, considering treatment initiation timing and potential intrinsic effects of the delivery systems. Methods: Formulations were prepared and characterized in terms of hydrodynamic size, polydispersity index, and AmB content. Antileishmanial activity was assessed in two independent in vivo experiments, with topical monotherapy administered five days per week for four weeks, starting either 10 or 30 days post-infection, representing early and established chronic stages of infection, respectively. Results: All formulations exhibited nanoscale dimensions and high homogeneity, with the lipid system demonstrating superior AmB solubilization. Both PCL-AmB and Oil_AmB reduced parasite load in the footpad, with Oil_AmB also reducing parasite load in draining lymph nodes. Conclusions: PCL-AmB and Oil_AmB reduced lesions and parasite burden in L. amazonensis-infected mice. Treatment timing was critical, with early Oil_AmB also reducing parasite loads in draining lymph nodes. These findings suggest that topical AmB formulations may provide a promising alternative for CL treatment, though further studies are required to optimize efficacy and administration schedules.

Leishmania species are responsible for a broad spectrum of diseases, denominated Leishmaniasis, affecting over 12 million people worldwide. During the last decade, there have been impressive efforts for sequencing the genome of most of the pathogenic Leishmania spp. as well as hundreds of strains, but large-scale proteomics analyses did not follow these achievements and the Leishmania proteome remained mostly uncharacterized. Here, we report a comprehensive comparative study of the proteomes of strains representing L. braziliensis, L. panamensis and L. guyanensis species. Proteins extracted by SDS-mediated lysis were processed following the multi-enzyme digestion-filter aided sample preparation (FASP) procedure and analysed by high accuracy mass spectrometry. \"Total Protein Approach\" and \"Proteomic Ruler\" were applied for absolute quantification of proteins. Principal component analysis demonstrated very high reproducibility among biological replicates and a very clear differentiation of the three species. Our dataset comprises near 7000 proteins, representing the most complete Leishmania proteome yet known, and provides a comprehensive quantitative picture of the proteomes of the three species in terms of protein concentration and copy numbers. Analysis of the abundance of proteins from the major energy metabolic processes allow us to highlight remarkably differences among the species and suggest that these parasites depend on distinct energy substrates to obtain ATP. Whereas L. braziliensis relies the more on glycolysis, L. panamensis and L. guyanensis seem to depend mainly on mitochondrial respiration. These results were confirmed by biochemical assays showing opposite profiles for glucose uptake and O.sub.2 consumption in these species. In addition, we provide quantitative data about different membrane proteins, transporters, and lipids, all of which contribute for significant species-specific differences and provide rich substrate for explore new molecules for diagnosing purposes. Data are available via ProteomeXchange with identifier PXD017696.

Leishmaniasis is a complex group of infectious and parasitic diseases that afflict many thousands of individuals across five continents. Leishmaniasis treatment remains a challenge because it relies on drugsknown for their high toxicity and limited efficacy, making itimperative to identify new molecules that offer greater effectiveness and safety. This study sought to explore the impact of seven synthetic guanidine derivatives (LQOF-G1, LQOF-G2, LQOF-G6, LQOF-G7, LQOF-G32, LQOF-G35 and LQOF-G36) onthe parasite Leishmania (Viannia) braziliensis and in vitro macrophage infection by this parasite, as well as cytotoxic approaches in vitro models of mammalian host cells and tissues. The synthesized compounds showed purity ≥ 99.65% and effectively inhibited parasite growth. LQOF-G1 proved the most potent, yielding the best half-maximal inhibitory concentration (IC50) values against promastigotes (4.62 μmol/L), axenic amastigotes (4.27 μmol/L), and intracellular amastigotes (3.65 μmol/L). Notably, the antileishmanial activity of LQOF-G1, LQOF-G2, and LQOF-G6 was related to immunomodulatory effects, evidenced by alterations in TNF-α, IL-12, IL-10, nitric oxide (NO), and reactive oxygen species (ROS) levels in the supernatant of culture macrophages infected with L. (V.) braziliensis and coincubated with these compounds. LQOF-G2 and LQOF-G36 compounds exhibited vasodilator and spasmolytic effects at higher concentrations (≥100 μmol/L). Generally, LQOF-G1, LQOF-G2, and LQOF-G32 compounds were found to be nontoxic to assessed organs and cells. No toxic effects were observed in human cell lines, such as HEK-293, CaCo-2 and A549, at concentrations ≥ 500 μmol/L. Collectively, data have shown unequivocal evidence of the effectiveness of these compounds against L. (V.) braziliensis parasite, one of the causative agents of Tegumentary Leishmaniasis and Mucocutaneous Leishmaniasis in America.

Proteases are virulence factors with a recognized impact on the Leishmania spp. life cycle. This study considers a set of analyses measuring phenotypic factors of L. (V.) braziliensis clinical isolates as promastigotes growth curves, murine peritoneal macrophages infection, inflammatory mediators production, and serine proteases gene expression (subtilisin 13: S13, subtilisin 28: S28, oligopeptidase B: OPB) assessing these isolates’ fitness on in vitro conditions. Parasites had different behavior during the early growth phase from day zero to day three, and all isolates reached the stationary growth phase between days four and seven. Macrophages infection showed two tendencies, one of decreased infection rate and number of parasites per macrophage (Infection Index <1000) and another with a constant infection index (≥1400). TNF-α (≥10 pg/mL) detected in infections by 75% of isolates, IL-6 (≥80 pg/mL) by 30% of isolates and low levels of NO (≥0.01µM) in almost all infections. Gene expression showed higher values of S13 (≥2RQ) in the intracellular amastigotes of all the isolates evaluated. On the contrary, S28 expression was low (≤1RQ) in all isolates. OPB expression was different between promastigotes and intracellular amastigotes, being significantly higher (≥2RQ) in the latter form of 58% of the isolates. Predictive structural assays of S13 and OPB were performed to explore temperature influence on gene expression and the encoded proteases. Gene expression data is discussed based on in silico predictions of regulatory regions that show plasticity in the linearity index of secondary structures of S13 and OPB 3’-untranslated regions of mRNA, dependent on temperature changes. While hairpin structures suggest an active region of mRNA for both genes above 26°C, pseudoknot structure found in S13 is an indication of a particular profile of this gene at mammalian host temperatures (37°C). Furthermore, the predicted 3D structures are in accordance with the influence of these temperatures on the catalytic site stability of both enzymes, favoring their action over peptide substrates. Data gathered here suggest that L. (V.) braziliensis serine proteases can be influenced by the temperature conditions affecting parasite fitness throughout its life cycle.

In American Tegumentary Leishmaniasis production of cytokines, reactive oxygen species and nitric oxide (NO) by host macrophages normally lead to parasite death. However, some Leishmania braziliensis strains exhibit natural NO resistance. NO-resistant strains cause more lesions and are frequently more resistant to antimonial treatment than NO-susceptible ones, suggesting that NO-resistant parasites are endowed with specific mechanisms of survival and persistence. To tests this, we analyzed the effect of pro- and antioxidant molecules on the infectivity in vitro of L. braziliensis strains exhibiting polar phenotypes of resistance or susceptibility to NO. In addition, we conducted a comprehensive quantitative mass spectrometry-based proteomics analysis of those parasites. NO-resistant parasites were more infective to peritoneal macrophages, even in the presence of high levels of reactive species. Principal component analysis of protein concentration values clearly differentiated NO-resistant from NO-susceptible parasites, suggesting that there are natural intrinsic differences at molecular level among those strains. Upon NO exposure, NO-resistant parasites rapidly modulated their proteome, increasing their total protein content and glutathione (GSH) metabolism. Furthermore, NO-resistant parasites showed increased glucose analogue uptake, and increased abundance of phosphotransferase and G6PDH after nitrosative challenge, which can contribute to NADPH pool maintenance and fuel the reducing conditions for the recovery of GSH upon NO exposure. Thus, increased glucose consumption and GSH-mediated redox capability may explain the natural resistance of L. braziliensis against NO.

Visceral leishmaniasis (VL) is a neglected disease caused by Leishmania parasites. Although significant morbidity and mortality in tropical and subtropical regions of the world are associated with VL, the low investment for developing new treatment measures is chronic. Moreover, resistance and treatment failure are increasing for the main medications, but the emergence of resistance phenotypes is poorly understood at the protein level. Here, we analyzed the development of resistance to miltefosine upon experimental selection in a L. infantum strain. Time to miltefosine resistance emergence was ~six months and label-free quantitative mass-spectrometry-based proteomics analyses revealed that this process involves a remodeling of components of the membrane and mitochondrion, with significant increase in oxidative phosphorylation complexes, particularly on complex IV and ATP synthase, accompanied by increased energy metabolism mainly dependent on β-oxidation of fatty acids. Proteins canonically involved in ROS detoxification did not contribute to the resistant process whereas sterol biosynthesis enzymes could have a role in this development. Furthermore, changes in the abundance of proteins known to be involved in miltefosine resistance such as ABC transporters and phospholipid transport ATPase were detected. Together, our data show a more complete picture of the elements that make up the miltefosine resistance phenotype in L. infantum.

Background Anopheles aquasalis is a dipteran of the family Culicidae that is widely distributed in the coastal regions of South and Central America. This species acts as a vector of Plasmodium vivax , an important etiological agent of malaria, which represents a serious public health problem. In mosquitoes, trypsin-like serine proteases are important in blood meal digestion, immune responses and reproductive functions. The study of peptidases expressed in the mosquito midgut is essential to understanding the mechanisms of parasite-host interaction and the physiological process of nutrient digestion. Methods Our study aimed to identify and characterize the proteolytic activities in the midgut of sugar-fed An. aquasalis females using zymographic analyses (substrate-SDS-PAGE), in-solution assays and mass spectrometry. Results Here, we used a zymographic analysis to further biochemically characterize the proteolytic profile of the midgut of sugar-feeding An. aquasalis females. The trypsin peptidases migrated between ~17 and ~76 kDa and displayed higher proteolytic activities between pH 7.5 and 10 and at temperatures between 37 °C and 50 °C. Four putative trypsin-like serine peptidases were identified using mass spectrometry and data mining. The molecular masses of these peptidases were similar to those observed using zymography, which suggested that these peptidases could be responsible for some of the observed proteolytic bands. Conclusions Taken together, our results contribute to the gene annotation of the unknown genome of this species, to the tissue location of these peptidases, and to the functional prediction of these crucial enzymes, which all impact further studies of this species.

Background Culex quinquefasciatus is a hematophagous insect from the Culicidae family that feeds on the blood of humans, dogs, birds and livestock. This species transmits a wide variety of pathogens between humans and animals. The midgut environment is the first location of pathogen-vector interactions for blood-feeding mosquitoes and the expression of specific peptidases in the early stages of feeding could influence the outcome of the infection. Trypsin-like serine peptidases belong to a multi-gene family that can be expressed in different isoforms under distinct physiological conditions. However, the confident assignment of the trypsin genes that are expressed under each condition is still a challenge due to the large number of trypsin-coding genes in the Culicidae family and most likely because they are low abundance proteins. Methods We used zymography for the biochemical characterization of the peptidase profile of the midgut from C. quinquefasciatus females fed on sugar. Protein samples were also submitted to SDS-PAGE followed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis for peptidase identification. The peptidases sequences were analyzed with bioinformatics tools to assess their distinct features. Results Zymography revealed that trypsin-like serine peptidases were responsible for the proteolytic activity in the midgut of females fed on sugar diet. After denaturation in SDS-PAGE, eight trypsin-like serine peptidases were identified by LC-MS/MS. These peptidases have structural features typical of invertebrate digestive trypsin peptidases but exhibited singularities at the protein sequence level such as: the presence of different amino acids at the autocatalytic motif and substrate binding regions as well as different number of disulfide bounds. Data mining revealed a group of trypsin-like serine peptidases that are specific to C. quinquefasciatus when compared to the culicids genomes sequenced so far. Conclusion We demonstrated that proteomics approaches combined with bioinformatics tools and zymographic analysis can lead to the functional annotation of trypsin-like serine peptidases coding genes and aid in the understanding of the complexity of peptidase expression in mosquitoes.