Design, synthesis and identification of novel molecular hybrids based on naphthoquinone aromatic hydrazides as potential trypanocide and leishmanicidal agents.

In pursuit of potential agents to treat Chagas disease and leishmaniasis, we report the design, synthesis, and identification novel naphthoquinone hydrazide-based molecular hybrids. The compounds were subjected to in vitro trypanocide and leishmanicidal activities. N'-(1,4-Dioxo-1,4-dihydronaphthalen-2-yl)-3,5-dimethoxybenzohydrazide (13) showed the best performance against Trypanosoma cruzi (IC50 1.83 µM) and Leishmania amazonensis (IC50 9.65 µM). 4-Bromo-N'-(1,4-dioxo-1,4-dihydronaphthalen-2-yl)benzohydrazide (16) exhibited leishmanicidal activity (IC50 12.16 µM). Regarding trypanocide activity, compound 13 was low cytotoxic to LLC-MK2 cells (SI = 95.28). Furthermore, through molecular modeling studies, the cysteine proteases cruzain, rhodesain and CPB2.8 were identified as the potential biological targets.

Steppe lemmings and Chinese hamsters as new potential animal models for the study of the Leishmania subgenus Mundinia (Kinetoplastida: Trypanosomatidae).

Leishmania, the dixenous trypanosomatid parasites, are the causative agents of leishmaniasis currently divided into four subgenera: Leishmania, Viannia, Sauroleishmania, and the recently described Mundinia, consisting of six species distributed sporadically all over the world infecting humans and/or animals. These parasites infect various mammalian species and also cause serious human diseases, but their reservoirs are unknown. Thus, adequate laboratory models are needed to enable proper research of Mundinia parasites. In this complex study, we compared experimental infections of five Mundinia species (L. enriettii, L. macropodum, L. chancei, L. orientalis, and four strains of L. martiniquensis) in three rodent species: BALB/c mouse, Chinese hamster (Cricetulus griseus) and steppe lemming (Lagurus lagurus). Culture-derived parasites were inoculated intradermally into the ear pinnae and progress of infection was monitored for 20 weeks, when the tissues and organs of animals were screened for the presence and quantity of Leishmania. Xenodiagnoses with Phlebotomus duboscqi were performed at weeks 5, 10, 15 and 20 post-infection to test the infectiousness of the animals throughout the experiment. BALB/c mice showed no signs of infection and were not infectious to sand flies, while Chinese hamsters and steppe lemmings proved susceptible to all five species of Mundinia tested, showing a wide spectrum of disease signs ranging from asymptomatic to visceral. Mundinia induced significantly higher infection rates in steppe lemmings compared to Chinese hamsters, and consequently steppe lemmings were more infectious to sand flies: In all groups tested, they were infectious from the 5th to the 20th week post infection. In conclusion, we identified two rodent species, Chinese hamster (Cricetulus griseus) and steppe lemming (Lagurus lagurus), as candidates for laboratory models for Mundinia allowing detailed studies of these enigmatic parasites. Furthermore, the long-term survival of all Mundinia species in steppe lemmings and their infectiousness to vectors support the hypothesis that some rodents have the potential to serve as reservoir hosts for Mundinia.

Py-CoMFA, docking, and molecular dynamics simulations of Leishmania (L.) amazonensis arginase inhibitors.

Leishmaniasis is a disease caused by a protozoan of the genus Leishmania, affecting millions of people, mainly in tropical countries, due to poor social conditions and low economic development. First-line chemotherapeutic agents involve highly toxic pentavalent antimonials, while treatment failure is mainly due to the emergence of drug-resistant strains. Leishmania arginase (ARG) enzyme is vital in pathogenicity and contributes to a higher infection rate, thus representing a potential drug target. This study helps in designing ARG inhibitors for the treatment of leishmaniasis. Py-CoMFA (3D-QSAR) models were constructed using 34 inhibitors from different chemical classes against ARG from L. (L.) amazonensis (LaARG). The 3D-QSAR predictions showed an excellent correlation between experimental and calculated pIC50 values. The molecular docking study identified the favorable hydrophobicity contribution of phenyl and cyclohexyl groups as substituents in the enzyme allosteric site. Molecular dynamics simulations of selected protein-ligand complexes were conducted to understand derivatives' interaction modes and affinity in both active and allosteric sites. Two cinnamide compounds, 7g and 7k, were identified, with similar structures to the reference 4h allosteric site inhibitor. These compounds can guide the development of more effective arginase inhibitors as potential antileishmanial drugs.

Susceptibility of Leishmania to novel pentavalent organometallics: Investigating impact on DNA and membrane integrity in antimony(III)-sensitive and -resistant strains.

The aim the present study was to investigate the impact of novel pentavalent organobismuth and organoantimony complexes on membrane integrity and their interaction with DNA, activity against Sb(III)-sensitive and -resistant Leishmania strains and toxicity in mammalian peritoneal macrophages. Ph3M(L)2 type complexes were synthesized, where M = Sb(V) or Bi(V) and L = deprotonated 3-(dimethylamino)benzoic acid or 2-acetylbenzoic acid. Both organobismuth(V) and organoantimony(V) complexes exhibited efficacy at micromolar concentrations against Leishmania amazonensis and L. infantum but only the later ones demonstrated biocompatibility. Ph3Sb(L1)2 and Ph3Bi(L1)2 demonstrated distinct susceptibility profiles compared to inorganic Sb(III)-resistant strains of MRPA-overexpressing L. amazonensis and AQP1-mutated L. guyanensis. These complexes were able to permeate the cell membrane and interact with the Leishmania DNA, suggesting that this effect may contribute to the parasite growth inhibition via apoptosis. Taken altogether, our data substantiate the notion of a distinct mechanism of uptake pathway and action in Leishmania for these organometallic complexes, distinguishing them from the conventional inorganic antimonial drugs.

In vitro antileishmanial activities of hydro-methanolic crude extracts and solvent fractions of Clematis simensis fresen leaf, and Euphorbia abyssinica latex.

As a result of increasing drug resistance, crossover resistance development, prolonged therapy, and the absence of different agents with innovative methods for implementation, the efficacy of recent antileishmanial medications is severely declining. So, it is vital to look for other medications from botanical remedies that have antileishmanial activity. The latex of Euphorbia abyssinica (E abyssinica) and the leaves of Clematis simensis fresen (C simensis) were macerated in methanol (80%). In vitro antileishmanial activity of the preparation was tried on promastigotes of Leishmania aethiopica (L aethiopica) and Leishmania donovani (L donovani) using resazurin assay, and fluorescence intensity was measured. One percent of dimethyl sulfoxide (DMSO) and media as negative control and amphotericin B as positive control were used. Additionally, hemolytic & phytochemical tests of the preparation were done. The mean and standard errors of each extract were evaluated and interpreted for statistical significance using one-way analysis of variance. From sigmoidal dose-response curves of % inhibition, half maximal inhibitory concentration (IC50) values were determined by GraphPad Prism and Microsoft Excel; outcomes were presented as mean ±â€…standard error of mean of triplicate trials. P < .05 was statistical significance. The phytochemical screening of C simensis and E abyssinica confirmed the existence of steroids, phenols, tannins, saponins, alkaloids, terpenoids, flavonoids and glycosides. C simensis possesses antileishmanial activity with IC50 outcomes of 46.12 ±â€…0.03 and 8.18 ±â€…0.10 µg/mL on the promastigotes of L aethiopica and L donovani, respectively. However, E abyssinica showed stronger activity with IC50 outcomes of 16.07 ±â€…0.05 µg/mL and 4.82 ±â€…0.07 µg/mL on L aethiopica and L donovani, respectively. C simensis and E abyssinica have a less hemolytic effect on human red blood cells at low concentrations. The outcomes from this investigation demonstrated that the preparation of C simensis and E abyssinica indicated significant antileishmanial activity. Therefore, further in vivo assessment of antileishmanial, cytotoxicity activity and quantitative identification of secondary metabolites are highly recommended.

The non-pathogenic protozoon Leishmania tarentolae interferes with the activation of NLRP3 inflammasome in human cells: new perspectives in the control of inflammation.

Background: Innate immune responses against infectious agents can act as triggers of inflammatory diseases. On the other hand, various pathogens have developed mechanisms for the evasion of the immune response, based on an inhibition of innate immunity and inflammatory responses. Inflammatory diseases could thus be controlled through the administration of pathogens or pathogen-derived molecules, capable of interfering with the mechanisms at the basis of inflammation. In this framework, the NLRP3 inflammasome is an important component in innate antimicrobial responses and a major player in the inflammatory disease. Parasites of the genus Leishmania are master manipulators of innate immune mechanisms, and different species have been shown to inhibit inflammasome formation. However, the exploitation of pathogenic Leishmania species as blockers of NLRP3-based inflammatory diseases poses safety concerns. Methods: To circumvent safety issues associated with pathogenic parasites, we focused on Leishmania tarentolae, a species of Leishmania that is not infectious to humans. Because NLRP3 typically develops in macrophages, in response to the detection and engulfment microorganisms, we performed our experiments on a monocyte-macrophage cell line (THP-1), either wild type or knockout for ASC, a key component of NLRP3 formation, with determination of cytokines and other markers of inflammation. Results: L. tarentolae was shown to possess the capability of dampening the formation of NLRP3 inflammasome and the consequent expression of pro-inflammatory molecules, with minor differences compared to effects of pathogenic Leishmania species. Conclusion: The non-pathogenic L. tarentolae appears a promising pro-biotic microbe with anti-inflammatory properties or a source of immune modulating cellular fractions or molecules, capable of interfering with the formation of the NLRP3 inflammasome.

4-Quinolinylhydrazone analogues kill Leishmania (Leishmania) amazonensis by inducing apoptosis and mitochondria-dependent pathway cell death.

Despite efforts, available alternatives for the treatment of leishmaniasis are still scarce. In this work we tested a class of 15 quinolinylhydrazone analogues and presented data that support the use of the most active compound in cutaneous leishmaniasis caused by Leishmania amazonensis. In general, the compounds showed activity at low concentrations for both parasitic forms (5.33-37.04 µM to promastigotes, and 14.31-61.98 µM to amastigotes). In addition, the best compound (MHZ15) is highly selective for the parasite. Biochemical studies indicate that the treatment of promastigotes with MHZ15 leads the loss of mitochondrial potential and increase in ROS levels as the primary effects, which triggers accumulation of lipid droplets, loss of plasma membrane integrity and apoptosis hallmarks, including DNA fragmentation and phosphatidylserine exposure. These effects were similar in the intracellular form of the parasite. However, in this parasitic form there is no change in plasma membrane integrity in the observed treatment time, which can be attributed to metabolic differences and the resilience of the amastigote. Also, ultrastructural changes such as vacuolization suggesting autophagy were observed. The in vivo effectiveness of MHZ15 in the experimental model of cutaneous leishmaniasis was carried out in mice of the BALB/c strain infected with L. amazonensis. The treatment by intralesional route showed that MHZ15 acted with great efficiency with significantly reduction in the parasite load in the injured paws and draining lymph nodes, without clinical signs of distress or compromise of animal welfare. In vivo toxicity was also evaluated and null alterations in the levels of hepatic enzymes aspartate aminotransferase, and alanine aminotransferase was observed. The data presented herein demonstrates that MHZ15 exhibits a range of favorable characteristics conducive to the development of an antileishmanial agent.

Leishmania (Sauroleishmania) tarentolae versus pathogenic species: comparative evaluation of protease activity, glycoconjugates, resistance to complement and metabolome composition.

BACKGROUND: Leishmania tarentolae is a non-pathogenic species found in lizards representing an important model for Leishmania biology. However, several aspects of this Sauroleishmania remain unknown to explain its low level of virulence. OBJECTIVES: We reported several aspects of L. tarentolae biology including glycoconjugates, proteolytic activities and metabolome composition in comparison to pathogenic species (Leishmania amazonensis, Leishmania braziliensis, Leishmania infantum and Leishmania major). METHODS: Parasites were cultured for extraction and purification of lipophosphoglycan (LPG), immunofluorescence probing with anti-gp63 and resistance against complement. Parasite extracts were also tested for proteases activity and metabolome composition. FINDINGS: Leishmania tarentolae does not express LPG on its surface. It expresses gp63 at lower levels compared to pathogenic species and, is highly sensitive to complement-mediated lysis. This species also lacks intracellular/extracellular activities of proteolytic enzymes. It has metabolic differences with pathogenic species, exhibiting a lower abundance of metabolites including ABC transporters, biosynthesis of unsaturated fatty acids and steroids, TCA cycle, glycine/serine/threonine metabolism, glyoxylate/dicarboxylate metabolism and pentose-phosphate pathways. MAIN CONCLUSIONS: The non-pathogenic phenotype of L. tarentolae is associated with alterations in several biochemical and molecular features. This reinforces the need of comparative studies between pathogenic and non-pathogenic species to elucidate the molecular mechanisms of virulence during host-parasite interactions.

Expression and functional characterization of chimeric recombinant bovine follicle-stimulating hormone produced in Leishmania tarentolae.

Assisted reproductive techniques are routinely used in livestock species to increase and enhance productivity. Ovarian hyperstimulation is a process that currently relies on administering pituitary-derived follicle-stimulating hormone (FSH) or equine chorionic gonadotropin in combination with other hormones to promote the maturation of multiple follicles and thereby achieve superovulation. The use of partially purified preparations of FSH extracted from natural sources is associated with suboptimal and variable results. Recombinant FSH (rFSH) has been produced in a variety of heterologous organisms. However, attaining a bioactive rFSH of high quality and at low cost for use in livestock remains challenging. Here we report the production and characterization of a single chain bovine rFSH consisting of the ß- and α-subunit fused by a polypeptide linker (scbFSH) using Leishmania tarentolae as heterologous expression system. This unicellular eukaryote is non-pathogenic to mammals, can be grown in bioreactors using simple and inexpensive semisynthetic media at 26°C and does not require CO2 or bovine serum supplementation. Stable cell lines expressing scbFSH in an inducible fashion were generated and characterized for their productivity. Different culture conditions and purification procedures were evaluated, and the recombinant product was biochemically and biologically characterized, including bioassays in an animal model. The results demonstrate that L. tarentolae is a suitable host for producing a homogeneous, glycosylated and biologically active form of scbFSH with a reasonable yield.

Extracellular Vesicles: Translational Agenda Questions for Three Protozoan Parasites.

The protozoan parasites Plasmodium falciparum, Leishmania spp. and Trypanosoma cruzi continue to exert a significant toll on the disease landscape of the human population in sub-Saharan Africa and Latin America. Control measures have helped reduce the burden of their respective diseases-malaria, leishmaniasis and Chagas disease-in endemic regions. However, the need for new drugs, innovative vaccination strategies and molecular markers of disease severity and outcomes has emerged because of developing antimicrobial drug resistance, comparatively inadequate or absent vaccines, and a lack of trustworthy markers of morbid outcomes. Extracellular vesicles (EVs) have been widely reported to play a role in the biology and pathogenicity of P. falciparum, Leishmania spp. and T. cruzi ever since they were discovered. EVs are secreted by a yet to be fully understood mechanism in protozoans into the extracellular milieu and carry a cargo of diverse molecules that reflect the originator cell's metabolic state. Although our understanding of the biogenesis and function of EVs continues to deepen, the question of how EVs in P. falciparum, Leishmania spp. and T. cruzi can serve as targets for a translational agenda into clinical and public health interventions is yet to be fully explored. Here, as a consortium of protozoan researchers, we outline a plan for future researchers and pose three questions to direct an EV's translational agenda in P. falciparum, Leishmania spp. and T. cruzi. We opine that in the long term, executing this blueprint will help bridge the current unmet needs of these medically important protozoan diseases in sub-Saharan Africa and Latin America.

Metalloenzyme Inhibitors against Zoonotic Infections: Focus on Leishmania and Schistosoma.

The term "zoonosis" denotes diseases transmissible among vertebrate animals and humans. These diseases constitute a significant public health challenge, comprising 61% of human pathogens and causing an estimated 2.7 million deaths annually. Zoonoses not only affect human health but also impact animal welfare and economic stability, particularly in low- and middle-income nations. Leishmaniasis and schistosomiasis are two important neglected tropical diseases with a high prevalence in tropical and subtropical areas, imposing significant burdens on affected regions. Schistosomiasis, particularly rampant in sub-Saharan Africa, lacks alternative treatments to praziquantel, prompting concerns regarding parasite resistance. Similarly, leishmaniasis poses challenges with unsatisfactory treatments, urging the development of novel therapeutic strategies. Effective prevention demands a One Health approach, integrating diverse disciplines to enhance diagnostics and develop safer drugs. Metalloenzymes, involved in parasite biology and critical in different biological pathways, emerged in the last few years as useful drug targets for the treatment of human diseases. Herein we have reviewed recent reports on the discovery of inhibitors of metalloenzymes associated with zoonotic diseases like histone deacetylases (HDACs), carbonic anhydrase (CA), arginase, and heme-dependent enzymes.

GRASP negatively regulates the secretion of the virulence factor gp63 in Leishmania.

Metalloprotease-gp63 is a virulence factor secreted by Leishmania. However, secretory pathway in Leishmania is not well defined. Here, we cloned and expressed the GRASP homolog from Leishmania. We found that Leishmania expresses one GRASP homolog of 58 kDa protein (LdGRASP) which localizes in LdRab1- and LPG2-positive Golgi compartment in Leishmania. LdGRASP was found to bind with COPII complex, LdARF1, LdRab1 and LdRab11 indicating its role in ER and Golgi transport in Leishmania. To determine the function of LdGRASP, we generated LdGRASP knockout parasites using CRISPR-Cas9. We found fragmentation of Golgi in Ld:GRASPKO parasites. Our results showed enhanced transport of non-GPI-anchored gp63 to the cell surface leading to higher secretion of this form of gp63 in Ld:GRASPKO parasites in comparison to Ld:WT cells. In contrast, we found that transport of GPI-anchored gp63 to the cell surface is blocked in Ld:GRASPKO parasites and thereby inhibits its secretion. The overexpression of dominant-negative mutant of LdRab1 or LdSar1 in Ld:GRASPKO parasites significantly blocked the secretion of non-GPI-anchored gp63. Interestingly, we found that survival of transgenic parasites overexpressing Ld:GRASP-GFP is significantly compromised in macrophages in comparison to Ld:WT and Ld:GRASPKO parasites. These results demonstrated that LdGRASP differentially regulates Ldgp63 secretory pathway in Leishmania.

Leishmania vaccine development: A comprehensive review.

Infectious diseases like leishmaniasis, malaria, HIV, tuberculosis, leprosy and filariasis are responsible for an immense burden on public health systems. Among these, leishmaniasis is under the category I diseases as it is selected by WHO (World Health Organization) on the ground of diversity and complexity. High cost, resistance and toxic effects of Leishmania traditional drugs entail identification and development of therapeutic alternative. Since the natural infection elicits robust immunity, consistence efforts are going on to develop a successful vaccine. Clinical trials have been conducted on vaccines like Leish-F1, F2, and F3 formulated using specific Leishmania antigen epitopes. Current strategies utilize individual or combined antigens from the parasite or its insect vector's salivary gland extract, with or without adjuvant formulation for enhanced efficacy. Promising animal data supports multiple vaccine candidates (Lmcen-/-, LmexCen-/-), with some already in or heading for clinical trials. The crucial challenge in Leishmania vaccine development is to translate the research knowledge into affordable and accessible control tools that refines the outcome for those who are susceptible to infection. This review focuses on recent findings in Leishmania vaccines and highlights difficulties facing vaccine development and implementation.

Sampling is decisive to determination of Leishmania (Viannia) species.

BACKGROUND: Accuracy of molecular tools for the identification of parasites that cause human cutaneous leishmaniasis (CL) could largely depend on the sampling method. Non-invasive or less-invasive sampling methods such as filter paper imprints and cotton swabs are preferred over punch biopsies and lancet scrapings for detection methods of Leishmania based on polymerase chain reaction (PCR) because they are painless, simple, and inexpensive, and of benefit to military and civilian patients to ensure timely treatment. However, different types of samples can generate false negatives and there is a clear need to demonstrate which sample is more proper for molecular assays. METHODOLOGY: Here, we compared the sensitivity of molecular identification of different Leishmania (Viannia) species from Peru, using three types of sampling: punch biopsy, filter paper imprint and lancet scraping. Different composite reference standards and latent class models allowed to evaluate the accuracy of the molecular tools. Additionally, a quantitative PCR assessed variations in the results and parasite load in each type of sample. PRINCIPAL FINDINGS: Different composite reference standards and latent class models determined higher sensitivity when lancet scrapings were used for sampling in the identification and determination of Leishmania (Viannia) species through PCR-based assays. This was consistent for genus identification through kinetoplastid DNA-PCR and for the determination of species using FRET probes-based Nested Real-Time PCR. Lack of species identification in some samples correlated with the low intensity of the PCR electrophoretic band, which reflects the low parasite load in samples. CONCLUSIONS: The type of clinical sample can directly influence the detection and identification of Leishmania (Viannia) species. Here, we demonstrated that lancet scraping samples consistently allowed the identification of more leishmaniasis cases compared to filter paper imprints or biopsies. This procedure is inexpensive, painless, and easy to implement at the point of care and avoids the need for anesthesia, surgery, and hospitalization and therefore could be used in resource limited settings for both military and civilian populations.

Canine leishmaniasis in the Americas: etiology, distribution, and clinical and zoonotic importance.

Canine leishmaniasis is a widespread disease on the American continent, with cases reported from Uruguay to the USA and Canada. While numerous Leishmania spp. have been reported in dogs in this region, Leishmania infantum and Leishmania braziliensis are the most common etiological agents of canine leishmaniasis from a continental perspective. Nonetheless, other species may predominate locally in some countries. The participation of dogs in the transmission cycle of various Leishmania spp. has long been speculated, but evidence indicates that their role as reservoirs of species other than L. infantum is negligible. Various native wildlife (e.g., small rodents, marsupials, sloths, and monkeys) are, in fact, the primary hosts of Leishmania spp. in the Americas. In this review, an updated list of Leishmania spp. infecting dogs in the Americas is presented, along with their distribution and clinical and zoonotic importance.

A novel chemically defined medium for the biotechnological and biomedical exploitation of the cell factory Leishmania tarentolae.

The development of media for cell culture is a major issue in the biopharmaceutical industry, for the production of therapeutics, immune-modulating molecules and protein antigens. Chemically defined media offer several advantages, as they are free of animal-derived components and guarantee high purity and a consistency in their composition. Microorganisms of the genus Leishmania represent a promising cellular platform for production of recombinant proteins, but their maintenance requires supplements of animal origin, such as hemin and fetal bovine serum. In the present study, three chemically defined media were assayed for culturing Leishmania tarentolae, using both a wild-type strain and a strain engineered to produce a viral antigen. Among the three media, Schneider's Drosophila Medium supplemented with Horseradish Peroxidase proved to be effective for the maintenance of L. tarentolae promastigotes, also allowing the heterologous protein production by the engineered strain. Finally, the engineered strain was maintained in culture up to the 12th week without antibiotic, revealing its capability to produce the recombinant protein in the absence of selective pressure.

Unmasking the Mechanism behind Miltefosine: Revealing the Disruption of Intracellular Ca2+ Homeostasis as a Rational Therapeutic Target in Leishmaniasis and Chagas Disease.

Originally developed as a chemotherapeutic agent, miltefosine (hexadecylphosphocholine) is an inhibitor of phosphatidylcholine synthesis with proven antiparasitic effects. It is the only oral drug approved for the treatment of Leishmaniasis and American Trypanosomiasis (Chagas disease). Although its precise mechanisms are not yet fully understood, miltefosine exhibits broad-spectrum anti-parasitic effects primarily by disrupting the intracellular Ca2+ homeostasis of the parasites while sparing the human hosts. In addition to its inhibitory effects on phosphatidylcholine synthesis and cytochrome c oxidase, miltefosine has been found to affect the unique giant mitochondria and the acidocalcisomes of parasites. Both of these crucial organelles are involved in Ca2+ regulation. Furthermore, miltefosine has the ability to activate a specific parasite Ca2+ channel that responds to sphingosine, which is different to its L-type VGCC human ortholog. Here, we aimed to provide an overview of recent advancements of the anti-parasitic mechanisms of miltefosine. We also explored its multiple molecular targets and investigated how its pleiotropic effects translate into a rational therapeutic approach for patients afflicted by Leishmaniasis and American Trypanosomiasis. Notably, miltefosine's therapeutic effect extends beyond its impact on the parasite to also positively affect the host's immune system. These findings enhance our understanding on its multi-targeted mechanism of action. Overall, this review sheds light on the intricate molecular actions of miltefosine, highlighting its potential as a promising therapeutic option against these debilitating parasitic diseases.

Aurantoside L, a New Tetramic Acid Glycoside with Anti-Leishmanial Activity Isolated from the Marine Sponge Siliquariaspongia japonica.

A new tetramic acid glycoside, aurantoside L (1), was isolated from the sponge Siliquariaspongia japonica collected at Tsushima Is., Nagasaki Prefecture, Japan. The structure of aurantoside L (1) composed of a tetramic acid bearing a chlorinated polyene system and a trisaccharide part was elucidated using spectral analysis. Aurantoside L (1) showed anti-parasitic activity against L. amazonensis with an IC50 value of 0.74 µM.

Promising Antileishmanial Activity of Micromeria nervosa Essential Oil: In Vitro and In Silico Studies.

The present study aimed to evaluate the leishmanicidal potential of the essential oil (EO) of Micromeria (M.) nervosa and to investigate its molecular mechanism of action by qPCR. Furthermore, in silicointeraction study of the major M. nervosa EO compounds with the enzyme cytochrome P450 sterol 14α-demethylase (CYP51) was also performed. M. nervosa EO was analyzed by gas chromatography-mass spectrometry (GC-MS). Results showed that α-pinene (26.44%), t-cadinol (26.27%), caryophyllene Oxide (7.73 ± 1.04%), and α-Cadinene (3.79 ± 0.12%) are the major compounds of M. nervosa EO. However, limited antioxidant activity was observed, as this EO was ineffective in neutralizing DPPH free radicals and in inhibiting ß-carotene bleaching. Interestingly, it displayed effective leishmanicidal potential against promastigote (IC50 of 6.79 and 5.25 µg/mL) and amastigote (IC50 of 8.04 and 7.32 µg/mL) forms of leishmania (L.) infantum and L. major, respectively. Molecular mechanism investigation showed that M. nervosa EO displayed potent inhibition on the thiol regulatory pathway. Furthermore, a docking study of the main components of the EO with cytochrome P450 sterol 14α-demethylase (CYP51) enzyme revealed that t-cadinol exhibited the best binding energy values (-7.5 kcal/mol), followed by α-cadinene (-7.3 kcal/mol) and caryophyllene oxide (-7 kcal/mol). These values were notably higher than that of the conventional drug fluconazole showing weaker binding energy (-6.9 kcal/mol). These results suggest that M. nervosa EO could serve as a potent and promising candidate for the development of alternative antileishmanial agent in the treatment of leishmaniasis.

Characterization of the First Secreted Sorting Nexin Identified in the Leishmania Protists.

Proteins of the sorting nexin (SNX) family present a modular structural architecture with a phox homology (PX) phosphoinositide (PI)-binding domain and additional PX structural domains, conferring to them a wide variety of vital eukaryotic cell's functions, from signal transduction to membrane deformation and cargo binding. Although SNXs are well studied in human and yeasts, they are poorly investigated in protists. Herein, is presented the characterization of the first SNX identified in Leishmania protozoan parasites encoded by the LdBPK_352470 gene. In silico secondary and tertiary structure prediction revealed a PX domain on the N-terminal half and a Bin/amphiphysin/Rvs (BAR) domain on the C-terminal half of this protein, with these features classifying it in the SNX-BAR subfamily of SNXs. We named the LdBPK_352470.1 gene product LdSNXi, as it is the first SNX identified in Leishmania (L.) donovani. Its expression was confirmed in L. donovani promastigotes under different cell cycle phases, and it was shown to be secreted in the extracellular medium. Using an in vitro lipid binding assay, it was demonstrated that recombinant (r) LdSNXi (rGST-LdSNXi) tagged with glutathione-S-transferase (GST) binds to the PtdIns3P and PtdIns4P PIs. Using a specific a-LdSNXi antibody and immunofluorescence confocal microscopy, the intracellular localization of endogenous LdSNXi was analyzed in L. donovani promastigotes and axenic amastigotes. Additionally, rLdSNXi tagged with enhanced green fluorescent protein (rLdSNXi-EGFP) was heterologously expressed in transfected HeLa cells and its localization was examined. All observed localizations suggest functions compatible with the postulated SNX identity of LdSNXi. Sequence, structure, and evolutionary analysis revealed high homology between LdSNXi and the human SNX2, while the investigation of protein-protein interactions based on STRING (v.11.5) predicted putative molecular partners of LdSNXi in Leishmania.