A Multi-Color Immunofluorescence Assay to Distinguish Intracellular From External Leishmania Parasites.

Leishmaniasis, a neglected tropical disease, is caused by the intracellular protozoan parasite Leishmania. Upon its transmission through a sandfly bite, Leishmania binds and enters host phagocytic cells, ultimately resulting in a cutaneous or visceral form of the disease. The limited therapeutics available for leishmaniasis, in combination with this parasite's techniques to evade the host immune system, call for exploring various methods to target this infection. To this end, our laboratory has been characterizing how Leishmania is internalized by phagocytic cells through the activation of multiple host cell signaling pathways. This protocol, which we use routinely for our experiments, delineates how to infect mammalian macrophages with either promastigote or amastigote forms of the Leishmania parasite. Subsequently, the number of intracellular parasites, external parasites, and macrophages can be quantified using immunofluorescence microscopy and semi-automated analysis protocols. Studying the pathways that underlie Leishmania uptake by phagocytes will not only improve our understanding of these host-pathogen interactions but may also provide a foundation for discovering additional treatments for leishmaniasis. Key features • This protocol visualizes and quantifies multiple intracellular forms of Leishmania. • It offers flexibility at various points for researchers to introduce modifications according to their study needs.

Vaccine Designing Technology against Leishmaniasis: Current Challenges and Implication.

Leishmaniasis, a debilitating disease caused by protozoan parasites of the genus Leishmania and transmitted by the bite of a female sandfly, continues to present significant challenges despite ongoing research and collaboration in vaccine development. The intricate interaction between the parasite's life cycle stages and the host's immunological response, namely the promastigote and amastigote forms, adds complexity to vaccine design. The quest for a potent vaccine against Leishmaniasis demands a comprehensive understanding of the immune mechanisms that confer long-lasting protection, which necessitates extensive research efforts. In this pursuit, innovative approaches such as reverse vaccinology and computer-aided design offer promising avenues for unraveling the intricacies of host-pathogen interactions and identifying effective vaccine candidates. However, numerous obstacles, including limited treatment options, the need for sustained antigenic presence, and the prevalence of co-infections, particularly with HIV, impede progress. Nevertheless, through persistent research endeavours and collaborative initiatives, the goal of developing a highly efficacious vaccine against Leishmaniasis can be achieved, offering hope through the latest Omics data development with immunoinformatics approaches for effective vaccine design for the prevention of this disease.

Antileishmanial effect of podophyllotoxin and podophyllin on Leishmania major in vitro and in vivo.

Therapeutic research is very important in the prevention and treatment of leishmaniasis due to problems such as drug resistance, scarring and disease recurrence. The aim of this study was to determine how Leishmania major responds to the anti-leishmaniasis properties of podophyllotoxin and podophyllin. Cultured Leishmania promastigotes were exposed to different concentrations of podophyllotoxin and podophyllin for 24 and 48 h. Then, during the animal phase, Balb/c mice were experimentally injected with Leishmania promastigotes. After wounding, the effects of 0.5% podophyllotoxin and 25% podophyllin on reducing wound diameter and the number of amastigotes in the wound were evaluated. Podophyllotoxin and podophyllin were 83% and 59% lethal to Leishmania major promastigotes at the highest concentrations (200 µg/ml) and time (48 h). In the in vivo study, the mean lesion diameter at the end of treatment in the negative control group was 15.10 mm compared to 14.21 mm and 11.55 mm in the 25% podophyllin and 0.5% podophyllotoxin groups, respectively. Although both agents reduced the size of mice wounds and the number of amastigotes in the wounds, podophyllotoxin was more effective in this regard. Based on the results, podophyllotoxin and podophyllin can be used as leishmaniasis drugs after further research.

Anti-Leishmania major activity of Calotropis procera extract by increasing ROS production and upregulating TNF-α, IFN-γ and iNOS mRNA expression under in vitro conditions.

BACKGROUND: Leishmaniasis, caused by protozoan parasites of the genus Leishmania, is a neglected tropical disease with 700,000 to 1,000,000 global new cases annually. Adverse effects associated with expense, long-term treatment and drug resistance have made conventional therapies unfavorable, encouraging the search for alternative drugs based on plant products. In this study, the effect of Calotropis procera (Asclepiadaceae) extract against viability of promastigotes and amastigotes of Leishmania major was evaluated in vitro. METHODS: The extract from the leaves of C. procera seedlings was prepared using a methanol maceration method. The colorimetric cell viability 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the growth-inhibitory effect of the extract on promastigotes. The level of reactive oxygen species (ROS) in promastigote cultures was determined after treatment with the extract using the 2',7'-dichlorofluorescein diacetate (DCFH-DA) method and compared with untreated cultures (control). After exposure to the extract the expression levels of tumor necrosis factor-α (TNF-α), interferon gamma (IFN-γ) and inducible nitric oxide synthase (iNOS) genes were determined and compared to control in peripheral blood mononuclear cells (PBMCs) infected with L. major. RESULTS: Based on the MTT assay, the C. procera extract significantly reduced the proliferation of L. major promastigotes with IC50 values of 377.28 and 222.44 µg/mL for 24 and 72 h, respectively (p < 0.01). After treatment with 222.44 and 377.28 µg/mL of C. procera extract, ROS production in L. major promastigote cultures increased 1.2- to 1.65-fold and 2- to 4-fold compared to the control, respectively (p < 0.05). C. procera extract induced significant increases in gene expression of TNF-α (2.76-14.83 fold), IFN-γ (25.63-threefold) and iNOS (16.32-3.97 fold) in infected PBMCs compared to control (p < 0.01). CONCLUSIONS: On the basis of its anti-leishmanial activity, C. procera can be considered as a promising new plant source for the potential treatment of leishmaniasis.

Recognition of Immunoreactive Proteins in Leishmania infantum Amastigote-Like and Promastigote Using Sera of Visceral Leishmaniasis Patients: a Preliminary Study.

PURPOSE: Visceral leishmaniasis (VL) is a systemic and parasitic disease that is usually fatal if left untreated. VL is endemic in different parts of Iran and is caused mainly by Leishmania infantum. This study aimed to recognition immunoreactive proteins in amastigote-like and promastigote stages of L. infantum (Iranian strain) by antibodies present in the sera of VL patients. METHODS: Total protein extract from amastigote-like and promastigote cells was separated by two-dimensional electrophoresis (2DE). To detect the immunoreactive proteins, 2DE immunoblotting method was performed using different pools of VL patients' sera. RESULTS: Approximately 390 and 430 protein spots could be separated in 2DE profiles of L. infantum amastigote-like and promastigote stages, respectively. In immunoblotting method, approximately 295 and 135 immunoreactive proteins of amastigotes-like reacted with high antibody titer serum pool and low antibody titer serum pool, respectively. Approximately 120 and 85 immunoreactive proteins of promastigote extract were recognized using the high antibody titer sera pool and low antibody titer sera, respectively. CONCLUSION: The present study has recognized a number of antigenic diversity proteins based on the molecular weight and pH in amastigote-like and promastigote stages of L. infantum. These results provide us a new concept for further analysis development in the field of diagnosis biomarkers and vaccine targets.

Comparison of biosynthetic zinc oxide nanoparticle and glucantime cytotoxic effects on Leishmania major (MRHO/IR/75/ER).

Currently, zinc oxide (ZnO) particles are used in nanotechnology to destroy a wide range of microorganisms. Although pentavalent antimony compounds are used as antileishmanial drugs, they are associated with several limitations and side effects. Therefore, it is always desirable to try to find new and effective treatments. The aim of this research is to determine the antileishmanial effect of ZnO particles in comparison to the Antimoan Meglumine compound on promastigotes and amastigotes of Leishmania major (MRHO/IR/75/ER). After the extraction and purification of macrophages from the peritoneal cavity of C57BL/6 mice, L. major parasites were cultured in Roswell Park Memorial Institute-1640 culture medium containing fetal bovine serum (FBS) 10% and antibiotic. In this experimental study, the effect of different concentrations of nanoparticles was investigated using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) colorimetric method, in comparison to the glucantime on promastigotes, amastigotes and healthy macrophages in the culture medium. The amount of light absorption of the obtained color from the regeneration of tetrazolium salt to the product color of formazan by the parasite was measured by an enzyme-linked immunosorbent assay (ELISA) reader, and the IC50 value was calculated. IC50 after 24 h of incubation was calculated as IC50 = 358.6 µg/mL. The results showed, that the efficacy of ZnO nanoparticles was favorable and dose-dependent. The concentration of 500 µg/mL of ZnO nanoparticles induced 84.67% apoptosis after 72. Also, the toxicity of nanoparticles was less than the drug. Nanoparticles exert their cytotoxic effects by inducing apoptosis. They can be suitable candidates in the pharmaceutical industry in the future.

Trypanosomiasis: An emerging disease in Alpine swift (Tachymarptis melba) nestlings in Switzerland?

Alpine swifts (Tachymarptis melba) are sub-Saharan migratory birds, which, in Switzerland, nest in colonies that have been continuously monitored for over 40 years. In the summer of 2022, despite favourable environmental conditions, an unexpectedly high number of sudden mortalities (30-80%) occurred in 20 to 45-day-old nestlings from several nesting sites, of which 3 were monitored in detail. Nestlings submitted for post-mortem analysis (n = 5) were in good body condition but exhibited extensive subcutaneous haematomas (n = 5), myocardial petechiae (n = 2) and stunted growth of primary feathers (n = 1). In all birds, 4-5 µm large, amastigote-like protozoans were identified in skeletal and cardiac muscle sections. These tissues tested positive in a PCR targeting the 18S-rRNA gene of Trypanosoma spp. Amplified sequences showed 99.63% identity with sequences of Trypanosoma corvi (JN006854 and AY461665) and Trypanosoma sp. (AJ620557, JN006841). 72 blood smears of 45-day-old nestlings from two colonies were assessed, of which 20 contained trypomastigote forms, some with high parasitaemia (highest average of 56.4 in 10 high power fields, 400x magnification). Trypomastigote morphometrics (n = 36; mean total length = 30.0 µm; length of free flagellum = 5.8 µm) were consistent with those of T. bouffardi. These findings suggest that an avian trypanosomiasis causing mass nestling mortality could be an emerging disease in Swiss Alpine swift populations.

Synthesis and evaluation of hybrid sulfonamide-chalcones with potential antileishmanial activity.

Leishmaniasis is an emerging tropical infectious disease caused by a protozoan parasite of the genus Leishmania. In this work, the molecular hybridization between a trimethoxy chalcone and a sulfonamide group was used to generate a series of sulfonamide-chalcones. A series of eight sulfonamide-chalcone hybrids were made with good yields (up to 95%). These sulfonamide-chalcones were tested against promastigotes of Leishmania amazonensis and cytotoxicity against mouse macrophages, which showed good antileishmanial activity with IC50 = 1.72-3.19 µM. Three of them (10c, 10g, and 10h) were also highly active against intracellular amastigotes and had a good selectivity index (SI > 9). Thus, those three compounds were docked in the cytosolic tryparedoxin peroxidase (cTXNPx) enzyme of the parasite, and molecular dynamics simulations were carried out. This enzyme was selected as a target protein for the sulfonamide-chalcones due to the fact of the anterior report, which identified a strong and stable interaction between the chalcone NAT22 (6) and the cTXNPx. In addition, a prediction of the drug-likeness, and the pharmacokinetic profile of all compounds were made, demonstrating a good profile of those chalcones.

Phytochemical study of Seriphidium khorassanicum (syn. Artemisia khorassanica) aerial parts: sesquiterpene lactones with anti-protozoal activity.

Two new eudesmane-type sesquiterpene lactones, 1ß,3α,8α-trihydroxy-11ß,13-dihydroeudesma-4(15)-en-12,6α-olide (1) and 1ß,4α,8α-trihydroxy-11ß,13-dihydroeudesma-12,6α-olide (2), and an unprecedented elemane-type sesquiterpene lactone, 1ß,2ß,8α-trihydroxy-11ß,13-dihydroelema-12,6α-olide (3) along with a known eudesmanolide artapshin (4) were isolated from Seriphidium khorassanicum. Structures were elucidated by NMR, HR-ESI-MS, and ECD spectral data analysis. The anti-protozoal activity was evaluated against Leishmania major promastigotes and amastigote-infected macrophages. They showed dose- and time-dependent activity against L. major amastigotes with IC50 values in the range of 4.9 to 25.3 µM being favourably far below their toxicity against normal murine macrophages with CC50 values ranging from 432.5 to 620.7 µM after 48 h of treatment. Compound 3 exhibited the strongest activity and the highest selectivity index (SI) with IC50 of 4.9 ± 0.6 µM and SI of 88.2 comparable with the standard drug, meglumine antimoniate (Glucantime), with IC50 and SI values of 15.5 ± 2.1 µM and 40.0, respectively.

Recent patents in the treatment and prevention of leishmaniasis.

Leishmaniasis, a neglected tropical disease, is caused by protozoal parasites of the genus Leishmania. Clinical manifestations vary from asymptomatic to lethal grade depending on the type of the disease. The currently available antileishmanial drugs suffer from considerable limitations. There is a dire need for better and safer drugs and/or vaccines to eradicate this disease. There are enormous developments ongoing in this field. Newer combinations of existing drugs and newer drugs targeting these intracellular parasites as well as their vectors are being tried to control the disease. Attempts to develop vaccines to enhance the immunity of the patient have shown some promise. This article is a peep into the recent patent developments in this field.

Identification of potent inhibitors for Leishmania donovani homoserine kinase: an integrated in silico and kinetic study.

Leishmaniasis is caused by ∼20 species of Leishmania that affects millions in endemic areas. Available therapies are not sufficient to effectively control the disease, cause severe side effects and eventually lead to drug resistance, making the discovery of novel therapeutic molecules an immediate need. Molecular target-based drug discovery, where the target is a defined molecular gene, protein or a mechanism, is a rationale driven approach for novel therapeutics. Humans obtain the essential amino acid such as threonine from dietary sources, while Leishmania synthesize it de-novo. Enzymes of the threonine biosynthesis pathway, including the rate limiting Homoserine kinase (HSK) which converts L-homoserine into ortho-phospho homoserine are thus attractive targets for rationale driven therapy. The absence of HSK in humans and its presence in Leishmania donovani enhances the opportunity to exploit HSK as a molecular target for anti-leishmanials therapeutic development. In this study, we utilize structure-based high throughput drug discovery (SBDD), followed by biochemical validation and identified two potential inhibitors (RH00038 and S02587) from Maybridge chemical library that targets L. donovani HSK. These two inhibitors effectively induced the mortality of Leishmania donovani in both amastigote and promastigote stages, with one of them being specific to parasite and twice as effective as the standard therapeutic molecule.

Antileishmanial activity of 5-nitroindazole derivatives.

Background: Currently, there is no safe and effective vaccine against leishmaniasis and existing therapies are inadequate due to high toxicity, cost and decreased efficacy caused by the emergence of resistant parasite strains. Some indazole derivatives have shown in vitro and in vivo activity against Trichomonas vaginalis and Trypanosoma cruzi. On that basis, 20 indazole derivatives were tested in vitro against Leishmania amazonensis. Objective: To evaluate the in vitro activity of twenty 2-benzyl-5-nitroindazolin-3-one derivatives against L. amazonensis. Design: For the selection of promising compounds, it is necessary to evaluate the indicators for in vitro activity. For this aim, a battery of studies for antileishmanial activity and cytotoxicity were implemented. These results enabled the determination of the substituents in the indazole derivatives responsible for activity and selectivity, through the analysis of the structure-activity relationship (SAR). Methods: In vitro cytotoxicity against mouse peritoneal macrophages and growth inhibitory activity in promastigotes were evaluated for 20 compounds. Compounds that showed adequate selectivity were tested against intracellular amastigotes. The SAR from the results in promastigotes was represented using the SARANEA software. Results: Eight compounds showed selectivity index >10% and 50% inhibitory concentration <1 µM against the promastigote stage. Against intracellular amastigotes, four were as active as Amphotericin B. The best results were obtained for 2-(benzyl-2,3-dihydro-5-nitro-3-oxoindazol-1-yl) ethyl acetate, with 50% inhibitory concentration of 0.46 ± 0.01 µM against amastigotes and a selectivity index of 875. The SAR study showed the positive effect on the selectivity of the hydrophilic fragments substituted in position 1 of 2-benzyl-5- nitroindazolin-3-one, which played a key role in improving the selectivity profile of this series of compounds. Conclusion: 2-bencyl-5-nitroindazolin-3-one derivatives showed selective and potent in vitro activity, supporting further investigations on this family of compounds as potential antileishmanial hits.

In vitro effect of alpha-bisabolol and its synthetic derivatives on macrophages, promastigotes, and amastigotes of Leishmania amazonensis and Leishmania infantum.

Cutaneous and visceral leishmaniasis are public health problems in Africa, Asia, Europe, and America. The treatment has a high cost and toxicity. Thus, this work aims to evaluate the leishmanicidal activity of alpha-bisabolol and its three synthetic derivatives, P1, P2, and P3, on the promastigotes and amastigotes Leishmania infantum and L. amazonensis forms. Alpha-bisabolol showed the lowest IC50 with 3.43 for L. amazonensis promastigotes, while P1 was the most toxic for L. infantum with an IC50 of 9.10. The derivative P3 was better for the amastigote form, with an IC50 of 3.39 for L. amazonensis. All the compounds effectively decreased the intracellular load of amastigote and its ability to turn promastigote again. Thus, alpha-bisabolol and its three synthetic derivatives were effective in their leishmanicidal activity. Therefore, it can be an option for developing new treatments against leishmaniasis.

Detergent-free parasite transformation and replication assay for drug screening against intracellular Leishmania amastigotes.

Leishmaniasis is an infectious disease caused by protozoan species in the genera Leishmania and Endotrypanum. Current antileishmanial drugs are limited due to adverse effects, variable efficacy, the development of resistant parasites, high cost, parenteral administration and lack of availability in endemic areas. Therefore, active searching for new antileishmanial drugs has been done for years, mainly by academia. Drug screening techniques have been a challenge since the intracellular localization of Leishmania amastigotes implies that the host cell may interfere with the quantification of the parasites and the final estimation of the effect. One of the procedures to avoid host cell interference is based on its detergent-mediated lysis and subsequent transformation of viable amastigotes into promastigotes, their proliferation and eventual quantification as an axenic culture of promastigotes. However, the use of detergent involves additional handling of cultures and variability. In the present work, cultures of intracellular amastigotes were incubated for 72 h at 26 °C after exposure to the test compounds and the transformation and proliferation of parasites took place without need of adding any detergent. The assay demonstrated clear differentiation of negative and positive controls (average Z´ = 0.75) and 50% inhibitory concentrations of compounds tested by this method and by the gold standard enumeration of Giemsa-stained cultures were similar (p = 0.5002) and highly correlated (r = 0.9707). This simplified procedure is less labor intensive, the probability of contamination and the experimental error are reduced, and it is appropriate for the automated high throughput screening of compounds.

Anti-Leishmania activity of the Mayan medicinal plant Thouinia paucidentata Radlk extracts.

Mexico's Yucatan Peninsula is an endemic area of cutaneous leishmaniasis, locally known as the chiclero's ulcer, and Mayan traditional medicine which refers to the use of Thouinia paucidentata Radlk, known as k'an chuunup. Aqueous and organic leaves extracts were evaluated against promastigotes and amastigotes of Leishmania mexicana. Toxicity tests of extracts were performed using Vero and J774A.1 macrophage cell lines. The composition of the most active extracts was analysed by GC-MS. The n-hexane and ethyl acetate extracts showed potent anti-Leishmania activity against the promastigote form, and remarkably, n-hexane extract exhibited potent activity against the amastigote form. Both extracts showed low toxicity on Vero both not on J774A.1 cells. Analysis of both bioactive extracts identified as more abundant compounds, germacrene D-4-ol and thunbergen in n-hexane, and thunbergol in ethyl acetate extracts. Our study presents T. paucidentata as anti-Leishmania phytomedicine supporting its medicinal use and contributes to the understanding of its phytochemical composition.

In vitro and in vivo antileishmanial effects of Nectaroscordum koelzi extract against Leishmania major.

Due to the increasing resistance to common medicinal compounds, the use of medicinal plants has received special attention. Therefore, the current survey was designed to study the antileishmanial effects of Nectaroscordum koelzi Trautv. methanolic extract against Leishmania major. In this study, after preparing the methanolic extract of N. koelzi, its effect on the amastigotes of L. major and triggering the nitric oxide (NO) were measured. Then, the in vivo effect of the methanol extract on cutaneous leishmaniasis in mice was evaluated. The best anti-amastigote effect was for the methanol extract of N. koelzi along with meglumine antimony with 50% inhibitory concentrations value of 17.4 µg/ml (p < 0.001). The 50% cytotoxic concentrations values of methanol extract, meglumine antimoniate, and methanol extract + meglumine antimoniate were 596.3, 784.6, and 296.4 µg/ml, respectively. Macrophages treated with the methanolic extract markedly (p < 0.001) induced the release of nitric oxide. After 28 days of treatment, lesions were completely (p < 0.001) healed in mice treated with the methanolic extract (100 mg/kg) + meglumine antimoniate (25 mg/kg). N. koelzi methanolic extract mainly in combination with meglumine antimoniate showed favorable antileishmanial effects on L. major, concluding that the methanolic extract of N. koelzi can be used for the production of new leishmanicidal agents agaist cutaneous leishmaniasis. Although we revealed that NO trigerring and inhibition of infection in host cells are the antileishmanial mechanism action of N. koelzi methanolic extract, more studies must be performed to clear the mechanisms and its safety.

The hows and whys of amastigote flagellum motility in Trypanosoma cruzi.

The protist Trypanosoma cruzi exhibits several extracellular stages characterized by the presence of a long and motile flagellum and one intracellular life cycle stage termed amastigote, which possesses a tiny flagellum barely exiting the flagellar pocket. This stage was so far described as replicative but immotile cells. Unexpectedly, the recent work of M. M. Won, T. Krüger, M. Engstler, and B. A. Burleigh (mBio 14:e03556-22, 2023, https://doi.org/10.1128/mbio.03556-22) revealed that this short flagellum actually displays beating activity. This commentary explores how such a short flagellum could be constructed and why it could affect the parasite's survival inside the mammalian host.

Antileishmanial, cellular mechanisms, and cytotoxic effects of green synthesized zinc nanoparticles alone and in combined with glucantime against Leishmania major infection.

BACKGROUND: We decided to investigate the antileishmanial, cellular mechanisms, and cytotoxic effects of green synthesized Zinc nanoparticles (ZnNPs) alone and combined with glucantime against Leishmania major infection. METHODS: The effect of green synthesized ZnNP on L. major amastigote was studied through macrophage cells. The mRNA expression level of iNOS and IFN-γ followed by the exposure of J774-A1 macrophage cells to ZnNPs was assessed by Real-time PCR. The Caspase-3-like activity of promastigotes exposed to ZnNPs was studied. Effects of ZnNPs alone and combined with glucantime (MA) were studied on cutaneous leishmaniasis in BALB/c mice. RESULTS: ZnNPs displayed the spherical shape with sizes ranging from 30 to 80 nm. The obtained IC50 values for ZnNPs, MA, and ZnNPs + MA were 43.2, 26.3, and 12.6 µg/mL, respectively; indicating the synergistic effects of ZnNPs in combination with MA. CL lesions had completely improved in the mice received with ZnNPs in combination with MA. The mRNA expression level of iNOS, TNF-α, and IFN-γ was dose-dependently (p < 0.01) upregulated; whereas it was downregulated in IL-10. ZnNPs markedly stimulated the caspase-3 activation with no significant toxicity on normal cells. CONCLUSION: Based on these in vitro and in vivo results, green synthesized ZnNPs, mainly along with MA, showed that has the potential to be introduced as a new drug for CL therapy. Triggering of NO production, and inhibition of infectivity rate are revealed as mechanisms of action ZnNPs on L. major. But, supplementary investigations are necessary to clear the efficacy and safety of these agents.

Leishmanicidal and immunomodulatory activities of the formononetin (a natural isoflavone) against Leishmania tropica.

OBJECTIVE: This work aimed to examine the leishmanicidal, cellular mechanisms and cytotoxicity effects of formononetin (FMN), a natural isoflavone, against Leishmania tropica. We used the MTT assay to determine the leishmanicidal effects of FMN against promastigotes and its cytotoxicity effects on J774-A1 macrophage cells. The Griess reaction assay and quantitative real-time PCR were used to determine the nitric oxide (NO) and the mRNA expression levels of IFN-γ and iNOS in infected J774-A1 macrophage cells. RESULTS: FMN significantly (P < 0.001) decreased the viability and number of promastigotes and amastigotes forms. The 50% inhibitory concentrations value for FMN and glucantime was 9.3 and 14.3 µM for promastigote and amastigote, respectively. We found that the macrophages exposed with FMN especially at concentrations of 1/2 IC50 and IC50 significantly activated the NO release and the mRNA expression levels of IFN-γ, iNOS. The findings of the current research showed the favorable antileishmanial effects formononetin, a natural isoflavone, against various stages of L. tropica through inhibition of infectivity rate of macrophage cells and triggering the NO production and cellular immunity. However, supplementary works are essential to evaluate the ability and safety of FMN in animal model before use in the clinical phase.

One-pot synthesis of polyhydroquinoline-1,2,3-triazole hybrids in deep eutectic solvent as anti-leishmanial agents and molecular modeling studies.

The novel hybrids with 1,2,3-triazole and polyhydroquinoline scaffolds were successfully synthesized by multicomponent reaction of propargyloxybenzaldehyde, 1,3-cyclohexadione, ethylacetoacetate and ammonium acetate followed through click reaction in the presence of deep eutectic solvent ChCl/ZnCl2 as an efficient catalyst. Their anti-leishmanial activity was evaluated against amastigote and promastigote forms of L. tropica, L. major, and two different species of L. infantum. Furthermore, to determine the cytotoxicity of the hybrids, they were evaluated against the murine macrophage cell line J774.A1. Based on the results, three hybrids showed the highest antileishmanial activity. However, they revealed low cytotoxicity. Hybrid 6j was the most potent compound against both the forms of all leishmanial types, with IC50 = 13.5 and 11.9 µg/mL for L. major, 37.5 and 25 µg/mL for L. tropica, 17.5 and 20 µg/mL for L. infantum (MCAN/IR//96/LON49) and 35.5 and 30 µg/mL for L. infantum (MCAN/ES/98/LIM-877), respectively. Finally, molecular docking and molecular dynamics simulations were also performed to identify possible mechanism antileishmanial activity.Communicated by Ramaswamy H. Sarma.