In vitro growth inhibitory activity of Medicines for Malaria Venture pathogen box compounds against Leishmania aethiopica

Introduction Leishmania aethiopica (L. aethiopica) is responsible for different forms of cutaneous leishmaniasis (CL) in Ethiopia. Treatment heavily depends on limited drugs, together with drawbacks like toxicity and microbial resistance. The current research aimed to investigate in vitro growth inhibitory activity of Medicines for Malaria Ventures - Pathogen Box (MMV - PB) compounds against L. aethiopica clinical isolate. Methodology Four hundred MMV – PB compounds were screened against L. aethiopica using resazurin based colourimetric assay. Compounds with > 70% inhibition were further tested using macrophage based intracellular amastigote assay. Cytotoxic and hemolytic activity of candidate hits were assessed on THP1- cells and sheep red blood cells (RBCs), respectively. In vitro drug interaction study was also conducted for the most potent hit using the combination index method. Results At the test concentration of 1 μM, twenty-three compounds showed > 50% inhibition of promastigotes parasite growth, of which 11 compounds showed > 70% inhibition. The 50% growth inhibition (IC50) of the 11 compounds was ranged from 0.024 to 0.483 μM in anti-promastigote assay and from 0.064 to 0.899 μM in intracellular amastigote assay. Candidate compounds demonstrated good safety on sheep RBCs and THP-1 cell lines. MMV688415 demonstrated a slight hemolytic activity on sheep RBC (5.3% at 25 μM) and THP-1 cell line (CC20 = 25 μM) while MMV690102 inhibited half of THP-1 cells at 36.5 μM (selectivity index = 478). No synergistic activity was observed from the combinations of MMV690102 and amphotericin B (CI > 1), and MMV690102 and Pentamidine (CI > 1) at lower and higher combination points. Conclusion The present study identified a panel of compounds that can be used as a novel starting point for lead optimization. MMV690102 appears to be the most potent inhibitor against L. aethiopica promastigotes and amastigotes. Future works should investigate the antileishmanial mechanism of action and in vivo antileishmanial activities of identified hits.

In Vitro Susceptibility to Miltefosine of Leishmania infantum (syn. L. chagasi) Isolates from Different Geographical Areas in Brazil

Treatment of visceral leishmaniasis in Brazil still relies on meglumine antimoniate, with less than ideal efficacy and safety, making new therapeutic tools an urgent need. The oral drug miltefosine was assayed in a phase II clinical trial in Brazil with cure rates lower than previously demonstrated in India. The present study investigated the susceptibility to miltefosine in 73 Brazilian strains of Leishmania infantum from different geographic regions, using intracellular amastigote and promastigote assays. The EC50 for miltefosine of 13 of these strains evaluated in intracellular amastigotes varied between 1.41 and 4.57 μM. The EC50 of the 73 strains determined in promastigotes varied between 5.89 and 23.7 μM. No correlation between in vitro miltefosine susceptibility and the presence of the miltefosine sensitive locus was detected among the tested strains. The relatively low heterogeneity in miltefosine susceptibility observed for the 73 strains tested in this study suggests the absence of decreased susceptibility to miltefosine in Brazilian L. infantum and does not exclude future clinical evaluation of miltefosine for VL treatment in Brazil.

Antiprotozoal and Antibacterial Activity of Ravenelin, a Xanthone Isolated from the Endophytic Fungus Exserohilum rostratum

The natural compound ravenelin was isolated from the biomass extracts of Exserohilum rostratum fungus, and its antimicrobial, antiplasmodial, and trypanocidal activities were evaluated. Ravenelin was isolated by column chromatography and HPLC and identified by NMR and MS. The susceptibility of Gram-positive and Gram-negative bacteria strains to ravenelin was determined by microbroth dilution assay. Cytotoxicity was evaluated in hepatocarcinoma cells (HepG2) and BALB/c peritoneal macrophages by using MTT. SYBR Green I-based assay was used in the asexual stages of Plasmodium falciparum. Trypanocidal activity was tested against the epimastigote and intracellular amastigote forms of Trypanosoma cruzi. Ravenelin was active against Gram-positive bacteria strains, with emphasis on Bacillus subtilis (MIC value of 7.5 µM). Ravenelin’s antiparasitic activities were assessed against both the epimastigote (IC50 value of 5 ± 1 µM) and the intracellular amastigote forms of T. cruzi (IC50 value of 9 ± 2 µM), as well as against P. falciparum (IC50 value of 3.4 ± 0.4 µM). Ravenelin showed low cytotoxic effects on both HepG2 (CC50 > 50 µM) and peritoneal macrophage (CC50 = 185 ± 1 µM) cells with attractive selectivity for the parasites (SI values > 15). These findings indicate that ravenelin is a natural compound with both antibacterial and antiparasitic activities, and considerable selectivity indexes. Therefore, ravenelin is an attractive candidate for hit-to-lead development.

Identification of Leucinostatins from Ophiocordyceps sp. as Antiparasitic Agents Against Trypanosoma cruzi

Safe and effective treatments for Chagas disease, a potentially fatal parasitic infection associated with cardiac and gastrointestinal pathology and caused by the kinetoplastid parasite Trypanosoma cruzi, have yet to be developed. Benznidazole and nifurtimox, which are currently the only available drugs against T. cruzi, are associated with severe adverse effects and questionable efficacy in the late stage of the disease. Natural products have proven to be a rich source of new chemotypes for other infectious agents. We utilized a microscopy-based high-throughput phenotypic screen to identify inhibitors of T. cruzi from a library of natural product samples obtained from fungi procured through a Citizen Science Soil Collection Program (https://whatsinyourbackyard.org/), and the Great Lakes (USA) benthic environment. We identified five leucinostatins (A, B, F, NPDG C and NPDG D) as potent inhibitors of the intracellular amastigote form of T. cruzi. Leucinostatin B also showed in vivo efficacy in a mouse model of Chagas disease. Given prior reports that leucinostatins A and B have antiparasitic activity against the related kinetoplastid T. brucei, our findings suggest a potential cross-trypanocidal compound class and provide a platform for further chemical derivatization of a potent chemical scaffold against T. cruzi.

A Systematic Literature Review of Curcumin With Promising Antileishmanial Activity

Background: Curcumin (CUR) is a bright yellow chemical and it is used as an additive in foods. Recently CUR and its associated bioactive compounds have received much attention in the literature review. The aim of this systematic review is to overview antileishmanial properties of CUR and its mechanism, perhaps the results of this study will be used for therapeutic and preventive purposes. Methods: Following the PRISMA guidelines, international databases were systematically searched for studies published until September 2019. Articles related to the subject were selected and included in this systematic review. Results: A total of 15 articles met our eligibility criteria. Then, the effect of CUR and its associated bioactive compounds on Leishmania species was evaluated. In most studies CUR/derivatives were tested on L. major and in vitro condition. Most investigations were conducted on the promastigote rather than the more relevant intracellular amastigote stage. Our results showed that CUR overcomes the inhibitory effect of nitric oxide (NO) on Leishmania parasites. Conclusions: This review indicated that CUR derivatives instead of alone CUR showed a high potential to serve as an effective herbal drug against leishmaniasis. Moreover, we concluded that the antileishmanial activity of CUR/bioactive compounds is mostly due to increasing oxidative stress and inducing apoptosis.

A Triazole Hybrid of Neolignans as a Potential Antileishmanial Agent by Triggering Mitochondrial Dysfunction

In the search for new compounds with antileishmanial activity, we synthesized a triazole hybrid analogue of the neolignans grandisin and machilin G (LASQUIM 25), which was previously found highly active against both promastigotes and intracellular amastigote forms of Leishmania amazonensis. In this work, we investigated the leishmanicidal effects of LASQUIM 25 to identify the mechanisms involved in the cell death of L. amazonensis promastigotes. Transmission electron microscopy (TEM) analysis showed marked effects of LASQUIM 25 (IC50 = 7.2 µM) on the morphology of promastigote forms, notably on mitochondria. The direct action of the triazole derivative on the parasite was noticed over time from 2 h to 48 h, and cells displayed several ultrastructural alterations characteristic of apoptotic cells. Also, flow cytometric analysis (FACS) after TMRE staining detected changes in mitochondrial membrane potential after LASQUIM 25 treatment (64.83% labeling versus 83.38% labeling in nontreated cells). On the other hand, FACS after PI staining in 24 h-treatment showed a slight alteration in the integrity of the cell membrane, a necrotic event (16.76% necrotic cells versus 3.19% staining in live parasites). An abnormal secretion of lipids was observed, suggesting an exocytic activity. Another striking finding was the presence of autophagy-related lysosome-like vacuoles, suggesting an autophagic cell death that may arise as consequence of mitochondrial stress. Taken together, these results suggest that LASQUIM 25 leishmanicidal mechanisms involve some degree of mitochondrial dysregulation, already evidenced by the treatment with the IC50 of this compound. This effect may be due to the presence of a methylenedioxy group originated from machilin G, whose toxicity has been associated with the capacity to generate electrophilic intermediates.

Genomic and Metabolomic Polymorphism among Experimentally Selected Paromomycin-Resistant Leishmania donovani Strains

ABSTRACT Understanding the mechanism(s) underpinning drug resistance could lead to novel treatments to reverse the increased tolerance of a pathogen. In this study, paromomycin (PMM) resistance (PMMr) was induced in three Nepalese clinical strains of Leishmania donovani with different inherent susceptibilities to antimony (Sb) drugs by stepwise exposure of promastigotes to PMM. Exposure to PMM resulted in the production of mixed populations of parasites, even though a single cloned population was used at the start of selection. PMM 50% inhibitory concentration (IC50) values for PMMr parasites varied between 104 and 481 μM at the promastigote stage and 32 and 195 μM at the intracellular amastigote stage. PMM resistance was associated with increased resistance to nitric oxide at the amastigote stage but not the promastigote stage (P < 0.05). This effect was most marked in the Sb-resistant (Sbr) PMMr clone, in which PMM resistance was associated with a significant upregulation of glutathione compared to that in its wild type (P < 0.05), although there was no change in the regulation of trypanothione (detected in its oxidized form). Interestingly, PMMr strains showed an increase in either the keto acid derivative of isoleucine (Sb intermediate PMMr) or the 2-hydroxy acids derived from arginine and tyrosine (Sb susceptible PMMr and Sbr PMMr). These results are consistent with the recent finding that the upregulation of the branched-chain amino acid aminotransferase and d-lactate dehydrogenase is linked to PMMr. In addition, we found that PMMr is associated with a significant increase in aneuploidy during PMM selection in all the strains, which could allow the rapid selection of genetic changes that confer a survival advantage.

Identification of Leishmania donovani inhibitors from pathogen box compounds of Medicine for Malaria Venture

Leishmaniasis is a collective term used to describe various pathological conditions caused by an obligate intracellular protozoan of the genus Leishmania. It is one of the neglected diseases and has been given low attention in drug discovery researches to narrow the existing gap in safety and efficacy of the currently used drugs to treat leishmaniasis. The challenge is further exacerbated by the emergence of drug resistance by the parasites. Aiming to look for potential anti-leishmanial hits and leads, we screened MMV Pathogen Box against clinically isolated L. donovani strain. Compounds were screened against promastigote, and then against amastigote stages; of which, 35 compounds showed >50% inhibition on promastigotes in the initial screen (1 μM). Out of these compounds, 9 compounds showed >70% inhibition with median inhibitory concentration (IC50) ranges from 12 nM to 491 nM on anti-promastigote assay and 53 to 704 nM on intracellular amastigote assay. Identified compounds demonstrated good safety on THP-1 cell lines and sheep RBCs, and appropriate physico-chemical property suitable for further drug development. Two compounds (MMV690102 and MMV688262) were identified as lead compounds. Among these compounds, anti-tubercular agent MMV688262 (delamanid) showed synergistic effect with amphotericin B, indicating the prospect of this compound for combination therapy. The current study indicates the presence of additional hits which may hold promise as starting points for anti-leishmanial drug discovery and in-depth structure activity relationship studies. Future works also needs to investigate antiamastigotes activity of remaining ‘hits’, which were not covered in the present study.Authors summaryVisceral leishmaniasis is a major public health problem in endemic regions. Different drugs have been used to treat visceral leishmaniasis. However, the available drugs are either toxic, non-compliance to the patient, painful upon administration, low in efficacy, or costly. New chemical entities that overcome the limitations of existing drugs are therefore desperately needed. Screening of 400 pathogen box compounds against of Leishmania donovani clinical isolate resulted in identification of 35 compounds with >50% inhibition against promastigotes at 1 μM. Out of these compounds, 9 showed >70% inhibition with median inhibitory concentration ranges from 12 nM to 491 nM on anti-promastigote assay, and 53 to 704 nM on intracellular amastigote assay. Our work identified new compounds which hold promise for further drug development.

Endlicheria bracteolata (Meisn.) Essential Oil as a Weapon Against Leishmania amazonensis: In Vitro Assay

The difficulties encountered and the numerous side effects present in the treatment of cutaneous leishmaniasis have encouraged the research for new compounds that can complement or replace existing treatment. The growing scientific interest in the study of plants, which are already used in folk remedies, has led our group to test Endlicheria bracteolata essential oil against Leishmania amazonensis. Several species of the Lauraceae family, or their compounds, have relevant antiprotozoal activities Therefore, the biological potential on L. amazonensis forms from the essential oil of Endlicheria bracteolata leaves was verified for the first time in that work. The antileishmanial activity was evaluated against promastigotes and intracellular amastigotes, and cytotoxicity were performed with J774.G8, which were incubated with different concentrations of E. bracteolata essential oil. Transmission electron microscopy and flow cytometry were performed with E. bracteolata essential oil IC50. Promastigote forms showed E. bracteolata essential oil IC50 of 7.945 ± 1.285 µg/mL (24 h) and 6.186 ± 1.226 µg/mL (48 h), while for intracellular amastigote forms it was 3.546 ± 1.184 µg/mL (24 h). The CC50 was 15.14 ± 0.090 µg/mL showing that E. bracteolata essential oil is less toxic to macrophages than to parasites. Transmission electron microscopy showed that E. bracteolata essential oil treatment is capable of inducing mitochondrial damage to promastigote and intracellular amastigote forms, while flow cytometry showed ΔѰm disruption in treated parasites. These results could bring about new possibilities to develop products based on E. bracteolata essential oil to treat cutaneous leishmaniasis, especially for people who cannot receive the conventional therapy.

Cinnamomum zeylanicum, Origanum vulgare, and Curcuma longa Essential Oils: Chemical Composition, Antimicrobial and Antileishmanial Activity

The resistance mechanisms of bacteria and protozoans have evidenced the need of discover new compounds with potential pharmaceutical activity against pathogenic microorganisms. Medicinal plants have been for centuries a promising alternative as sources of new drugs. The objective of this work was to evaluate the chemical composition, antimicrobial and antileishmanial activities of Cinnamomum zeylanicum, Origanum vulgare, and Curcuma longa essential oils. Chemical analysis was performed by gas chromatography-mass spectrometry. Antimicrobial activity was performed by disk diffusion and minimum inhibitory concentration (MIC) test. Antileishmanial activity was performed against antipromastigote and intracellular amastigote of Leishmania amazonensis. Cytotoxic and nitrite production were realized in BALB/c peritoneal macrophages. The major compounds of the essential oils were cinnamic aldehyde (46.30%) in C. zeylanicum, cis-p-menth-2-en-1-ol (33.88%) and linalyl acetate (13.90%) in O. vulgare, and turmerone (55.43%) in C. longa. The MIC showed significant antimicrobial activity of C. longa essential oil against S. aureus (83.3 ± 14.43 µg/mL). Antipromastigote activity showed IC50 values >500 µg/mL to C. zeylanicum, 308.4 ± 1.402 µg/mL to O. vulgare, and 405.5 ± 1.119 µg/mL to C. longa essential oil. Activity against intracellular amastigote of L. amazonensis showed IC50 of 63.3 ± 1.369 µg/mL and cytotoxic was not observed, resulting in selectivity index higher than 15.79 to parasite. C. longa essential oil decreased nitrite production in peritoneal macrophages, but not in Leishmania-infected cells. The chemical composition of the three essential oils is directly associated to its potential biological action, as the antimicrobial activity. C. longa presented a potent antileishmanial activity against promastigote and intracellular amastigote of L. amazonensis, although this activity is not linked to nitric oxide, since C. longa essential oil inhibits its production.