Evaluation of α,β-unsaturated ketones as antileishmanial agents

In this study, we assessed the antileishmanial activity of 126 α,β-unsaturated ketones. The compounds NC901, NC884, and NC2459 showed high leishmanicidal activity for both the extracellular (50% effective concentration [EC(50)], 456 nM, 1,122 nM, and 20 nM, respectively) and intracellular (EC(50), 1,870 nM, 937 nM, and 625 nM, respectively) forms of Leishmania major propagated in macrophages, with little or no toxicity to mammalian cells. Bioluminescent imaging of parasite replication showed that all three compounds reduced the parasite burden in the murine model, with no apparent toxicity

Development of Thiophene Compounds as Potent Chemotherapies for the Treatment of Cutaneous Leishmaniasis Caused by Leishmania major

Leishmania major (L. major) is a protozoan parasite that causes cutaneous leishmaniasis. About 12 million people are currently infected with an annual incidence of 1.3 million cases. The purpose of this study was to synthesize a small library of novel thiophene derivatives, and evaluate its parasitic activity, and potential mechanism of action (MOA). We developed a structure–activity relationship (SAR) study of the thiophene molecule 5A. Overall, eight thiophene derivatives of 5A were synthesized and purified by silica gel column chromatography. Of these eight analogs, the molecule 5D showed the highest in vitro activity against Leishmania major promastigotes (EC50 0.09 ± 0.02 µM), with an inhibition of the proliferation of intracellular amastigotes higher than 75% at only 0.63 µM and an excellent selective index. Moreover, the effect of 5D on L. major promastigotes was associated with generation of reactive oxygen species (ROS), and in silico docking studies suggested that 5D may play a role in inhibiting trypanothione reductase. In summary, the combined SAR study and the in vitro evaluation of 5A derivatives allowed the identification of the novel molecule 5D, which exhibited potent in vitro anti-leishmanial activity resulting in ROS production leading to cell death with no significant cytotoxicity towards mammalian cells

In vitro and in vivo characterization of potent antileishmanial methionine aminopeptidase 1 inhibitors

Leishmania major is the causative agent of cutaneous leishmaniasis (CL). No human vaccine is available for CL, and current drug regimens present several drawbacks, such as emerging resistance, severe toxicity, medium effectiveness, and/or high cost. Thus, the need for better treatment options against CL is a priority. In the present study, we validate the enzyme methionine aminopeptidase 1 of L. major (MetAP1Lm), a metalloprotease that catalyzes the removal of N-terminal methionine from peptides and proteins, as a chemotherapeutic target against CL infection. The in vitro antileishmanial activities of eight novel MetAP1 inhibitors (OJT001 to OJT008) were investigated. Three compounds, OJT006, OJT007, and OJT008, demonstrated potent antiproliferative effects in macrophages infected with L. major amastigotes and promastigotes at submicromolar concentrations, with no cytotoxicity against host cells. Importantly, the leishmanicidal effect in transgenic L. major promastigotes overexpressing MetAP1Lm was diminished by almost 10-fold in comparison to the effect in wild-type promastigotes. Furthermore, the in vivo activities of OJT006, OJT007, and OJT008 were investigated in L. major-infected BALB/c mice. In comparison to the footpad parasite load in the control group, OJT008 decreased the footpad parasite load significantly, by 86%, and exhibited no toxicity in treated mice. We propose MetAP1 inhibitor OJT008 as a potential chemotherapeutic candidate against CL infection caused by L. major infection

Ruthenium-azole complexes as chemotherapeutic agents against Leishmaniasis

Leishmaniasis is a neglected disease that affects millions of people worldwide. Poor efficacy and high toxicity of the limited treatment options demonstrate the need for the development of new chemotherapeutic agents to treat this disease. In order to respond this concern, the development of novel Ru-azole complexes as chemotherapeutic agents by inhibiting the cytochrome P-450 dependent C14-&agr;-demethylation of lanosterol to ergosterol which is essential for the parasite survival. In addition, Ruthenium would enhance the activity of the parental drug, by helping ketoconazole/clotrimazole to cross the parasite membrane, together presenting low toxicity. The viability and citotoxicity of the Ru-azole compunds was tested against Leishmania major and different mammalian cells lines. Based on these experiments Ru-azole complexes represent excellent leads for the development of new chemotherapeutic agents to treat leishmaniasis

Novel Possibilities for the Treatment and Prevention of Cutaneous Leishmaniasis

Protozoan parasites from the genus Leishmania cause broad clinical manifestations known as leishmaniasis, which affect millions of people worldwide. Cutaneous leishmaniasis (CL) is mainly caused by either Leishmania major or L. tropica parasites, which produce localized cutaneous ulcers, often leading to scarring and social stigma. Currently, the disease has reached hyperendemicity levels in the Middle East due to conflict and human displacement and is one the most common forms of the disease in the Old World. Furthermore, the first choice of treatment in that region continues to be pentavalent antimonials, which are costly and highly toxic, and current vector control measures alone are not sufficient to stop disease transmission. CL remains largely neglected, with no prophylactic or therapeutic vaccine available, and existing drug treatments are expensive, have toxic side effects, and resistant parasite strains have been reported. Hence, further therapeutic and preventive interventions against the disease are necessary. In this regard, previous studies from our laboratory, reported promising in vitro anti-leishmanicidal findings of a novel series of organometallic compounds containing RuII and clotrimazole (CTZ) termed AM160 or AM162 [1]. For the first part of the present study, we determine the efficacy of AM160 or AM162, in BALB/c infected mice with L. major metacyclic promastigotes to alleviate CL. AM162 treatment (4 mg/kg/day) reduced the lesion size in the murine model of CL by a 68%, a markedly better effect than CTZ alone or a RuII complex not containing CTZ (C3). In addition, in a hyper infection experiment, AM162 treatment at 6 mg/kg/day significantly (p=0.0146) reduced CL footpad lesions and parasite load in comparison with vehicle control group, with no significant toxicity observed. Moreover, we investigated the potential mechanism of cell death inflicted by RuII CTZ complexes on L. major promastigotes. AM160 and AM162 exhibited pro-apoptotic-like properties, implicating phospholipids externalization, mitochondrial depolarization and DNA fragmentation, as its mechanism to induce cell death in L. major parasites; encompassing early and late hallmarks of apoptosis. Overall, these findings indicate that AM162 complex is an efficient compound for the treatment of CL in a murine model. Furthermore, the second part of this dissertation focuses on the development of a vaccine based on terminal, non-reducing, and linear α-Galactopyranosyl (α-Gal) epitopes that are abundantly found on the plasma membrane glycolipids of L. major known as glycoinositolphospholipids (GIPLs). Previous studies have suggested that sugars are promising vaccine candidates against leishmaniasis, since most parasite species have a cell surface coat composed of immunogenic sugars, including linear α-Gal epitopes, which are absent in humans. Here, we evaluated three neoglycoproteins (NGPs), containing synthetic α-Gal epitopes covalently attached to bovine serum albumin (BSA), as vaccine candidates against L. major, using α1,3-galactosyltransferase-knockout (α1,3GalT-KO) mice. These transgenic mice, similarly to humans, do not express non-reducing, linear α-Gal epitopes in their cells and are, therefore, capable of producing high levels of anti-α-Gal antibodies. We observed that Galα(1,6)Galβ-BSA (NGP5B), but not Galα(1,4)Galβ-BSA (NGP12B) or Galα(1,3)Galβ-BSA (NGP17B), was able to significantly reduce the size of footpad lesions by 96% in comparison to control groups. Furthermore, we observed a robust humoral and cellular immune response with production of high levels of protective lytic anti-α-Gal antibodies and induction of Th1 cytokines. When tested in transgenic mice, which like humans, lack α-Gal epitopes in their cells, NGP5B was able to induce a significant partial protection against L. major infection by significantly reducing mouse footpad lesions and parasite burden. Altogether, we propose NGP5B as a promising preventive vaccine, and AM162 as a treatment option for CL caused by L. major

High-Throughput of Indoline Derivatives against Leishmania major and Trypanozoma cruzi

Leishmania major and Trypanozoma cruzi are obligate intracellular parasites responsible for Leishmaniasis and Chaga’s disease, respectively. These neglected diseases are endemic in many regions such as Africa and South America, and are becoming a serious problem in the blood and tissue banks in the U.S. due to high immigration of people from endemic regions. Current treatments are characterized by their low efficacy against parasites and high toxicity in humans. Therefore, the discovery of new ways to treat these parasitoses has become urgent. Drug Screening of novel compounds serves as a starting point for the design of new therapies and for understanding the interaction between a drug and its target. In this study, experiments utilizing transgenic L. major strain Friedlin clone V1 (expressing luciferase gene) and T. cruzi Y strain (wild type) were performed testing the viability of these parasites in the presence of Indoline derivatives inhibitors. Additionally, mammalian cells were also incubated with these compounds to test for toxicity. All five compounds displayed toxicity against L. major promastigotes at concentrations between 200 μM to 6.25 μM. Moreover, these compounds were not toxic to the human cell line, U2-OS (osteoblasts), at these concentrations. Currently, experiments are in progress testing the viability of T. cruzi epimastigotes against these compounds

Evaluation of ␣,␤-Unsaturated Ketones as Antileishmanial Agents

In this study, we assessed the antileishmanial activity of 126 ␣,␤-unsaturated ketones. The compounds NC901, NC884, and NC2459 showed high leishmanicidal activity for both the extracellular (50% effective concentration [EC 50 ], 456 nM, 1,122 nM, and 20 nM, respectively) and intracellular (EC 50 , 1,870 nM, 937 nM, and 625 nM, respectively) forms of Leishmania major propagated in macrophages, with little or no toxicity to mammalian cells. Bioluminescent imaging of parasite replication showed that all three compounds reduced the parasite burden in the murine model, with no apparent toxicity. L eishmania major is the causative agent of cutaneous leishmaniasis (CL) disease (1). Worldwide, the incidence of CL is estimated to be 0.7 to 1.2 million new cases per year (2). Other than the drug miltefosine, which was approved by the FDA in 2014, most of the currently used antileishmanial drugs were developed in the 1940s (3, 4). However, drug-resistant strains of the parasite have emerged. Furthermore, toxicity to the host and the high costs of these drugs limit their wider application and use (4, 5). Therefore, the burden of leishmaniasis and limited effective treatments clearly point to the need for new drugs. In view of the current interest in examining different antineoplastic agents for their antiprotozoal properties (6-8), we assessed the antileishmanial activity of ␣,␤-unsaturated ketones (enones) provided by J. R. Dimmock from the University of Saskatchewan in Canada (1, 9). Enones react preferentially with cellular thiols, in contrast to amino or hydroxyl functional groups present in protein and DNA (10, 11); therefore, interactions with nucleic acids, which can lead to adverse genotoxic effects, should be absent in enones (12). Since thiol-dependent metabolism is the main detoxifying mechanism in trypanosomatids, we hypothesized that enones are attractive candidates for examination as potential antileishmanial agents. Hence, in this study, we screened the enone library for antiparasitic and cytotoxic activity. The compounds were dissolved in dimethyl sulfoxide (DMSO) and tested at concentrations ranging from 500 M to 1 nM (1, 9). The parasites tested were a firefly luciferase-expressing line of L. major promastigotes described previously (Lmj-FV1-LUC-TK [L. major strain Friedlin {MHOM/JL/ 80/Friedlin}], clone V1) and cultured as previously described The in vitro infectivity experiments were carried out to determine the activity of NC901, NC884, and NC2459 against L. major intracellular amastigotes. Peritoneal macrophages isolated from BALB/c mice were infected with L. major metacyclic promastigotes for 24 h, followed by treatment with the NC lead compounds for an additional 48 h ( Additionally, the activity of the compounds against L. major was investigated in a murine model of CL. The first set of experiments To determine the relative toxicity of the compounds, the weights of the mice were measured twice a week (data not shown). There was no significant weight loss for any of the groups except for the mice receiving amphotericin B. Additionally, no mice died as a result of the toxicity of the compounds. Hyperinfection with L. major was tested. Twenty-six female BALB/c mice organized in four groups of five and one group of six mice were infected with 10 6 L. major metacyclic promastigotes Our findings demonstrate the ability of these compounds to reduce L. major replication in vivo without any obvious toxic side effects. NC2459 evidently exhibited a much smaller amount of swelling than that in all other groups by decreasing the footpad lesion by 99.99