Anti-leishmanial Therapeutics Research Articles

Synthesis and in vitro evaluation shows disquaramide compounds are a promising class of anti-leishmanial drugs.

An increasing number of treatment failures with current pharmaceutics, as well as a lack of a vaccine, demonstrates the need to develop new treatment options for leishmaniasis. Herein, we describe the synthesis and in vitro analysis of 24 disquaramide compounds targeting the Leishmania major parasite. Of the compounds that were evaluated, six of them ( 13 , 19 , 20 , 22 , 24 , and 26 ) were capable of significantly decreasing the number of parasites by up to 42% compared to the control by day four. This demonstrates that disquaramides either impair parasite replication or have leishmancidal effects. Additionally, none of the disquaramide compounds tested displayed host cell cytotoxicity. These experiments provide evidence that disquaramides have the potential to be effective anti-leishmanial therapeutics.

Generation of 2D-QSAR and pharmacophore models for fishing better anti-leishmanial therapeutics

Leishmaniasis, a life-threatening tropical disease that is endemic in nearly 100 countries, contributes to millions of deaths each year. However, very few antileishmanial compounds are available in the market and that too possess many drawbacks. Hence, the therapeutic arsenal requires potential and novel anti-leishmanial compounds to treat Leishmaniasis. In the present study, quantitative structure activity relationship (QSAR) model and Pharmacophore model were developed with a set of antileishmanial compounds collected from literature and commercial antileishmanial drugs. A ligand-based pharmacophore model was developed using active compound as template and it was used for searching the purchasable compound dataset of ZINC database for matching compounds. Thirteen novel, readily purchasable compounds were obtained from this approach, which shows good predicted activity, ADME and druglikeness. These compounds can be regarded as potential candidates to be developed as novel antileishmanial drugs with improved activity and reduced side effects.

Generation of 2D-QSAR and pharmacophore models for fishing better anti-leishmanial therapeutics

Generation of 2D-QSAR and pharmacophore models for fishing better anti-leishmanial therapeutics

Metabolic Pathways of Leishmania Parasite: Source of Pertinent Drug Targets and Potent Drug Candidates.

Leishmaniasis is a tropical disease caused by a protozoan parasite Leishmania that is transmitted via infected female sandflies. At present, leishmaniasis treatment mainly counts on chemotherapy. The currently available drugs against leishmaniasis are costly, toxic, with multiple side effects, and limitations in the administration route. The rapid emergence of drug resistance has severely reduced the potency of anti-leishmanial drugs. As a result, there is a pressing need for the development of novel anti-leishmanial drugs with high potency, low cost, acceptable toxicity, and good pharmacokinetics features. Due to the availability of preclinical data, drug repurposing is a valuable approach for speeding up the development of effective anti-leishmanial through pointing to new drug targets in less time, having low costs and risk. Metabolic pathways of this parasite play a crucial role in the growth and proliferation of Leishmania species during the various stages of their life cycle. Based on available genomics/proteomics information, known pathways-based (sterol biosynthetic pathway, purine salvage pathway, glycolysis, GPI biosynthesis, hypusine, polyamine biosynthesis) Leishmania-specific proteins could be targeted with known drugs that were used in other diseases, resulting in finding new promising anti-leishmanial therapeutics. The present review discusses various metabolic pathways of the Leishmania parasite and some drug candidates targeting these pathways effectively that could be potent drugs against leishmaniasis in the future.

Antileishmanial Activity of the Genus Piper: A Systematic Review

Background: Leishmaniases are anthropozoonotic vector-borne diseases, caused by the protozoan Leishmania spp. These diseases have significant morbidity and mortality worldwide and, as there are currently no vaccines available for their treatment, chemotherapy remains the mainstay for anti-leishmanial therapeutics. However, the severe side effects, reduced bioavailability, high cost and chemoresistance, amongst other problems, limit the use of available drugs. In recent years, natural compounds have shown promise as anti-leishmanial agents, especially those extracted from medicinal plants. The genus Piper has been used in traditional medicine and widely explored for its biological properties and bioactive phytocompounds.
 Aim: The aim of this study was to conduct a systematic literature review of the biological activity of the genus Piper against the etiological agents of leishmaniasis, to provide a perspective for effective and safe phytotherapics, new drugs or potentially active prototype chemical substances.
 Methodology: This systematic review was prepared in accordance with PRISMA guidelines. The databases used for this review were SciELO, Pubmed, ScienceDirect and Google Scholar, using a temporal profile of 2009 to 2020.
 Conclusion: In this review, we summarize a wide range of isolated compounds, extracts and essential oils of the genus Piper that are worth screening given their potential for development as effective anti-leishmanial drugs.

Palladium-mediated synthesis and biological evaluation of C-10b substituted Dihydropyrrolo[1,2-b]isoquinolines as antileishmanial agents.

The development of new molecules for the treatment of leishmaniasis is, a neglected parasitic disease, is urgent as current anti-leishmanial therapeutics are hampered by drug toxicity and resistance. The pyrrolo[1,2-b]isoquinoline core was selected as starting point, and palladium-catalyzed Heck-initiated cascade reactions were developed for the synthesis of a series of C-10 substituted derivatives. Their invitro leishmanicidal activity against visceral (L.donovani) and cutaneous (L.amazonensis) leishmaniasis was evaluated. The best activity was found, in general, for the 10-arylmethyl substituted pyrroloisoquinolines. In particular, 2ad (IC50=3.30μM, SI>77.01) and 2bb (IC50=3.93μM, SI>58.77) were approximately 10-fold more potent and selective than the drug of reference (miltefosine), against L.amazonensis on invitro promastigote assays, while 2ae was the more active compound in the invitro amastigote assays (IC50=33.59μM, SI>8.93). Notably, almost all compounds showed low cytotoxicity, CC50>100μg/mL in J774cells, highest tested dose. In addition, we have developed the first Perturbation Theory Machine Learning (PTML) algorithm able to predict simultaneously multiple biological activity parameters (IC50, Ki, etc.) vs. any Leishmania species and target protein, with high values of specificity (>98%) and sensitivity (>90%) in both training and validation series. Therefore, this model may be useful to reduce time and assay costs (material and human resources) in the drug discovery process.

Review on natural products as an alternative to contemporary anti-leishmanial therapeutics

Leishmaniasis is a vector-borne protozoan disease caused by an intracellular parasite which globally affects 350 million people. This infectious disease mainly affects undeveloped and developing countries across the world. Chemotherapeutic therapies mainly depend on pentavalent antimonials, miltefosine, stibogluconate, amphotericin B and paromomycin. However, these medications exhibit several side-effects including toxicity, cost and emerging drug resistance and this has proved to be an obstacle in the eradication of the disease. Considering the alarming drug resistance, there is an urgent need for a novel and potentially effective approach against leishmaniasis. In the course of identification of new therapies, plant-based compounds were found to be an alternative that can either be used directly or with structural modifications. Various plants have been known for ages to be the source of phytochemicals with high medicinal values. These phytochemicals have been extracted by different techniques and have shown efficacy against numerous diseases. This review study concentrates on the application of various plant source drugs and their modified analogs for the treatment of Leishmania. These natural compounds have also been tagged with nanoparticles for better delivery, efficacy, and bioavailability. These modified nanoformulations have the potential to become a better drug delivery system in the future. Thus, this review provides an overview of the current chemotherapeutic therapies, plant-based therapies and the application of nanotechnology for better efficacy.

Identification of potential inhibitors targeted for strengthening search of anti-leishmanial therapeutics

Visceral leishmaniasis (VL: a fatal disease) is reported to occur at the rate of 70,000 to 90,000 per year, according to WHO. VL is caused by Leishmania donovani species causing 90% cases of VL. Current treatment regime is not satisfactory due to substantial toxic side effects of existing antileishmanial therapy and the emergence of drug resistance from different part of the world. These issues make existing therapy unsuitable management of VL. There are various studies dedicated to overcoming the shortcomings in antileishmanial therapeutics. Likewise, in this study, we have targeted HSP100 (heat shock protein) of L. donovani for identification of potent antileishmanial. HSP100 protein is released when the Leishmania parasite encounters heat stress in macrophage of mammalian host making it an effective target. This in silico study was carried out with 10 unexplored ligands and Miltefosine (an established anti-leishmanial drug used as control) via targeting L. donovani HSP100. We report that two test ligands Diospyrin and Benzoxaborole had outstanding docking potential with L. donovani HSP100 and were chosen for further analysis of drug-likeness and ADMET (absorption distribution, metabolism, and excretion) properties. These two ligands docking scores were better than the antileishmanial Miltefosine and had better ADMET properties as a drug candidate. The result gave us a lead and confirmation to support our hypothesis that HSP100 can be a potential drug target of L. donovani and Diospyrin and Benzoxaborole can be potent antileishmanial molecules.

A novel nanoliposomal formulation of the FDA approved drug Halofantrine causes cell death of Leishmania donovani promastigotes in vitro

Leishmaniasis is a notorious disease affecting mainly poor people. Visceral Leishmaniasis (VL) is a clinical form of Leishmaniasis which mainly affects people living below poverty line. There are no available vaccines against VL and chemotherapy still remains the mainstay for treatment. However, the use of the current therapeutic agents are limited due to issues of toxicity, side effects and cost effectiveness. Thus, repositioning of FDA approved drugs for anti-leishmanial therapeutics and encapsulation of the same in nanoliposomes may pave the way of future anti-leishmanial therapeutics. In this work, liposome encapsulated Halofantrine (Halolipo) was prepared by extrusion of hydrated lipid film method. The Halolipo was characterized by Dynamic Light Scattering and Transmission Electron Microscopy. The effect of Halolipo on Leishmania donovani Ag83 promastigotes was assessed by MTT assay and qualitative analysis of ROS generation and mitochondrial membrane depolarization was performed. The cell death was visualized by dual staining with Acridine orange and Ethidium bromide followed by fluorescence microscopy. The encapsulation of Halofantrine drug in liposome was confirmed by characteristic peak at 260 nm by UV–vis spectrophotometer. The formulated halolipo was 20 nm in size and stable for > 4 weeks at 4 °C. Maximum encapsulation was observed at a ratio of 1:200 (Lipid:Halofantrine) Halofantrine is released from Halolipo at both pH 7.2 and 5.5. Halolipo causes decrease in cell viability of L. donovani Ag83 promastigotes and leading to cellular stress as evident from increased ROS generation and mitochondrial membrane depolarization. These events culminate into cell death as observed by dual staining of L. donovani promastigotes by Acridine orange and Ethidium bromide. The novel Halolipo is a promising anti-leishmanial agent and may be used for future anti-leishmanial therapeutics.

Anti-leishmanial Nanotherapeutics: A Current Perspective.

Leishmaniasis is a dreaded disease caused by protozoan parasites belonging to the genus Leishmania which results in significant morbidity and mortality worldwide. There are no vaccines available currently for the treatment of Leishmaniasis and chemotherapy still remains the mainstay for anti-leishmanial therapeutics. However, toxicity, reduced bioavailability, high cost and chemoresistance are the principal problems which limit the use of the available drugs. In this context, anti-leishmanial nanotherapeutics may show the way for effective treatment of this dreaded disease. We carried out extensive literature search of bibliographic database using keywords strictly within the scope of the present study for peer reviewed research articles. We focused specifically on articles related to the application of nanotechnology in drug development, drug delivery and vaccine delivery for anti-leishmanial therapeutics. This study shows the immense potential of the application of nanotechnology in the field of anti-leishmanial therapeutics. This will aid the targeted delivery of different drugs which is expected to increase the bioavailability, reduce toxicity and also address the problem of chemoresistance. We surmise that exciting research in the field of anti-leishmanial nanotherapeutics is already showing the promise for effective applicability. Though direct use of nanoparticles as therapeutic agents does not seem to be a good option, the application of nanotechnology in this field for vaccine development is still in its early days. The nano based drug delivery system for anti-leishmanial therapeutics has evolved considerably over the past ten years and holds the potential to drastically change the landscape of anti-leishmanial therapeutics.

RETRACTED ARTICLE: Nucleic acid sensing activates the innate cytosolic surveillance pathway and promotes parasite survival in visceral leishmaniasis

Microbial pattern recognition critically contributes to innate response, both at extracellular and intracellular cytosolic surveillance pathway (CSP) interface. However, the role of pattern recognition by host innate receptors in CSP is poorly understood in Leishmania donovani infection. Here, we have demonstrated that cytosolic targeting of L.donovani DNA (Ld-DNA) inhibits macrophage responsiveness to IFNɣ, through decreased MHC-II expression and lowered pSTAT1 (Y701) levels, involving host three-prime repair exonuclease-1 (TREX-1). The Ld-DNA potently induced type-1 IFNs, i.e. significant over-production of IFNβ through activation of the IRF pathway. Interestingly, knockdown of TRIF or MyD88 expression in macrophages had no effect on cytosolic Ld-DNA transfection-mediated IFN-β production, indicating involvement of a TLR independent pathway. Contrastingly, Ld-DNA failed to induce IFNβ in both TBK-1 and IRF3KO knockout macrophages. Although IFNβ was not induced by Ld-DNA in STING- knockout macrophages, STING alone was not enough for the induction. Evidently, besides STING, Ld-DNA recognition for induction of IFNβ critically required cytosolic cyclic GMP-AMP synthase (cGAS). Furthermore, the cGAS dependent targeting of Ld-DNA induced IFNβ over-production that contributed to antimony resistance in L.donovani infection. We provide the first evidence that enhanced cytosolic sensing of Ld-DNA in infection by antimony resistant (SBR-LD), but not antimony sensitive L.donovani strains (SBS-LD), was critically regulated by host MDRs, multi drug resistant associated protein 1 (MRP 1) and permeability glycoprotein (P-gp) in macrophages. Collectively, our results disclose Ld-DNA as a vital pathogen associated molecular pattern (PAMP) driving host Type-I IFN responses and antimony resistance. The findings may help in future development of policies for novel anti-leishmanial therapeutics.

Computational elucidation of novel antagonists and binding insights by structural and functional analyses of serine hydroxymethyltransferase and interaction with inhibitors

The crystallographic structure of the Leishmania donovani serine hydroxymethyltransferase (LdSHMT), an attractive target for the design of leishmanicidal agents, is still unknown. Serine hydroxymethyltranferase (SHMT) is a key enzyme in the purine biosynthesis pathway, which is essential for the synthesis of DNA in leishmania parasite. Aim of this study is to find out the novel drug candidate against this parasitic protozoan. The present study describes the development of a robust homology model of LdSHMT to forecast interaction phenomenon with inhibitory molecules using structure-based drug designing strategy. Here we report a robust three-dimensional structure (98.4% of the residues were in the most favored region and additional allowed region) which consist of N terminal small domain (residues 1–46), N terminal large domain (residues 46–298) and C terminal domain (residues 299–457). The second N-terminal domain or the large domain (residues 46–298) binds PLP, has most of the active site residues and folds into α–β–α type sandwich containing eight α-helices wrapped around seven mixed β-sheets and third is the C-terminal domain (residues 299–457) folds into an α–β sandwich. Docking studies of diverse reported structures with modeled protein exhibited good binding affinities. Purvalanol, revealed a good binding affinity (−29.78) and residues involved in interaction are ASP208, Arg243, His236 and Lys237 forming hydrogen bonds, which signifies the presence of strong interaction with the target protein. Methotrexate-alpha-arginine and fluoresceinated have also exhibited good binding affinity −38.85 and −37.27 respectively. Pemetrexed, which has been earlier implicated in treatment of lung cancer, also exhibited a good binding affinity (−32.76).The residues which played a crucial role in the formation of strong H bonds are Ser102, Gly101, Arg243, Lys360, Asn361 and Lys 237. Apart from these molecules, several other molecules also showed strong interaction with LdSHMT. Overall, the present study is believed to provide valuable information to design a new compound with improved activity for antileishmanial therapeutics development.

Cross-resistance of Leishmania infantum isolates to nitric oxide from patients refractory to antimony treatment, and greater tolerance to antileishmanial responses by macrophages.

Visceral leishmaniasis is a life-threatening disease characterized by intense parasitism of the spleen, liver, and bone marrow. Antimonials have served as front-line antileishmanial therapeutics for decades, but the increasing failure rates under antimonial treatment have challenged the continued use of these drugs. Pentavalent antimonials are known to reinforce the killing mechanisms of macrophages, although the associated mechanism remains unclear. Here, for the first time, we determined whether Leishmania infantum strains isolated from patients refractory to antimony treatment (relapse cases) were cross-resistant to antimonials, liposomal amphotericin B, and/or nitric oxide, and also whether these strains modulate macrophage infection. We selected four clinical isolates from relapse cases and two clinical isolates from antimony-responsive patients (control group) for the present study. The L. infantum promastigotes from all four relapse cases were resistant to trivalent antimonial treatment and nitric oxide, while only one isolate was resistant to liposomal amphotericin B. We evaluated whether the resistant strains from relapse cases showed enhanced infectivity and amastigote survival in macrophages, or macrophage-killing mechanisms in macrophages activated by lipopolysaccharide plus interferon gamma. Infection indexes calculated using macrophages infected with isolates from relapse were higher than those observed with control strains that were stimulated independently. Macrophage infection was higher with L. infantum isolates from relapse cases and correlated with enhanced interleukin 1-β production but showed similar nitrite production. Our results demonstrate that L. infantum field isolates from relapse cases were resistant to antimonials and nitric oxide and that these parasites stimulated inflammatory cytokines and were resistant to macrophage-killing mechanisms, factors that may contribute to disease severity.

Computational Elucidation of Structural Basis for Ligand Binding withLeishmania donovaniAdenosine Kinase

Enzyme adenosine kinase is responsible for phosphorylation of adenosine to AMP and is crucial for parasites which are purine auxotrophs. The present study describes development of robust homology model of Leishmania donovani adenosine kinase to forecast interaction phenomenon with inhibitory molecules using structure-based drug designing strategy. Docking calculation using reported organic small molecules and natural products revealed key active site residues such as Arg131 and Asp16 for ligand binding, which is consistent with previous studies. Molecular dynamics simulation of ligand protein complex revealed the importance of hydrogen bonding with active site residues and solvent molecules, which may be crucial for successful development of drug candidates. Precise role of Phe168 residue in the active site was elucidated in this report that provided stability to ligand-protein complex via aromatic-π contacts. Overall, the present study is believed to provide valuable information to design a new compound with improved activity for antileishmanial therapeutics development.

Inhibitors of Leishmania GDP-Mannose Pyrophosphorylase Identified by High-Throughput Screening of Small-Molecule Chemical Library

The current treatment for leishmaniasis is based on chemotherapy, which relies on a handful of drugs with serious limitations, such as high cost, toxicity, and a lack of efficacy in regions of endemicity. Therefore, the development of new, effective, and affordable antileishmanial drugs is a global health priority. Leishmania synthesizes a range of mannose-rich glycoconjugates that are essential for parasite virulence and survival. A prerequisite for glycoconjugate biosynthesis is the conversion of monosaccharides to the activated mannose donor, GDP-mannose, the product of a reaction catalyzed by GDP-mannose pyrophosphorylase (GDP-MP). The deletion of the gene encoding GDP-MP in Leishmania led to a total loss of virulence, indicating that the enzyme is an ideal drug target. We developed a phosphate sensor-based high-throughput screening assay to quantify the activity of GDP-MP and screened a library containing approximately 80,000 lead-like compounds for GDP-MP inhibitors. On the basis of their GDP-MP inhibitory properties and chemical structures, the activities of 20 compounds which were not toxic to mammalian cells were tested against ex vivo amastigotes and in macrophage amastigote assays. The most potent compound identified in the primary screen (compound 3), a quinoline derivative, demonstrated dose-dependent activity in both assays (50% inhibitory concentration = 21.9 microM in the macrophage assay) and was shown to be nontoxic to human fibroblasts. In order to elucidate signs of an early structure-activity relationship (SAR) for this class of compounds, we obtained and tested analogues of compound 3 and undertook limited medicinal chemistry optimization, which included the use of a number of SAR probes of the piperazinyl aryl substituent of compound 3. We have identified novel candidate compounds for the design and synthesis of antileishmanial therapeutics.

Intracellular parasite kill: Flow cytometry and NO detection for rapid discrimination between anti-leishmanial activity and macrophage activation

Transgenic Leishmania expressing fluorescent reporter proteins such as green fluorescent protein (GFP) have opened the way for a flow cytometry (FACS)-based method to assess the killing of Leishmania parasites inside their macrophage host. Compared with counting parasites in microscopic preparations, the assessment of anti-leishmanial effects by FACS analysis promises both strict objectivity and significant reduction of labour-per-sample while scanning thousands of cells within seconds. Compared with other semi-automated methods based on host cell lysis and biochemical quantification of released parasites, the procedure is more direct and simple, reducing handling artefacts. An assay system is described using highly pure murine bone marrow-derived macrophages infected in vitro as a suspension culture with GFP-transfected Leishmania major promastigotes. The cells were rested for 24 h, allowing intracellular promastigotes to transform into amastigotes, and then exposed to macrophage-activating agents (IFN-γ, LPS) or standard anti-leishmanial therapeutics. Within 48 h, the GFP signal from parasitized macrophages became indiscernible by FACS analysis, both in activated host cells and in cultures treated with the anti-leishmanials. In cultures activated with rIFN-γ+LPS this coincided with the release of nitric oxides, but this was not the case in cultures treated with anti-leishmanials. Furthermore, by adding propidium iodide immediately before FACS analysis, the effect of treatment on the viability of the host cell was assessed at the same time. The combination of FACS analysis, and PI and NO detection offers a rapid and objective means of testing for intracellular anti-leishmanial effects and general cytotoxicity and gives a first indication of whether the former is due to direct leishmanicidal activity or indirect functions via macrophage activation.

A COOH-terminal domain regulates the activity of Leishmania proton pumps LDH1A and LDH1B

Leishmania donovani requires actively transporting proton efflux pumps to survive the acidic environment of macrophage phagolysosomal vacuoles and to maintain an electrogenic H + gradient for nutrient uptake. The L. donovani genome contains a differentially expressed pair of genes, LDH1 A and LDH1 B, with homology to yeast H +-ATPases that are 98% identical in sequence with amino acid differences concentrated at the COOH-terminus (15 of last 37 differ), a region implicated in regulation of yeast and plant proton pumps. Functional complementation of a Saccharomyces cerevisiae strain deficient in endogenous H +-ATPase activity, support of yeast growth at low pH, and ability to acidify media demonstrate that LDH1 A and LDH1 B encode proton pumps. LDH1 A and LDH1 B encode a COOH-terminal autoinhibitory domain as COOH-truncated peptides support increased rates of growth in yeast, enhanced media acidification, increased enzyme activity ( V max) and decreased K m. This regulatory domain mediates differing function properties; LDH1A, but not LDH1B, supports yeast growth at pH 3 and LDH1A shows a greater ability to acidify media. Deletion of the last eight amino acids from LDH1B permits growth at pH 3 and increases media acidification, swapping of the COOH-tails between LDH1A and LDH1B results in LDH1A (with LDH1B tail) unable to support yeast growth at pH 3 and LDH1B (with LDH1A tail) now able to support growth at pH 3. Replacement of the COOH-terminal eight amino acids of LDH1B with those from LDH1A also confers the ability to support growth at pH 3. The complementation system for the Leishmania proton pumps in yeast described here provides a means to dissect the functional properties of the two isoforms, a convenient supply of protein for structural analysis and a model amenable to screening proton pump inhibitors for potential anti-leishmanial therapeutics.