Antileishmanial Activities of (Z)-2-(Nitroimidazolylmethylene)-3(2H)-Benzofuranones: Synthesis, In Vitro Assessment, and Bioactivation by NTR 1 and 2.

The antileishmanial activity of a series of (Z)-2-(heteroarylmethylene)-3(2H)-benzofuranone derivatives, possessing 5-nitroimidazole or 4-nitroimidazole moieties, was investigated against Leishmania major promastigotes and some analogues exhibited prominent activities. Compounds with IC50 values lower than 20 µM were further examined against L. donovani axenic amastigotes. Evaluated analogues in 5-nitroimidazole subgroup demonstrated significantly superior activity (~17-88-folds) against L. donovani in comparison to L. major. (Z)-7-Methoxy-2-(1-methyl-5-nitroimidazole-2-ylmethylene)-3(2H)-benzofuranone (5n) showed the highest L. donovani anti-axenic amastigote activity with IC50 of 0.016 µM. The cytotoxicity of these analogues was determined using PMM peritoneal mouse macrophage and THP-1 human leukemia monocytic cell lines and high selectivity indices of 26 to 431 were obtained for their anti-axenic amastigote effect over the cytotoxicity on PMM cells. Further studies on their mode of action showed that 5-nitroimidazole compounds were bioactivated predominantly by nitroreductase 1 (NTR1) and 4-nitroimidazole analogues by both NTR1 and 2. It is likely that this bioactivation results in the production of nitroso and hydroxylamine metabolites that are cytotoxic for the Leishmania parasite.

Bidirectional cytokine-microRNA control: A novel immunoregulatory framework in leishmaniasis.

As effector innate immune cells and as a host to the protozoan parasite Leishmania, macrophages play a dual role in antileishmanial immunoregulation. The 2 key players in this immunoregulation are the macrophage-expressed microRNAs (miRNAs) and the macrophage-secreted cytokines. miRNAs, as small noncoding RNAs, play vital roles in macrophage functions including cytokines and chemokines production. In the reverse direction, Leishmania-regulated cytokines alter miRNAs expression to regulate the antileishmanial functions of macrophages. The miRNA patterns vary with the time and stage of infection. The cytokine-regulated macrophage miRNAs not only help parasite elimination or persistence but also regulate cytokine production from macrophages. Based on these observations, we propose a novel immunoregulatory framework as a scientific rationale for antileishmanial therapy.

Energy metabolism as a target for cyclobenzaprine: A drug candidate against Visceral Leishmaniasis.

Leishmaniases have a broad spectrum of clinical manifestations, ranging from a cutaneous to a progressive and fatal visceral disease. Chemotherapy is nowadays the almost exclusive way to fight the disease but limited by its scarce therapeutic arsenal, on its own compromised by adverse side effects and clinical resistance. Cyclobenzaprine (CBP), an FDA-approved oral muscle relaxant drug has previously demonstrated in vitro and in vivo activity against Leishmania sp., but its targets were not fully unveiled. This study aimed to define the role of energy metabolism as a target for the leishmanicidal mechanisms of CBP. Methodology to assess CBP leishmanicidal mechanism variation of intracellular ATP levels using living Leishmania transfected with a cytoplasmic luciferase. Induction of plasma membrane permeability by assessing depolarization with DiSBAC(2)3 and entrance of the vital dye SYTOX® Green. Mitochondrial depolarization by rhodamine 123 accumulation. Mapping target site within the respiratory chain by oxygen consumption rate. Reactive oxygen species (ROS) production using MitoSOX. Morphological changes by transmission electron microscopy. CBP caused on L. infantum promastigotes a decrease of intracellular ATP levels, with irreversible depolarization of plasma membrane, the collapse of the mitochondrial electrochemical potential, mild uncoupling of the respiratory chain, and ROS production, with ensuing intracellular Ca2+ imbalance and DNA fragmentation. Electron microscopy supported autophagic features but not a massive plasma membrane disruption. The severe and irreversible mitochondrial damage induced by CBP endorsed the bioenergetics metabolism as a relevant target within the lethal programme induced by CBP in Leishmania. This, together with the mild-side effects of this oral drug, endorses CBP as an appealing novel candidate as a leishmanicidal drug under a drug repurposing strategy.

In vitro activity and cell death mechanism induced by acrylonitrile derivatives against Leishmania amazonensis.

Leishmaniasis produces approximately-one million of new cases annually, making it one of the most important tropical diseases. As current treatments are not fully effective and are toxic, it is necessary to develop new therapies that are more effective and less toxic, and cause a controlled cell death, with which we can avoid the immunological problems caused by necrosis. In this work 32 acrylonitriles were studied in vitro against Leishmania amazonensis. Three compounds Q20 (12.41), Q29 (11.2) and Q31 (11.56) had better selectivity than the reference compound, miltefosine (11.14) against promastigotes of these parasites, for this reason they were selected to determine their mechanism of action to know the cell death type of they produce. The results of the mechanisms of action show that these three acrylonitriles tested produce chromatin condensation, decreased mitochondrial membrane potential, altered plasma permeability and production of reactive oxygen species. All these characteristic events seem to indicate programmed cell death. Therefore, this study demonstrates the activity of acrylonitriles derivatives as possible leishmanicidal agents.

Multiple mutations in RNA polymerase ß-subunit gene (rpoB) in Streptomyces incarnatus NRRL8089 enhance production of antiviral antibiotic sinefungin: modeling rif cluster region by density functional theory.

Streptomyces incarnatus NRRL8089 produces the antiviral, antifungal, antiprotozoal nucleoside antibiotic sinefungin. To enhance sinefungin production, multiple mutations were introduced to the rpoB gene encoding RNA polymerase (RNAP) ß-subunit at the target residues, D447, S453, H457, and R460. Sparse regression analysis using elastic-net lasso-ridge penalties on previously reported H457X mutations identified a numeric parameter set, which suggested that H457R/Y/F may cause production enhancement. H457R/R460C mutation successfully enhanced the sinefungin production by 3-fold, while other groups of mutations, such as D447G/R460C or D447G/H457Y, made moderate or even negative effects. To identify why the rif cluster residues have diverse effects on sinefungin production, an RNAP/DNA/mRNA complex model was constructed by homology modeling and molecular dynamics simulation. The 4 residues were located near the mRNA strand. Density functional theory-based calculation suggested that D447, H457, and R460 are in direct contact with ribonucleotide, and partially positive charges are induced by negatively charged chain of mRNA.

Potential therapeutic targets shared between leishmaniasis and cancer.

The association of leishmaniasis and malignancies in human and animal models has been highlighted in recent years. The misdiagnosis of coexistence of leishmaniasis and cancer and the use of common drugs in the treatment of such diseases prompt us to further survey the molecular biology of Leishmania parasites and cancer cells. The information regarding common expressed proteins, as possible therapeutic targets, in Leishmania parasites and cancer cells is scarce. Therefore, the current study reviews proteins, and investigates the regulation and functions of several key proteins in Leishmania parasites and cancer cells. The up- and down-regulations of such proteins were mostly related to survival, development, pathogenicity, metabolic pathways and vital signalling in Leishmania parasites and cancer cells. The presence of common expressed proteins in Leishmania parasites and cancer cells reveals valuable information regarding the possible shared mechanisms of pathogenicity and opportunities for therapeutic targeting in leishmaniasis and cancers in the future.

'Targeting the feast of a sleeping beast': Nutrient and mineral dependencies of encysted Acanthamoeba castellanii.

Acanthamoeba spp. cause a corneal infection, Acanthamoeba keratitis (AK), and a cerebral infection, granulomatous amoebic encephalitis (GAE). Though aggressive chemotherapy has been able to kill the active trophozoite form of Acanthamoeba, the encysted form of this parasite has remained problematic to resist physiological concentrations of drugs. The emergence of encysted amoeba into active trophozoite form poses a challenge to eradicate this parasite. Acanthamoeba trophozoites have active metabolic machinery that furnishes energy in the form of ATPs by subjecting carbohydrates and lipids to undergo pathways including glycolysis and beta-oxidation of free fatty acids, respectively. However, very little is known about the metabolic preferences and dependencies of an encysted trophozoite on minerals or potential nutrients that it consumes to live in an encysted state. Here, we investigate the metabolic and nutrient preferences of the encysted trophozoite of Acanthamoeba castellanii and the possibility to target them by drugs that act on calcium ion dependencies of the encysted amoeba. The experimental assays, immunostaining coupled with bioinformatics tools show that the encysted Acanthamoeba uses diverse nutrient pathways to obtain energy in the quiescent encysted state. These findings highlight potential pathways that can be targeted in eradicating amoebae cysts successfully.

Identification and characterization of the hemoglobin-binding domain of hemoglobin receptor in Leishmania.

Leishmania internalize hemoglobin (Hb) via a specific receptor (HbR) for their survival. To identify the Hb-binding domain of HbR, we cloned and expressed several truncated proteins of HbR and determined their ability to bind Hb. Our findings reveal that 90% of Hb-binding activity is retained in HbR41-80 in comparison with HbR1-471 . We synthesized a 40 amino acid peptide (SSEKMKQLTMYMIHEMVEGLEGRPSTVRMLPSFVYTSDPA) corresponding to HbR41-80 and found that it specifically binds Hb. Subsequently, we found that the HbR41-80 peptide completely blocks Hb uptake in both promastigote and amastigote forms of Leishmania and, thereby, inhibits the growth of the parasite. These results demonstrate that HbR41-80 is the Hb-binding domain of HbR, which might be used as a potential therapeutic agent to inhibit the growth of Leishmania.

Oral delivery and enhanced efficacy of antimonal drug through macrophage-guided multifunctional nanocargoes against visceral Leishmaniasis.

The present study aimed on the site specific delivery and enhanced in-vivo efficacy of antimonial drugs against the visceral leishmaniasis via macrophage targeted mannose anchored thiomer based nanoparticles. Mannose anchored thiolated nanoformulation [M-(CS-g-PEI)-TGA] was developed and evaluated in terms particle size, zeta-potential and entrapment efficacy. The TEM and EDX analysis was carried out to evaluate the morphology and successful entrapment of antimonial drug. Mucodhesion, permeation enhancement, oral pharmacokinetics, and in-vivo anti-leishmanial activity were carried out. The M-(CS-g-PEI)-TGA were found to be spherical having particle size of 287 ± 20 nm. Ex-vivo permeation indicated a 7.39-fold enhanced permeation of Meglumine Antimoniate with M-(CS-g-PEI)-TGA across Caco-2 cells compared to the Glucantime. Evaluation of in-vitro reduction in the parasitic burden via flow cytometric analysis indicated a 5.7-fold lower IC50 for M-(CS-g-PEI)-TGA compared to Glucantime. A 6.1-fold improvement in the oral bioavailability and 5.2-fold reduced parasitic burden in the L. donovani infected BALB/c mice model was observed with M-(CS-g-PEI)-TGA compared to Glucantime. The results encouraged the concept of M-(CS-g-PEI)-TGA nanoformulations as a promising strategy for oral therapy against visceral leishmaniasis.

Computationally designed synthetic peptides for transporter proteins imparts allostericity in Miltefosine resistant L. major.

The emergence of drug resistance is a major concern for combating against Cutaneous Leishmaniasis, a neglected tropical disease affecting 98 countries including India. Miltefosine is the only oral drug available for the disease and Miltefosine transporter proteins play a pivotal role in the emergence of drug-resistant Leishmania major. The cause of resistance is less accumulation of drug inside the parasite either by less uptake of the drug due to a decrease in the activity of P4ATPase-CDC50 complex or by increased efflux of the drug by P-glycoprotein (P-gp, an ABC transporter). In this paper, we are trying to allosterically modulate the behavior of resistant parasite (L. major) towards its sensitivity for the existing drug (Miltefosine, a phosphatidylcholine analog). We have used computational approaches to deal with the conservedness of the proteins and apparently its three-dimensional structure prediction through ab initio modeling. Long scale membrane-embedded molecular dynamics simulations were carried out to study the structural interaction and stability. Parasite-specific motifs of these proteins were identified based on the machine learning technique, against which a peptide library was designed. The protein-peptide docking shows good binding energy of peptides Pg5F, Pg8F and PC2 with specific binding to the motifs. These peptides were tested both in vitro and in vivo, where Pg5F in combination with PC2 showed 50-60% inhibition in resistant L. major's promastigote and amastigote forms and 80-90% decrease in parasite load in mice. We posit a model system wherein the data provide sufficient impetus for being novel therapeutics in order to counteract the drug resistance phenotype in Leishmania parasites.

Prenylquinones in Human Parasitic Protozoa: Biosynthesis, Physiological Functions, and Potential as Chemotherapeutic Targets.

Human parasitic protozoa cause a large number of diseases worldwide and, for some of these diseases, there are no effective treatments to date, and drug resistance has been observed. For these reasons, the discovery of new etiological treatments is necessary. In this sense, parasitic metabolic pathways that are absent in vertebrate hosts would be interesting research candidates for the identification of new drug targets. Most likely due to the protozoa variability, uncertain phylogenetic origin, endosymbiotic events, and evolutionary pressure for adaptation to adverse environments, a surprising variety of prenylquinones can be found within these organisms. These compounds are involved in essential metabolic reactions in organisms, for example, prevention of lipoperoxidation, participation in the mitochondrial respiratory chain or as enzymatic cofactors. This review will describe several prenylquinones that have been previously characterized in human pathogenic protozoa. Among all existing prenylquinones, this review is focused on ubiquinone, menaquinone, tocopherols, chlorobiumquinone, and thermoplasmaquinone. This review will also discuss the biosynthesis of prenylquinones, starting from the isoprenic side chains to the aromatic head group precursors. The isoprenic side chain biosynthesis maybe come from mevalonate or non-mevalonate pathways as well as leucine dependent pathways for isoprenoid biosynthesis. Finally, the isoprenic chains elongation and prenylquinone aromatic precursors origins from amino acid degradation or the shikimate pathway is reviewed. The phylogenetic distribution and what is known about the biological functions of these compounds among species will be described, as will the therapeutic strategies associated with prenylquinone metabolism in protozoan parasites.

In vitro assessment of 3-alkoxy-5-nitroindazole-derived ethylamines and related compounds as potential antileishmanial drugs.

Leishmaniasis is a widespread neglected tropical disease complex that is responsible of one million new cases per year. Current treatments are outdated and pose many problems that new drugs need to overcome. With the goal of developing new, safe, and affordable drugs, we have studied the in vitro activity of 12 different 5-nitroindazole derivatives that showed previous activity against different strains of Trypanosoma cruzi in a previous work. T. cruzi belongs to the same family as Leishmania spp., and treatments for the disease it produces also needs renewal. Among the derivatives tested, compounds 1, 2, 9, 10, 11, and 12 showed low J774.2 macrophage toxicity, while their effect against both intracellular and extracellular forms of the studied parasites was higher than the ones found for the reference drug Meglumine Antimoniate (Glucantime®). In addition, their Fe-SOD inhibitory effect, the infection rates, metabolite alteration, and mitochondrial membrane potential of the parasites treated with the selected drugs were studied in order to gain insights into the action mechanism, and the results of these tests were more promising than those found with glucantime, as the leishmanicidal effect of these new drug candidates was higher. The promising results are encouraging to test these derivatives in more complex studies, such as in vivo studies and other experiments that could find out the exact mechanism of action.

Biosynthesis of anti-leishmanial natural products in callus cultures of Artemisia scoparia.

Clinically, available synthetic chemotherapeutics in the treatment for leishmaniasis are associated with serious complications, such as toxicity and emergence of resistance. Natural products from plants can provide better remedies against the Leishmania parasite and can possibly minimize the associated side effects. In this study, various extracts of the callus cultures of Artimisia scoparia established in response to different plant growth regulators (PGRs) were evaluated for their anti-leishmanial effects against Leishmania tropica promastigotes, followed by an investigation of the possible mechanism of action through reactive apoptosis assay using fluorescent microscopy. Amongst the different callus extracts, higher anti-leishmanial activity (IC50:19.13 µg/mL) was observed in the callus raised in-vitro in the presence of 6-Benzylaminopurine (BA) plus 2,4-Dichlorophenoxyacetic Acid (2,4-D) at the concentration of 1.5 mg/L, each. Further, the results of apoptosis assay showed a large number of early-stage apoptotic (EA) and late-stage apoptotic (LA) cells in the Leishmania under the effect of callus extract grown in-vitro at BA plus 2,4-D. For the determination of the potent natural products in the callus extracts responsible for the anti-leishmanial activity, extracts were subjected to Gas chromatography-mass spectrometry (GC-MS) for the metabolite analysis. Nonetheless, higher levels of the metabolites, such as nerolidol (22%), pelletierine (18%), aspidin (15%) and ascaridole (11%) were detected in the callus grown in vitro at BA plus 2,4-D (1.5 mg/L, each). This protocol determines a novel method of production of anti-leishmanial natural products through callus cultures of A. scoparia, a medicinal plant.

Inhibitor-Induced Dimerization of an Essential Oxidoreductase from African Trypanosomes.

Trypanosomal and leishmanial infections claim tens of thousands of lives each year. The metabolism of these unicellular eukaryotic parasites differs from the human host and their enzymes thus constitute promising drug targets. Tryparedoxin (Tpx) from Trypanosoma brucei is the essential oxidoreductase in the parasite's hydroperoxide-clearance cascade. In vitro and in vivo functional assays show that a small, selective inhibitor efficiently inhibits Tpx. With X-ray crystallography, SAXS, analytical SEC, SEC-MALS, MD simulations, ITC, and NMR spectroscopy, we show how covalent binding of this monofunctional inhibitor leads to Tpx dimerization. Intra- and intermolecular inhibitor-inhibitor, protein-protein, and inhibitor-protein interactions stabilize the dimer. The behavior of this efficient antitrypanosomal molecule thus constitutes an exquisite example of chemically induced dimerization with a small, monovalent ligand that can be exploited for future drug design.

Sodium stibogluconate loaded nano-deformable liposomes for topical treatment of leishmaniasis: macrophage as a target cell.

Topical drug delivery against cutaneous leishmaniasis (CL) signifies an effective alternate for improving the availability and reducing the toxicity associated with the parenteral administration of conventional sodium stibogluconate (SSG) injection. The basic aim of the study was to develop nano-deformable liposomes (NDLs) for the dermal delivery of SSG against CL. NDLs were formulated by a modified thin film hydration method and optimized via Box-Behnken statistical design. The physicochemical properties of SSG-NDLs were established in terms of vesicle size (195.1 nm), polydispersity index (0.158), zeta potential (-32.8 mV), and entrapment efficiency (35.26%). Moreover, deformability index, in vitro release, and macrophage uptake studies were also accomplished. SSG-NDLs were entrapped within Carbopol gel network for the ease of skin application. The ex vivo skin permeation study revealed that SSG-NDLs gel provided 10-fold higher skin retention towards the deeper skin layers, attained without use of classical permeation enhancers. Moreover, in vivo skin irritation and histopathological studies verified safety of the topically applied formulation. Interestingly, the cytotoxic potential of SSG-NDLs (1.3 mg/ml) was higher than plain SSG (1.65 mg/ml). The anti-leishmanial activity on intramacrophage amastigote model of Leishmania tropica showed that IC50 value of the SSG-NDLs was ∼ fourfold lower than the plain drug solution with marked increase in the selectivity index. The in vivo results displayed higher anti-leishmanial activity by efficiently healing lesion and successfully reducing parasite burden. Concisely, the outcomes indicated that the targeted delivery of SSG could be accomplished by using topically applied NDLs for the effective treatment of CL.

Discovery of benzimidazole-based Leishmania mexicana cysteine protease CPB2.8ΔCTE inhibitors as potential therapeutics for leishmaniasis.

Chemotherapy is currently the only effective approach to treat all forms of leishmaniasis. However, its effectiveness is severely limited due to high toxicity, long treatment length, drug resistance, or inadequate mode of administration. As a consequence, there is a need to identify new molecular scaffolds and targets as potential therapeutics for the treatment of this disease. We report a small series of 1,2-substituted-1H-benzo[d]imidazole derivatives (9a-d) showing affinity in the submicromolar range (Ki  = 0.15-0.69 µM) toward Leishmania mexicanaCPB2.8ΔCTE, one of the more promising targets for antileishmanial drug design. The compounds confirmed activity in vitro against intracellular amastigotes of Leishmania infantum with the best result being obtained with derivative 9d (IC50  = 6.8 µM), although with some degree of cytotoxicity (CC50  = 8.0 µM on PMM and CC50  = 32.0 µM on MCR-5). In silico molecular docking studies and ADME-Tox properties prediction were performed to validate the hypothesis of the interaction with the intended target and to assess the drug-likeness of these derivatives.

Combined Approach of Backbone Amide Linking and On-Resin N-Methylation for the Synthesis of Bioactive and Metabolically Stable Peptides.

Rhabdopeptides are a large class of nonribosomal peptides from the bacteria Xenorhabdus and Photorhabdus with low micromolar activity against different protozoa, which are the causative agents of several tropical diseases. The development of a facile and flexible synthesis combining backbone amide linking with on-resin peralkylation for the synthesis of permethylated rhabdopeptides is described. This strategy allows the fast generation of permethylated naturally occurring and artificial rhabdopeptides for a structure-activity study. Furthermore, in vitro experiments revealed their superior properties regarding their stability and passive membrane diffusion.

Decoding the network of Trypanosoma brucei proteins that determines sensitivity to apolipoprotein-L1.

In contrast to Trypanosoma brucei gambiense and T. b. rhodesiense (the causative agents of human African trypanosomiasis), T. b. brucei is lysed by apolipoprotein-L1 (apoL1)-containing human serum trypanolytic factors (TLF), rendering it non-infectious to humans. While the mechanisms of TLF1 uptake, apoL1 membrane integration, and T. b. gambiense and T. b. rhodesiense apoL1-resistance have been extensively characterised, our understanding of the range of factors that drive apoL1 action in T. b. brucei is limited. Selecting our bloodstream-form T. b. brucei RNAi library with recombinant apoL1 identified an array of factors that supports the trypanocidal action of apoL1, including six putative ubiquitin modifiers and several proteins putatively involved in membrane trafficking; we also identified the known apoL1 sensitivity determinants, TbKIFC1 and the V-ATPase. Most prominent amongst the novel apoL1 sensitivity determinants was a putative ubiquitin ligase. Intriguingly, while loss of this ubiquitin ligase reduces parasite sensitivity to apoL1, its loss enhances parasite sensitivity to TLF1-dominated normal human serum, indicating that free and TLF1-bound apoL1 have contrasting modes-of-action. Indeed, loss of the known human serum sensitivity determinants, p67 (lysosomal associated membrane protein) and the cathepsin-L regulator, 'inhibitor of cysteine peptidase', had no effect on sensitivity to free apoL1. Our findings highlight a complex network of proteins that influences apoL1 action, with implications for our understanding of the anti-trypanosomal action of human serum.

Leishmania and other intracellular pathogens: selectivity, drug distribution and PK-PD.

New drugs and treatments for diseases caused by intracellular pathogens, such as leishmaniasis and the Leishmania species, have proved to be some of the most difficult to discover and develop. The focus of discovery research has been on the identification of potent and selective compounds that inhibit target enzymes (or other essential molecules) or are active against the causative pathogen in phenotypic in vitro assays. Although these discovery paradigms remain an essential part of the early stages of the drug R & D pathway, over the past two decades additional emphasis has been given to the challenges needed to ensure that the potential anti-infective drugs distribute to infected tissues, reach the target pathogen within the host cell and exert the appropriate pharmacodynamic effect at these sites. This review will focus on how these challenges are being met in relation to Leishmania and the leishmaniases with lessons learned from drug R & D for other intracellular pathogens.