Visceral leishmaniasis (VL) is a major threat to economically challenged sectors of the world, and there are only limited options in vaccines available at present. Vaccination using live attenuated strains is preferred for their retention of true antigens, however, the major concern about these vaccines is raised by their ability to protect from virulent challenge at the early time point and also bearing the risk of a potential outbreak of their own. In this study, we reported a metabolically deficient glutamine synthetase double knock-out live attenuated strain of Leishmania donovani (LdGS-/-) that, with a very low inoculum single dose, is highly effective in generating an IFN-γ+ Th1-mediated immunity during virulent challenge as early as 2 weeks post-challenge time point. Additionally, at this early time point, immunization with these mutant parasites also led to the differentiation of CD4+ T effector and T memory cells in the spleens of BALB/c mice, providing long-term memory cell based protection from virulent LdGS+/+ (wild type) infection. The levels of expression of pro-inflammatory and T memory inducing and maintenance cytokines were also found to be higher in animals immunized with LdGS-/- parasites and challenged with the wild type parasites at even as high as the 8th week post-challenge. In contrast, there was no significant increase in these cytokines when immunized with the same number of LdGS+/+ and challenged with wild type parasites. Hence, our findings present that LdGS-/- parasites are novel and valuable for designing a potential live attenuated vaccine candidate against the virulent infection of L. donovani.

Treatment failure in visceral leishmaniasis (VL; kala-azar) is an increasing problem worldwide, primarily due to drug resistance. Pentavalent antimonials were the mainstay of treatment for decades. However, during the last 20 years, their use has been superseded by other drugs in the Indian subcontinent (ISC) due to the emergence of resistance. Miltefosine, the only oral drug, was registered to treat visceral leishmaniasis (VL) in 2002 in ISC. Soon after its introduction, the drug was considered a treatment of choice in the kala-azar elimination program in the ISC. However, reports of relapses and a decline in its efficacy have raised significant concerns in recent years. Currently, a single-dose regimen of liposomal amphotericin B is the first-line treatment for VL in India. Understanding the factors responsible for the development of drug resistance will help manage the disease, especially in endemic areas. Treatment outcome is governed by several factors related to host-parasite and drug kinetics. Host-related factors include host immunity or host nutritional status; for example, enhanced Th1-type response is crucial for effective treatment outcomes. Factors related to the drug, such as long half-life, inappropriate dosing, or self-administration of the drug, may lead to the exposure of parasites to the subtherapeutic dosage and thus be responsible for the selection of drug-resistant parasites. Similarly, several parasite-related factors impact the outcome of the treatment. Leishmania parasites can adapt to various environmental shifts, including exposure to the drug in the field. For example, parasite virulence, fitness to host immune responses, oxidative stress evasion, host defense mechanism modulation, metabolic adaptations, and other unknown factors contribute to drug susceptibility and treatment response. This chapter covers various factors contributing to declining efficacy and relapses following miltefosine treatment. Further, the genomic and transcriptomic alterations responsible for the phenotypic alterations in miltefosine-resistant parasites are discussed.

2-Arylquinazolines with a range of alkyl polyamines as side chain/ring functional motifs at the 4th-position were considered for antileishmanial study with the rationale that related heterocyclic scaffolds and polyamine functionalities are present in drugs, clinical trial agents, natural products and anti-parasitic/leishmanial agents. Synthesis involves construction of the 2-arylquinazolin-4-one ring and deoxyamination via deoxychlorination followed by SNAr-based amination or a methodology of SNAr-deoxyamination driven by BOP-mediated hydroxyl-activation. Various alkyl-polyamines important for activities were incorporated. A total of 26 compounds were prepared and screened against Leishmania donovani (Ld) promastigote cells using the MTT assay. Most of the investigated series of compounds showed characteristic leishmanicidal properties. Several compounds showed pronounced leishmanicidal activities (IC50: 5-6.5 μM) with higher efficiency than the antileishmanial drug miltefosine (IC50: 10.5 μM), and relatively less cytotoxicity to macrophage host cells (SI: 9.27-13.5) compared to miltefosine (SI: 3.42). Important pharmacophoric skeletons were identified.

In the absence of adequate diagnosis and treatment, leishmaniasis remains a major public health concern on a global scale. Drug resistance remains a key obstacle in controlling and eliminating visceral leishmaniasis. The therapeutic gap due to lack of target-specific medicine and vaccine can be minimized by obtaining parasite’s genomic information. This study compared whole-genome sequence of paromomycin-resistant parasite (K133PMM) developed through in vitro adaptation and selection with sensitive Leishmania clinical isolate (K133WT). We found a large number of upstream and intergenic gene variations in K133PMM. There were 259 single nucleotide polymorphisms (SNPs), 187 insertion-deletion (InDels), and 546 copy number variations (CNVs) identified. Most of the genomic variations were found in the gene’s upstream and non-coding regions. Ploidy estimation revealed chromosome 5 in tetrasomy and 6, 9, and 12 in trisomy, uniquely in K133PMM. These contain the genes for protein degradation, parasite motility, autophagy, cell cycle maintenance, and drug efflux membrane transporters. Furthermore, we also observed reduction in ploidy of chromosomes 15, 20, and 23, in the resistant parasite containing mostly the genes for hypothetical proteins and membrane transporters. We chronicled correlated genomic conversion and aneuploidy in parasites and hypothesize that this led to rapid evolutionary changes in response to drug induced pressure, which causes them to become resistant.

Glutamine synthetase (GS) is one of the most important metabolic enzymes which catalyzes ligation of glutamate and ammonia to form glutamine. Previous studies from our lab had revealed significant differences in parasite and host GS enzyme which warranted us to further work on its relevance in parasite. To analyze glutamine synthetase function in Leishmania, we generated GS overexpressors and knockout mutants and evaluated their ability to grow in vitro in monocyte differentiated macrophage and in vivo by infections in BALB/c mice. GS knocked out strain showed significant growth retardation with delayed cell cycle progression and morphological alteration. Null mutants exhibited attenuated infectivity both in in vitro and in vivo experiments and the effect was reverted back when infected with GS complemented parasites. This indicated that the alterations in phenotype observed were indeed due to GS knockout. GS knockout also made the parasite increasingly sensitive to Miltefosine. Detailed investigation of mode of parasite death upon Miltefosine treatment by dual staining with Annexin-V conjugated FITC and propidium iodide, pointed towards apoptotic or necrotic mode of cell death. This is the first report to confirm that GS is essential for the survivability and infectivity of Leishmania donovani, and can be exploited as a potential drug-target.

Current therapy for visceral leishmaniasis (VL), compromised by drug resistance, toxicity, and high cost, demands for more effective, safer, and low-cost drugs. Artemisinin has been found to be an effectual drug alternative in experimental models of leishmaniasis. Comparative genome and transcriptome analysis of in vitro-adapted artesunate-resistant (K133AS-R) and -sensitive wild-type (K133WT) Leishmania donovani parasites was carried out using next-generation sequencing and single-color DNA microarray technology, respectively, to identify genes and interlinked pathways contributing to drug resistance. Whole-genome sequence analysis of K133WT vs. K133AS-R parasites revealed substantial variation among the two and identified 240 single nucleotide polymorphisms (SNPs), 237 insertion deletions (InDels), 616 copy number variations (CNVs) (377 deletions and 239 duplications), and trisomy of chromosome 12 in K133AS-R parasites. Transcriptome analysis revealed differential expression of 208 genes (fold change ≥ 2) in K133AS-R parasites. Functional categorization and analysis of modulated genes of interlinked pathways pointed out plausible adaptations in K133AS-R parasites, such as (i) a dependency on lipid and amino acid metabolism for generating energy, (ii) reduced DNA and protein synthesis leading to parasites in the quiescence state, and (iii) active drug efflux. The upregulated expression of cathepsin-L like protease, amastin-like surface protein, and amino acid transporter and downregulated expression of the gene encoding ABCG2, pteridine receptor, adenylatecyclase-type receptor, phosphoaceylglucosamine mutase, and certain hypothetical proteins are concordant with genomic alterations suggesting their potential role in drug resistance. The study provided an understanding of the molecular basis linked to artemisinin resistance in Leishmania parasites, which may be advantageous for safeguarding this drug for future use.

Glutamine synthetase from L. donovani (LdGS) has been identified as a potential antileishmanial target in our previous report based on biochemical and inhibition studies. With the aim to structurally explore LdGS, systematic in silico and in vitro studies have been employed in the present study to identify amino acids crucial for LdGS mediated catalysis. A comparative analysis with human GS (HsGS) was performed which revealed significant differences in the active site pocket of human and parasite GS enzyme. The important amino acids identified from the in silico analysis of the optimized complexes, were subjected to in silico and in vitro alanine scanning by site directed mutagenesis. The results indicated crucial conserved and non conserved residues required for GS activity. The role of these residues in maintenance of secondary and tertiary structure of GS enzyme was also explored. In silico virtual screening was performed which resulted in the identification of five hits i.e. ZINC83236243, ZINC77319454, ZINC83236244, ZINC83236734 and ZINC83236736, as potential LdGS selective inhibitors. The illustrated structural and functional details of enzyme provides a better understanding of the structural integrity of LdGS and can be further utilized for the development of parasite specific GS inhibitors for treatment of visceral leishmaniasis infections.

Since inception, the magic bullets developed against leishmaniasis traveled a certain path and then dropped down due to either toxicity or the emergence of resistance. The route of administration is also an important concern. We developed a series of water-soluble ferrocenylquinoline derivatives, targeting Leishmania donovani, among which CQFC1 showed the highest efficacy even in comparison to other drugs, in use or used, both in oral and intramuscular routes. It did not induce any toxicity to splenocytes and on hematopoiesis, induced protective cytokines, and did not hamper the drug-metabolizing enzymes in hosts. It acts through the reduction and the inhibition of parasites' survival enzyme trypanothione reductase of replicating amastigotes in hosts' reticuloendothelial tissues. Unlike conventional drugs, this molecule did not induce the resistance-conferring genes in laboratory-maintained resistant L. donovani lines. Experimentally, this easily bioavailable preclinical drug candidate overcame all of the limitations causing the discontinuation of the other conventional antileishmanial drugs.

Pyridoxal kinase (PdxK, EC 2.7.1.35) is an important enzyme of vitamin B6 salvage pathway which is required for phosphorylation of B6 vitamers. In the present study, pyridoxal kinase (pdxK) gene from Leishmania donovani (LdPdxK) was cloned and a 33 kDa protein was expressed and kinetically characterized. Site-directed mutagenesis was performed to determine the functional significance of conserved GXGD motif. Mutation of Thr229 to Ala did not affect the catalytic function of LdPdxK however Gly228, Gly230 and Asp231 were found to be indispensible for enzyme activity. To determine the role of LdPdxK in Leishmania promastigotes, LdPdxK overexpressing parasites were generated by episomal expression of the enzyme. The overexpression studies revealed the role of this enzyme in growth and infection of the parasite. In silico analysis of the human and parasite PdxK structure revealed significant differences in the active site region thus highlighting its potential as an antileishmanial drug target. Homology model of LdPdxK was built and was subjected to molecular dynamics simulations. Based on the above information, a pharmacophore was developed and shape based virtual screening was performed to identify potential and selective inhibitors against this essential enzyme. The current data suggests that LdPdxK could be a promising antileishmanial drug target.

Moringa oleifera is grown globally. It is a multipurpose tree and the seeds are rich in phytochemicals with antimicrobial activities. The crude powder of seeds clarify the turbid and metal contaminated water. M. oleifera (drumstick tree) seed coagulant protein (MoCP) was isolated to homogeneity from the crude extracts by carboxymethyl cellulose chromatography (CMC) and gel filtration. The molecular weight of the protein on gel filtration was 13 kDa and in SDS-PAGE it migrated as a single band under reducing conditions with molecular mass of 6.5 kDa (dimeric). Immobilized MoCP selectively binds cadmium from aqueous solutions (pH 2.0-7.0) with maximum binding at pH 6.0 in 180 min when tested at 10-600 minutes. It also bound the metal in the concentration range of 30-70mgL(-1). The adsorption kinetics was better described by pseudo second order and the data better explained by freundlich isotherm model than Langmuir isotherm model as in Freundlich model the correlation coefficient (R-2) is high and the calculated q(max) is very close to the experimental q(max) rather than Langmuir isotherm model. Furthermore, the nanoparticles of MoCP were prepared and characterized using transmission electron microscopy (TEM). The authenticity of the isolated protein and the nanopraticles prepared was confirmed by specific reactivity with the MoCP antibody raised earlier in our laboratory.

Leishmaniasis is a group of tropical diseases caused by protozoan parasites of the genus Leishmania. Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis, a fatal disease if left untreated. Chemotherapy for leishmaniasis is problematic as the available drugs are toxic, costly and shows drug resistance, hence, there is a necessity to look out for the novel drug targets, chemical entities and vaccine. Glutamine synthetase (GS) catalyzes the synthesis of glutamine from glutamate and ammonia. In the present study, we have identified and characterized GS from L. donovani. The nucleotide sequence encoding putative glutamine synthetase like sequence from L. donovani (LdGS, LDBPK_060370) was cloned. A 43.5 kDa protein with 6X-His tag at the C-terminal end was obtained by overexpression of LdGS in Escherichia coli BL21 (DE3) strain. Expression of native LdGS in promastigotes and recombinant L donovani glutamine synthetase (rLdGS) was confirmed by western blot analysis. An increase in expression of GS was observed at different phases of growth of the parasite. Expression of LdGS in promastigote and amastigote was confirmed by western blot analysis. Immunofluorescence studies of both the promastigote and amastigote stages of the parasite revealed the presence of LdGS in cytoplasm. GS exists as a single copy gene in parasite genome. Kinetic analysis of GS enzyme revealed K-m value of 26.3 +/- 0.4 mM for L- glutamate and V-max value of 2.15 +/- 0.07 U mg(-1). Present study confirms the presence of glutamine synthetase in L donovani and provides comprehensive overview of LdGS for further validating it as a potential drug target. (C) 2017 Elsevier Ltd. All rights reserved.

In the present study, glycyrrhizic acid (GA) the main component of Glycyrrhiza glabra was evaluated for its efficacy as antileishmanial agent and its mode of action explored. GA inhibits promastigotes and intracellular amastigotes in a dose dependent manner at an IC50 value of 34 ± 3.0 μM and 20 ± 4.2 μM respectively. GA was non-toxic against THP-1 macrophage host cell line. GA was found to inhibit recombinant Leishmania donovani HMG-CoA reductase (LdHMGR) enzyme at the half-maximum inhibitory concentration of 24 ± 4.3 μM indicating the sensitivity and specificity of GA towards the enzyme. However, GA could cause only 30% reduction in HMGR activity when measured in Leishmania promastigotes treated with 34 μM of GA. Interestingly western blot analysis revealed fivefold reduced HMGR expression in GLA treated promastigotes. To further study the mode of action of GA, we used transgenic parasites overexpressing LdHMGR. Results indicated that ∼2 fold resistance was exhibited by LdHMGR overexpressing promastigotes to GA with an IC50 value of 74 μM compared to the wild type parasite. This explained the specific binding of GA to LdHMGR enzyme. There was ∼2 fold depletion in ergosterol levels in wild type promastigotes compared to the HMGR overexpressors. This data was further validated by exogenous supplementation of GA treated cells with ergosterol and 40% reversal of growth inhibition was observed. The results obtained suggested that GA kills the parasite by affecting sterol biosynthetic pathway, especially by inhibiting the L. donovani HMGR and altering ergosterol levels. The finding from the current study shows that GA is a potential antileishmanial chemotherapeutic agent.