Leishmania donovani 6-phosphogluconolactonase: Crucial for growth and host infection?

The hexose monophosphate shunt is a crucial pathway in a variety of microorganisms owing to its vital metabolic products and intermediates such as NADPH, ribose 5-phosphate etc. The enzyme 6-phosphogluconolactonase catalyses the second step of this pathway, converting 6-phosphogluconolactone to 6-phosphogluconic acid. This enzyme has been known to have a significant involvement in growth, pathogenesis and sensitivity to oxidative stress in bacterial and protozoal pathogens. However, the functional role of kinetoplastid Leishmania donovani 6-phospohogluconolactonase (Ld6PGL) remains unexplored. L. donovani is the second largest parasitic killer and causative organism of life threatening visceral leishmaniasis. To understand its possible functional role in the parasite, the alleles of Ld6PGL were sequentially knocked-out followed by gene complementation. The Ld6PGL mutant cell lines showed decrease in transcriptional and translational expression as well as in the enzyme activity. In case of Ld6PGL null mutants, approximately 2-fold reduction was observed in growth. The null mutants also showed ∼38% decrease in infectivity, which recovered to ∼15% on complementation. Scanning electron microscopy showed a marked decrease in flagellar length in the knockout parasites. When treated with the standard drug miltefosine, the mutant strains had no significant change in the drug sensitivity. However, the Ld6PGL mutants were more susceptible to oxidative stress. Our findings suggest that 6PGL is required for parasite growth and infection, but it is not essential.

Molecular Docking and Dynamic Simulation to Identify α7nAChR Binding Affinity of Flavonoids for the Treatment of Alzheimer's Disease.

BACKGROUND: Alpha-7-nicotinic acetylcholine receptor (α7nAChR), a ligand-gated ion channel is one of the important parts of the cholinergic pathway in the brain and has a remarkable role in Alzheimer's disease (AD). It has been documented that the modulation of α7nAChR with the help of phytoconstituent can be helpful in the treatment of AD. METHOD: The binding efficacy of fifty flavonoids was evaluated for human α7nAChR using molecular docking. The best two flavonoids shortlisted from docking analysis were then subjected to molecular dynamic simulations for 100 ns to analyze conformational binding stability with the target protein. Further, the druggability of the selected flavonoids was checked using in silico ADMET studies. RESULT: The top two flavonoids selected based on binding affinity toward the binding site of α7nAChR from molecular docking were amentoflavone (-9.1 kcal/mol) and gallocatechin (-8.8 kcal/mol). The molecular dynamics simulation revealed that amentoflavone and gallocatechin have a stable state during overall simulation time, lesser root mean deviation (RMSD) and root mean square fluctuation (RMSF), and complex of both compounds with protein is stable until 100 ns. CONCLUSION: The two flavonoids amentoflavone and gallocatechin are potential lead molecules that could be utilized as effective agonists of α7nAChR to combat Alzheimer's disease. Future in vitro and in vivo analyses are required to confirm their effectiveness.

Targeting Parallel Topology of G-Quadruplex Structures by Indole- Fused Quindoline Scaffolds.

Preferential stabilization of G-quadruplex (G4) structures using small-molecule ligands has emerged as an effective approach to develop anticancer drugs. Herein, we report the synthesis of three indole-fused quindoline derivatives with varying lengths of side chains (InqEt1, InqEt2, and InqPr2) as selective ligands for promoter G4 structures. The ligands stabilize the parallel topology of c-MYC and c-KIT1 promoter G4 DNAs over telomeric and duplex DNAs, as evident from the circular dichroism melting and polymerase stop-assay experiments. The lead ligand, InqPr2, downregulates the gene expression of c-MYC and c-KIT in HeLa and HepG2 cells, respectively, leading to apoptotic cell death. Molecular modeling and dynamics studies support the 2:1 binding stoichiometry revealed from the Job plot analysis and show the ligand's structural features that enable the preferential binding to the parallel G4 structures over other topologies. Our studies show that indole-fused quindoline derivatives can be harnessed as new molecular scaffolds for selective targeting of parallel G4 topologies.

Autosomal dominant non-syndromic hearing loss maps to DFNA33 (13q34) and co-segregates with splice and frameshift variants in ATP11A, a phospholipid flippase gene.

Sequencing exomes/genomes have been successful for identifying recessive genes; however, discovery of dominant genes including deafness genes (DFNA) remains challenging. We report a new DFNA gene, ATP11A, in a Newfoundland family with a variable form of bilateral sensorineural hearing loss (SNHL). Genome-wide SNP genotyping linked SNHL to DFNA33 (LOD = 4.77), a locus on 13q34 previously mapped in a German family with variable SNHL. Whole-genome sequencing identified 51 unremarkable positional variants on 13q34. Continuous clinical ascertainment identified several key recombination events and reduced the disease interval to 769 kb, excluding all but one variant. ATP11A (NC_000013.11: chr13:113534963G>A) is a novel variant predicted to be a cryptic donor splice site. RNA studies verified in silico predictions, revealing the retention of 153 bp of intron in the 3' UTR of several ATP11A isoforms. Two unresolved families from Israel were subsequently identified with a similar, variable form of SNHL and a novel duplication (NM_032189.3:c.3322_3327+2dupGTCCAGGT) in exon 28 of ATP11A extended exon 28 by 8 bp, leading to a frameshift and premature stop codon (p.Asn1110Valfs43Ter). ATP11A is a type of P4-ATPase that transports (flip) phospholipids from the outer to inner leaflet of cell membranes to maintain asymmetry. Haploinsufficiency of ATP11A, the phospholipid flippase that specially transports phosphatidylserine (PS) and phosphatidylethanolamine (PE), could leave cells with PS/PE at the extracellular side vulnerable to phagocytic degradation. Given that surface PS can be pharmaceutically targeted, hearing loss due to ATP11A could potentially be treated. It is also likely that ATP11A is the gene underlying DFNA33.

Stabilization and fluorescence light-up of G-quadruplex nucleic acids using indolyl-quinolinium based probes.

G-Quadruplexes (G4s) are four-stranded motifs formed by G-rich nucleic acid sequences. These structures harbor significant biological importance as they are involved in telomere maintenance, transcription, and translation. Owing to their dynamic and polymorphic nature, G4 structures relevant for therapeutic applications need to be stabilized by small-molecule ligands. Some of these ligands turn on fluorescence upon binding to G4 structures, which provides a powerful detection platform for G4 structures. Herein, we report the synthesis of fluorescent ligands based on the indolyl-quinolinium moiety to specifically stabilize G4 structures and sense DNA. CD titration and melting experiments have shown that the lead ligand induces the formation of parallel G4 with preferential stabilization of the c-MYC and c-KIT1 promoter G4s over the telomeric, h-RAS1 G4, and duplex DNA. Fluorimetric titration data revealed fluorescence enhancement when these ligands interact with G4 DNA structures. The fluorescence lifetime experiment of the ligand with different DNAs revealed three excited state lifetimes (ns), which indicates more than one binding site. MD studies showed that the ligand exhibits 3 : 1 stoichiometry of binding with c-MYC G4 DNA and revealed the unique structural features, which impart selectivity toward parallel topology. The ligand was found to have low cytotoxicity and exhibited preferential staining of DNA over RNA. Collectively, the results presented here offer avenues to harness indolyl-quinolinium scaffolds for sensing and selective stabilization of G4 structures.

Synthesis of N-substituted indole derivatives as potential antimicrobial and antileishmanial agents.

Leishmaniasis and microbial infections are two of the major contributors to global mortality and morbidity rates. Hence, development of novel, effective and safer antileishmanial and antimicrobial agents having reduced side effects are major priority for researchers. Two series of N-substituted indole derivatives i.e. N-substituted indole based chalcones (12a-g) and N-substituted indole based hydrazide-hydrazones (18a-g, 19a-f, 21 a-g) were synthesized. The synthesized compounds were characterized by 1H NMR, 13C NMR, Mass and FT-IR spectral data. Further these derivatives were evaluated for their antimicrobial potential against Escherichia coli, Bacillus subtilis, Pseudomonas putida and Candida viswanathii, and antileishmanial potential against promastigotes of Leishmania donovani. Compounds 18b, 18d and 19d exhibited significant activity with an IC50 of 0.19 ± 0.03 µM, 0.14 ± 0.02 µM and 0.16 ± 0.06 µM against B. subtilis which was comparable to chloramphenicol (IC50 of 0.25 ± 0.03 µM). Compounds 12b and 12c exhibited an IC50 of 24.2 ± 3.5 µM and 21.5 ± 2.1 µM in the antileishmanial assay. Binding interactions of indole based hydrazide-hydrazones were studied with nitric oxide synthase in silico in order to understand the structural features responsible for activity.

Acetylcholinesterase and Aß Aggregation Inhibition by Heterometallic Ruthenium(II)-Platinum(II) Polypyridyl Complexes.

Two heteronuclear ruthenium(II)-platinum(II) complexes [Ru(bpy)2(BPIMBp)PtCl2]2+ (3) and [Ru(phen)2(BPIMBp)PtCl2]2+ (4), where bpy = 2,2'-bipyridine, phen = 1,10-phenanthroline, and BPIMBp = 1,4'-bis[(2-pyridin-2-yl)-1H-imidazol-1-ylmethyl]-1,1'-biphenyl, have been designed and synthesized from their mononuclear precursors [Ru(bpy)2(BPIMBp)]2+ (1) and [Ru(phen)2(BPIMBp)]2+ (2) as multitarget molecules for Alzheimer's disease (AD). The inclusion of the cis-PtCl2 moiety facilitates the covalent interaction of Ru(II) polypyridyl complexes with amyloid ß (Aß) peptide. These multifunctional complexes act as inhibitors of acetylcholinesterase (AChE), Aß aggregation, and Cu-induced oxidative stress and protect neuronal cells against Aß-toxicity. The study highlights the design of metal based anti-Alzheimer's disease (AD) systems.

Clinical characterization of influenza A and human respiratory syncytial virus among patients with influenza like illness.

Influenza A and Respiratory Syncytial Virus (RSV) has been recognized as a major cause of acute respiratory tract infection. H1N1 is one of the subtypes of influenza A, pandemic worldwide in July 2009, causing 18,449 deaths globally. To investigate the prevalence and clinical manifestation of the influenza A, H1N1pdm09, and RSV. Throat/nasal swab collected from the patients of all age group either outpatients/inpatients having respiratory illness from 2 to 5 days. The clinical data were recorded in a predesigned questionnaire. RNA was extracted and analyzed by real time PCR at a tertiary care center, 2009-2014. Total 4,352 samples tested for influenza A and H1N1. Out of 4,352, 32.2% (median positivity 21%; range 16-41% during 6 years) were positive for influenza A and 19% were H1N1 (median positivity 16.7%; range 8.7-23% during 6 years). Total 1653 samples were analyzed for RSV from 2011 to 2014, 12% were RSV positive (median positivity 11.35%; range 10-16.3% during 4 years). Pharyngitis, dyspnea were frequent symptoms in influenza A and H1N1 (P < 0.005) whereas bronchiolitis and pneumonia were commonly present in RSV (P < 0.005). The positivity of influenza A and H1N1 was higher in age-group 21-30, whereas RSV in infant and children. H1N1 and RSV were co-circulated and have common clinical symptoms particularly in lower age group. Therefore, laboratory confirmation is necessary for further disease prognosis. Age was an important risk factor that affects the positivity of influenza A, H1N1, and RSV. Different clinical manifestation of H1N1 and RSV will be helpful for early and accurate diagnosis. J. Med. Virol. 89:49-54, 2017. © 2016 Wiley Periodicals, Inc.

Biochemical and inhibition studies of glutamine synthetase from Leishmania donovani.

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 Km value of 26.3 ± 0.4 mM for l- glutamate and Vmax 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.

Glycyrrhizic acid attenuates growth of Leishmania donovani by depleting ergosterol levels.

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.

Honeycomb-like Ordered Assembly of DNA Induced by Flexible Binuclear Ruthenium(II)-Polypyridyl Complexes.

A series of binuclear ruthenium(II)-polypyridyl complexes of the type [Ru2 (N-N)4 (BPIMBp)]4+ , in which N-N is 2,2'-bipyridine (bpy; 1), 1,10-phenanthroline (phen; 2), dipyrido[3,2-d:2',3-f] quinoxaline (dpq; 3), dipyrido[3,2-a:2',3'-c] phenanzine (dppz; 4), and 1,4'-bis[(2-pyridin-2-yl)-1H-imidazol-1-yl)methyl]-1,1'-biphenyl (BPIMBp) is a bridging ligand, have been synthesized and characterized. These complexes are charged (4+) cations and flexible due to the -CH2 group of the bridging ligand and possess terminal ligands with variable intercalative abilities. The interaction of complexes 1-4 with calf thymus DNA (CT-DNA) was explored by using UV/Vis absorption spectroscopy, steady-state emission, emission quenching with K4 [Fe(CN)6 ], ethidium bromide displacement assay, Hoechst displacement assay, and viscosity measurements and revealed a groove-binding mode for all the complexes through a spacer and an intercalative mode for complexes 3 and 4. A decrease in the viscosity of DNA revealed bending and coiling of DNA, an initial step toward aggregation. Interestingly, a distinctive honeycomb-like ordered assembly of the DNA-complex species was visualized by fluorescence microscopy in the solution state. The use of SEM and AFM confirmed the disordered self-organization of the DNA-complex adduct on evaporation of the solvent. The small orderly nanosized DNA aggregates were confirmed by means of circular dichroism, dynamic light scattering (DLS), and TEM. These complexes are moderately cytotoxic against three different cell lines, namely, MCF-7, HeLa, and HL-60.

Design, synthesis and biological evaluation of 1,3,6-trisubstituted ß-carboline derivatives for cytotoxic and anti-leishmanial potential.

In the present study, 23 derivatives of 1,3,6-trisubstituted ß-carboline were synthesized and evaluated for cytotoxic potential against four human cancer cells, namely A-549, HeLa, Hep G2 and MCF-7 as well as anti-leishmanial activity against Leishmania donovani (MHOM/80/IN/Dd8) promastigotes. Among the studied compounds, compounds 13c and 13q showed potent cytotoxic activity better than the parent compound 10. For instance, compound 13c was found to be the most cytotoxic with IC50 of 4.72, 3.59, 3.65 and 4.17 µM against A-549, HeLa, Hep G2 and MCF-7 respectively, while for compound 13q, IC50 were 15.47, 5.30, 6.15 and 13.39 µM against the same cancer cells respectively. Further, these two compounds were found to be apoptotic in A-549 and MCF-7 cells when observed using Annexin V/propidium iodide staining under confocal microscope. All the compounds were also tested for anti-leishmanial potential. In which, compounds 13u and 13c were found to show moderate inhibition with IC50 of 23.5±9.0 and 68.0±0.0 µM respectively, while compound 10 was the most active with IC50 of 9.0±2.8 µM, suggesting the modification at C-6 detrimental for anti-leishmanial activity. Interestingly, amongst all, compound 13c was found to be the most active for cytotoxic and moderately active for anti-leishmanial activity which can be further developed as a lead for these disease areas.

Antileishmanial effect of mevastatin is due to interference with sterol metabolism.

Visceral leishmaniasis (VL) is one of the most severe forms of leishmaniasis which is fatal if left untreated. Sterol biosynthetic pathway in Leishmania is currently being explored for its therapeutic potential. In the present study, we have evaluated the antileishmanial efficacy of mevastatin, a known inhibitor of 3-hydroxy-3-methyl glutaryl-CoA reductase (HMGR) enzyme. Mevastatin inhibited Leishmania donovani promastigotes and intracellular amastigotes with an 50% inhibitory concentration (IC50) value of 23.8 ± 4.2 and 7.5 ± 1.1 µM, respectively, without exhibiting toxicity towards host cell line. Mevastatin also inhibited recombinant L. donovani HMGR (LdHMGR) enzyme activity with an IC50 value of 42.2 ± 3.0 µM. Kinetic analysis revealed that the inhibition of recombinant LdHMGR activity by mevastatin was competitive with HMG-CoA. Mevastatin-treated parasites exhibited 66% reduction in ergosterol levels with respect to untreated parasites. Incubation of mevastatin-treated L. donovani promastigotes with ergosterol resulted in revival of cell growth, whereas cholesterol supplementation failed to cause reversal in cell death. To further prove the specificity of mevastatin for HMGR enzyme, HMGR-overexpressing parasites were used which showed almost threefold resistance to mevastatin. It also induced morphological changes in the parasite accompanied by lipid body accumulation. Hence, antileishmanial effect of mevastatin was due to the inhibition of HMGR, which eventually leads to reduction in ergosterol levels and hence parasite death. The present study may have implications in the treatment of visceral form of leishmaniasis.

Procalcitonin kinetics as a prognostic marker in severe sepsis/septic shock.

BACKGROUND AND AIMS: To evaluate the prognostic value of change (fall) in serum procalcitonin level (PCT) in critically ill adults with severe sepsis/septic shock. METHODS: This was a prospective observational study in a general purpose Intensive Care Unit of a teaching Institute. PCT was measured at admission (D0) and after 72-96 h (D4) by electrochemi-luminescence immunoassay (BRAHMS PCT kit) in adults (>18 years) admitted with severe sepsis or septic shock. Change in procalcitonin values from D0 to D4 was correlated with the primary outcome, that is, 28 days mortality. All results are reported as median (interquartile range). RESULTS: A total of 171 (100 males) of 181 patients were included. The median age was 46 years (range 19-79). 137 patients were in septic shock and 34 in severe sepsis. The sequential organ failure assessment (SOFA) score in all patients was 11 (9-14).91 (53.2%) patients survived at 28 days (survivors). The baseline procalcitonin was similar in two groups (3.48 [1.04-15.85] vs. 5.27 [1.81-23.57] ng/ml in survivors and nonsurvivors [NS] respectively). The procalcitonin change was 1.58 (0.20-8.52) in survivors and 0.28 (-1.38-6.17) in NS (P = 0.01). The C-statistic of percentage change in procalcitonin from D0 to D4 to predict survival was 0.73 (95% confidence interval [CI]: 0.65-0.82) when compared to 0.78 (95% CI: 0.71-0.86) for change of SOFA score. For an absolute fall in procalcitonin of >1 ng/ml, a 70% fall predicted survival with 75% sensitivity and 64% specificity. CONCLUSIONS: In critically ill-patients with severe sepsis/septic shock, change (fall) in procalcitonin is associated with good outcome.

Leishmania donovani eukaryotic initiation factor 5A: molecular characterization, localization and homology modelling studies.

Eukaryotic translation initiation factor 5A (eIF5A) is a small acidic protein highly conserved from archaea to mammals. eIF5A is the only protein which undergoes a unique lysine residue modification to hypusine. Hypusinylation is important for the function of eIF5A which is reported to be essential for cell viability. eIF5A promotes formation of the first peptide bond at the onset of protein synthesis. However, its function in Leishmania donovani is unclear. The present study focuses on the characterization and localization of L. donovani eIF5A protein. The eIF5A gene contains an ORF of 501×bp encoding 166 amino acid residues with a predicted molecular mass and isoelectric point of 17.8 kDa and 4.83 respectively. A phylogenetic tree analysis revealed its close proximity to trypanosomes however it is distantly located from Trichomonas vaginalis and Plasmodium falciparum. The L. donovani eIF5A was expressed as a 6× His tagged protein whose identity was confirmed by western blot and MALDI. Biophysical investigation by CD revealed the predominant presence of 49% ß sheet structure which correlated well with secondary structure prediction. To gain insight into the role of eIF5A in L. donovani, we investigated the subcellular distribution of eIF5A. A GFP-fusion of L. donovani eIF5A was found to be localized in cytoplasm as confirmed by subcellular fractionation. Our studies indicated that eIF5A is primarily localized to cytoplasm and is undetectable in nuclear fraction. The homology model of eIF5A of L. donovani was built and the resulting model showed acceptable Ramachandran statistics. The model is reliable and can be used to study eIF5A binding with its effector molecules.

Exploring Leishmania donovani 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) as a potential drug target by biochemical, biophysical and inhibition studies.

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase (HMGR), an NADPH dependant enzyme catalyzes the synthesis of mevalonic acid from HMG-CoA required for isoprenoid biosynthesis. The HMGR gene from Leishmania donovani was cloned and expressed. Genome analysis of L. donovani revealed that HMGR gene having an open reading frame of 1305 bp encodes a putative protein of 434 amino acids. LdHMGR showed optimal activity at pH 7.2 and temperature 37 °C. Kinetic analysis of this enzyme revealed Km values of 35.7 ± 2.5 µM for (R,S)-HMG-CoA and 70 ± 7.9 µM for the cofactor NADPH. On tryptophan fluorescence quenching, the Stern Volmer constant (Ksv), binding constant (Ka) and protein:cofactor stoichiometry for interaction of NADPH cofactor with the enzyme were found to be 6.0 ± 0.7 M(-1), 0.17 µM and 0.72 respectively. Polyclonal anti-rat HMGR antibody detected a band of ∼45 kDa in all phases of promastigote growth. Biophysical analysis of the secondary structure of LdHMGR confirmed the presence of 25.7 ± 0.35% alpha helicity. Thermal denaturation studies showed extreme stability of the enzyme with 60% helical structure retained at 90 °C. Statins (simvastatin and atorvastatin) and non-statin (resveratrol) effectively inhibited the growth of L. donovani promastigotes as well as the catalytic activity of the recombinant LdHMGR. Atorvastatin was found to be most potent antileishmanial inhibitor with an IC50 value of 19.4 ± 3.07 µM and a very lower concentration of 315.5 ± 2.1 nM was enough to cause 50% recombinant LdHMGR enzyme inhibition suggesting direct interaction with the rate limiting enzyme of the ergosterol biosynthetic pathway. Exogenous supplementation of ergosterol in case of atorvastatin and resveratrol treated cells caused complete reversal of growth inhibition whereas simvastatin was found to be ergosterol refractory. Cholesterol supplementation however, failed to overcome growth inhibition in all the cases. Overall our study emphasizes on exploring LdHMGR as a potential drug target for the development of novel antileishmanial agents.

Mianserin, an antidepressant kills Leishmania donovani by depleting ergosterol levels.

In the present study, we have investigated the antileishmanial potential of mianserin, an antidepressant. Mianserin was found to inhibit both the promastigote and amastigote forms of the parasite in a dose dependant manner. The IC50 values for promastigotes and amastigotes were 21 µM and 46 µM respectively. Interestingly, mianserin failed to inhibit THP-1 differentiated macrophages up to 100 µM concentration thus, exhibiting parasite selectivity. When mianserin was incubated with recombinant Leishmania donovani 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) enzyme, it exhibited an IC50 value of 19.8 µM. Inhibition kinetics revealed competitive mode of enzyme inhibition as the Km increased with no change in Vmax. Further structural investigation of enzyme-inhibitor interaction revealed quenching of HMGR tryptophan intrinsic fluorescence with a K(sv) value of 3.025±0.37 M(-1) and an apparent binding constant of 0.0954 mM. We further estimated ergosterol levels which is a major component of Leishmania cell membrane. It is synthesized by HMGR enzyme, the first rate limiting enzyme of the sterol biosynthetic pathway. Analysis of ergosterol levels by HPLC revealed ∼2.5-fold depletion in mianserin treated promastigotes with respect to untreated parasites. This data was further validated by exogenous supplementation of mianserin treated cells with ergosterol and cholesterol. Reversal of growth inhibition was observed only upon ergosterol addition though it was refractory to cholesterol supplementation. Overall, our results demonstrate the possibility of repositioning of an antidepressant for the treatment of Visceral Leishmaniasis.

Association of microRNA-146a and its target gene IRAK1 polymorphism with enthesitis related arthritis category of juvenile idiopathic arthritis.

MicroRNAs (miRNAs) are non-coding RNA that modulate the expression of multiple target genes at the post-transcriptional level. Single-nucleotide polymorphisms (SNPs) in pre-miRNAs can alter miRNA expression, and polymorphism in target molecules can affect binding to target mRNA. Studies have shown an association between miR-146a gene polymorphism and autoimmune diseases. A target for miR-146a is IRAK1. We studied the SNPs of miRNA-146 and IRAK1 to see their association with susceptibility to juvenile idiopathic arthritis-enthesitis-related arthritis (JIA-ERA). One hundred and fifty patients with JIA-ERA (ILAR criteria) were included in the study. A total of 216 blood donors (201 male) with a mean age of 30.5 years served as controls. miR-146a (rs2910164) and its target IRAK1 (rs1059703) at exon-12 region and IRAK1 (rs3027898) at 3'UTR polymorphisms were analyzed using PCR-RFLP method. Among 150 patients, 133 were males and the mean age at onset of disease was 11 (4-16) years, mean disease duration was 4.5 (0.3-12) years. Twenty-two had uveitis and 21 had positive family history of spondyloarthropathy, 73 had enthesitis, 75 had inflammatory back pain, and all had arthritis. HLA B27 was present in 116 patients. Genotype frequency of miR-146a gene was in Hardy-Weinberg equilibrium in healthy controls. The genotype frequency for miR-146a was different in controls and patients [GG (51.85 vs. 50.0 %), GC (42.13 vs. 37.29 %) and CC (6.02 vs. 12.71 %), OR = 2.18; 95 % CI 1.02-4.68; p value = 0.0418]. The allele frequencies of IRAK1 (rs1059703) and IRAK1 (rs3027898) in males and genotype frequency in females were similar in controls and patients. The C allele of IRAK1 (rs1059703) was in linkage disequilibrium with T allele of IRAK1 (rs3027898). The CC genotype of the miR-146a rs2910164 polymorphism was significantly associated with the susceptibility to JIA-ERA.

Ketanserin, an antidepressant, exerts its antileishmanial action via inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) enzyme of Leishmania donovani.

Leishmaniasis is one of the major health problems existing globally. The current chemotherapy for leishmaniasis presents several drawbacks like toxicity and increased resistance to existing drugs, and hence, there is a necessity to look out for the novel drug targets and new chemical entities. Current trend in drug discovery arena is the "repurposing" of old drugs for the treatment of diseases. In the present study, an antidepressant, ketanserin, was found lethal to both Leishmania donovani promastigotes and intracellular amastigotes with no apparent toxicity to the cells. Ketanserin killed promastigotes and amastigotes with an IC50 value of 37 µM and 28 µM respectively, in a dose-dependent manner. Ketanserin was found to inhibit L. donovani recombinant 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) enzyme with an IC50 value of 43 µM. Ketanserin treated promastigotes were exogenously supplemented with sterols like ergosterol and cholesterol to rescue cell death. Ergosterol could recover the inhibition partially, whereas cholesterol supplementation completely failed to rescue the inhibited parasites. Further, HMGR-overexpressing parasites were generated by transfecting Leishmania promastigotes with an episomal pspα hygroα-HMGR construct. Wild-type and HMGR overexpressors of L. donovani were used to study the effect and mode of action of this inhibitor. The HMGR overexpressors showed twofold resistance to ketanserin. These observations suggest that the lethal effect of ketanserin is due to inhibition of HMGR, the rate-limiting enzyme of the ergosterol biosynthetic pathway. Since targeting of the sterol biosynthetic pathway enzymes may be useful therapeutically, the present study may have implications in treatment of leishmaniasis.

Antileishmanial compounds from Moringa oleifera Lam.

The antileishmanial activity of extracts and phytoconstituents of Moringa oleifera Lam. was investigated in vitro against promastigotes of Leishmania donavani. The 70% ethanolic extract of roots and the methanolic extract of leaves showed moderate inhibitory activity with IC50 values of 83.0 microg/ml and 47.5 microg/ml, respectively. Antileishmanial activity of the methanolic extract of leaves increased upon fractionation, as its ethyl acetate fraction was found to be more active with an IC50 value of 27.5 microg/ml. The most active antileishmanial compound niazinin, a thiocarbamate glycoside isolated from this fraction, showed an IC50 value of 5.25 microM. Results presented in this study indicate that extracts from M. oleifera may be developed as an adjuvant therapy for the treatment of leishmaniasis.