Computational exploration of natural compounds targeting Staphylococcus aureus: inhibiting AgrA promoter binding for antimicrobial intervention.

Staphylococcus aureus is a highly virulent nosocomial pathogen that poses a significant threat to individuals exposed to healthcare settings. Due to its sophisticated machinery for producing virulence factors, S. aureus can cause severe and potentially fatal infections in humans. This study focuses on the response regulator AgrA, which plays a crucial role in regulating the production of virulence factors in S. aureus. The objective is to identify natural compounds that can inhibit the binding of AgrA to its promoter site, thus inhibiting the expression of virulence genes. To achieve this, a pharmacophore model was generated using known drugs and applied to screen the ZINC natural product database. The resulting compounds were subjected to molecular docking-based virtual screening against the C-terminal DNA binding domain of AgrA. Three compounds, namely ZINC000077269178, ZINC000051012304, and ZINC000004266026, were shortlisted based on their strong affinity for key residues involved in DNA binding and transcription initiation. Subsequently, the unbound and ligand-bound complexes were subjected to a 200 ns molecular dynamics simulation to assess their conformational stability. Various analyses, including RMSD, RMSF, Rg, SASA, Principal Component Analysis, and Gibbs free energy landscape, were conducted on the simulation trajectory. The RMSD profile indicated similar fluctuations in both bound and unbound structures, while the Rg profile demonstrated the compactness of the protein without any unfolding during the simulation. Furthermore, Principal component analysis revealed that ligand binding reduced the overall atomic motion of the protein whereas free energy landscape suggested the energy variations obtained in complexes.Communicated by Ramaswamy H. Sarma.

In-silico design of an immunoinformatics based multi-epitope vaccine against Leishmania donovani.

BACKGROUND: Visceral Leishmaniasis (VL) is a fatal vector-borne parasitic disorder occurring mainly in tropical and subtropical regions. VL falls under the category of neglected tropical diseases with growing drug resistance and lacking a licensed vaccine. Conventional vaccine synthesis techniques are often very laborious and challenging. With the advancement of bioinformatics and its application in immunology, it is now more convenient to design multi-epitope vaccines comprising predicted immuno-dominant epitopes of multiple antigenic proteins. We have chosen four antigenic proteins of Leishmania donovani and identified their T-cell and B-cell epitopes, utilizing those for in-silico chimeric vaccine designing. The various physicochemical characteristics of the vaccine have been explored and the tertiary structure of the chimeric construct is predicted to perform docking studies and molecular dynamics simulations. RESULTS: The vaccine construct is generated by joining the epitopes with specific linkers. The predicted tertiary structure of the vaccine has been found to be valid and docking studies reveal the construct shows a high affinity towards the TLR-4 receptor. Population coverage analysis shows the vaccine can be effective on the majority of the world population. In-silico immune simulation studies confirms the vaccine to raise a pro-inflammatory response with the proliferation of activated T and B cells. In-silico codon optimization and cloning of the vaccine nucleic acid sequence have also been achieved in the pET28a vector. CONCLUSION: The above bioinformatics data support that the construct may act as a potential vaccine. Further wet lab synthesis of the vaccine and in vivo works has to be undertaken in animal model to confirm vaccine potency.

Unveiling the cytotoxicity of phytosynthesised silver nanoparticles using Tinospora cordifolia leaves against human lung adenocarcinoma A549 cell line.

Biosynthesis of silver nanoparticles (AgNPs) using plant extract is a cheap, easily accessible and natural process in which the phyto-constituents of the plants act as capping, stabilising and reducing agent. The present study explored the biosynthesis of AgNPs using aqueous leaf extract of Tinospora cordifolia and characterised via various techniques such as Fourier transform infrared, scanning electron microscopy, transmission electron microscopy (TEM), energy dispersive X-ray analysis and X-ray diffraction. Here, TEM confirmed the spherical morphology with 25-50 nm size of synthesised AgNPs. Further, anticancer efficiency of AgNPs synthesised using T. cordifolia leaves were evaluated against human lung adenocarcinoma cell line A549 by MTT, trypan blue assay, apoptotic morphological changes using Annexin V-FITC and Propidium iodide (PI), nuclear morphological changes by DAPI (4, 6-diamidino-2-phenylindole dihydrochloride) staining, reactive oxygen species generation and mitochondrial membrane potential determination. Results confirmed the AgNPs synthesised using T. cordifolia leaves are found to be highly toxic against human lung adenocarcinoma cell line A549.

Detection and characterization of an albumin-like protein in Leishmania donovani.

The protozoan parasite, Leishmania donovani, undergoes several molecular adaptations and secretes many effector molecules for host cell manipulation and successful parasitism. The current study identifies an albumin-like secretory protein, expressed in its extracellular promastigote forms. A leishmanial complementary DNA sequence of a partial gene has been cloned, and the encoded peptide (14 kD) is used for the production of polyclonal antibody. This targeted antibody identifies a large native protein (66.421 kD), expressed stage-specifically in promastigotes. Through electron microscopic studies, the native protein is found to be localized in the flagellar pocket and flagella and at the surface of the promastigotes. This native protein is purified with the same customized antibody for future characterization and sequencing. The sequence analysis reveals its homology with the mammalian serum albumin. It is evidenced from in silico studies that this albumin-like protein remains associated with long-chain fatty acids while in vitro studies indicate its close association with membrane cholesterol. Since antibody-mediated blocking compromises the parasite infectivity, these leishmanial albumin-like molecules are hereby proposed to play an instrumental role in the infectivity of L. donovani to peripheral blood monocyte cells. Thus, identification and characterization of an albumin-like protein in L. donovani promastigotes may be interpreted as a molecular adaptation candidate. It may be hypothesized that the parasite mimics the mammalian system for importing fatty acids into the intracellular amastigotes, facilitating its host cell infectivity.

Development of screen-printed electrode based immunosensor for the detection of HER2 antigen in human serum samples.

In this study, an immunosensor based on screen-printed electrode (SPE) has been developed for the detection of Human Epidermal Growth Factor Receptor-2 (HER2) antigen. The SPEs were fabricated and a sandwich enzyme linked immunosorbent assay (ELISA) format was followed for the construction of the immunosensor. The capture antibody (mouse anti-human ErbB2) was coated onto the electrode surface without any prior surface modification, followed by the addition of recombinant human HER2 antigen. Biotinylated goat anti-human ErbB2 was used as the detection antibody which was linked to streptavidin conjugated horseradish peroxidase (HRP). 3,3',5,5'-tetramethylbenzidine (TMB) was used as the substrate. The redox reaction was measured using cyclic voltammetry at scan rate of 50mV/s for the quantification of the antigen in solution. The biotin-avidin chemistry enabled the accurate detection of the antigen in nanogram levels. The amperometric signal obtained increased linearly with increase in the HER2 concentration and two-fold linear range was obtained between 5ng/ml-20ng/ml and 20-200ng/ml respectively. The limit of detection (LOD) and the limit of quantification (LOQ) of this immunosensor were found to be 4ng/ml and 5ng/ml respectively. The detection of HER2 in the serum samples of invasive and non-invasive breast cancer patients has been realized.

Cholesterol-lowering drug, in combination with chromium chloride, induces early apoptotic signals in intracellular L. donovani amastigotes, leading to death.

Leishmania establishes a successful parasitism by evading both oxidative and non-oxidative killing pathways, and its drug resistance against the currently available therapeutics demands for a safe and cheap drug. Since the parasite synthesizes ergosterol instead of cholesterol, using the same biochemical pathway and enzymes, an inhibitor of HMG-CoA-Reductase, Lovastatin, has been tried for its anti-Leishmanial effect. Lovastatin, being an inhibitor of HMG-CoA-Reductase, inhibits infection by cholesterol depletion, while chromium chloride complexes, at their higher concentrations, are reported to exhibit cytotoxicity. In intracellular amastigotes, cytotoxicity has been checked by assessing various manifestation of cell death, viz. DNA fragmentation, AnnexinV-FITC binding and JC-1 fluorescence ratio. Release of hydrogen peroxide (HPO) and nitric oxide (NO) has been assessed in live cell. Lovastatin and CrCl3.6H2O in combination has appeared to be ineffective on promastigotes but has induced cytotoxic effect on the intracellular amastigotes through up-regulation of cellular signalling mechanisms. CrCl 3.6H2O stimulates generation of NO, leading to reduction of the number of intracellular amastigote, while Lovastatin shows HPO-mediated killing of the same, keeping the host cell unaffected. This novel therapeutic approach, involving two known safe compounds in suboptimal doses, may resolve human visceral Leishmaniasis.

Comparative in-silico genome analysis of Leishmania (Leishmania) donovani: A step towards its species specificity.

Comparative genome analysis of recently sequenced Leishmania (L.) donovani was unexplored so far. The present study deals with the complete scanning of L. (L.) donovani genome revealing its interspecies variations. 60 distinctly present genes in L. (L.) donovani were identified when the whole genome was compared with Leishmania (L.) infantum. Similarly 72, 159, and 265 species specific genes were identified in L. (L.) donovani when compared to Leishmania (L.) major, Leishmania (L.) mexicana and Leishmania (Viannia) braziliensis respectively. The cross comparison of L. (L.) donovani in parallel with the other sequenced species of leishmanial led to the identification of 55 genes which are highly specific and expressed exclusively in L. (L.) donovani. We found mainly the discrepancies of surface proteins such as amastins, proteases, and peptidases. Also 415 repeat containing proteins in L. (L.) donovani and their differential distribution in other leishmanial species were identified which might have a potential role during pathogenesis. The genes identified can be evaluated as drug targets for anti-leishmanial treatment, exploring the scope for extensive future investigations.

N-acetyl cysteine enhances imatinib-induced apoptosis of Bcr-Abl+ cells by endothelial nitric oxide synthase-mediated production of nitric oxide.

INTRODUCTION: Imatinib, a small-molecule inhibitor of the Bcr-Abl kinase, is a successful drug for treating chronic myeloid leukemia (CML). Bcr-Abl kinase stimulates the production of H(2)O(2), which in turn activates Abl kinase. We therefore evaluated whether N-acetyl cysteine (NAC), a ROS scavenger improves imatinib efficacy. MATERIALS AND METHODS: Effects of imatinib and NAC either alone or in combination were assessed on Bcr-Abl(+) cells to measure apoptosis. Role of nitric oxide (NO) in NAC-induced enhanced cytotoxicity was assessed using pharmacological inhibitors and siRNAs of nitric oxide synthase isoforms. We report that imatinib-induced apoptosis of imatinib-resistant and imatinib-sensitive Bcr-Abl(+) CML cell lines and primary cells from CML patients is significantly enhanced by co-treatment with NAC compared to imatinib treatment alone. In contrast, another ROS scavenger glutathione reversed imatinib-mediated killing. NAC-mediated enhanced killing correlated with cleavage of caspases, PARP and up-regulation and down regulation of pro- and anti-apoptotic family of proteins, respectively. Co-treatment with NAC leads to enhanced production of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS). Involvement of eNOS dependent NO in NAC-mediated enhancement of imatinib-induced cell death was confirmed by nitric oxide synthase (NOS) specific pharmacological inhibitors and siRNAs. Indeed, NO donor sodium nitroprusside (SNP) also enhanced imatinib-mediated apoptosis of Bcr-Abl(+) cells. CONCLUSION: NAC enhances imatinib-induced apoptosis of Bcr-Abl(+) cells by endothelial nitric oxide synthase-mediated production of nitric oxide.

Leishmania donovani infection of human myeloid dendritic cells leads to a Th1 response in CD4+ T cells from healthy donors and patients with kala-azar.

The role played by dendritic cells (DCs) in Leishmania donovani infection is poorly understood. Here, we report that L. donovani amastigotes efficiently infect human peripheral-blood monocyte-derived DCs. Opsonization with normal human serum enhanced the infectivity of amastigotes and promastigotes only marginally. Surface attachment versus internalization was distinguished by incubation of DCs with live, fluorescein isothiocyanate-labeled parasites, followed by quenching with crystal violet. Infection with amastigotes was accompanied by DC maturation, as was evident from the up-regulation of maturation-associated cell-surface markers, the nuclear translocation of RelB, and the release of cytokines. Amastigote-primed DCs produced inflammatory cytokines in response to subsequent treatment with interferon- gamma or anti-CD40 monoclonal antibody. When cocultured, amastigote-infected DCs induced T helper cell type 1 (Th1) responses both in naive allogeneic CD4(+) T cells and in autologous CD4(+) T cells from patients with kala-azar and up-regulated the expression of T-bet. Our data reveal that infection with L. donovani amastigotes induces a Th1 cytokine milieu in both DCs and T cells.

Interaction of Leishmania parasites with dendritic cells and its functional consequences.

Interaction between dendritic cells (DC) and T cells is essential for the generation of cell mediated immunity and thus DC play a critical role in the initiation of immune responses against Leishmania parasites. Although macrophages (Mphi) are the major targets of all species of Leishmania, a number of studies demonstrated the infection of DC by Leishmania. DC specific intracellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN), has been reported to be the receptor for Leishmania amastigotes. The functional consequences in DC after Leishmania infections appear to depend on species of Leishmania. Some species of Leishmania enhance the surface expression of co-stimulatory molecules and CD40-ligand-induced IL-12 production in DC. On the other hand other species down-regulate co-stimulatory molecules and inhibit IL-12 production. The intrinsic differences among Leishmania species with regard to alteration of cell surface molecules and IL-12 production in DC may contribute to the healing and non-healing forms of the disease.

Present status of antileishmanial vaccines.

The term leishmaniasis refers collectively to various clinical syndromes that are caused by obligate intracellular protozoa of the genus Leishmania. Approximately 350 million people in 8 countries are estimated to be threatened by the disease. The World Health Organization estimated that there are 12 million cases of all forms of leishmaniasis worldwide, with over 500,000 new cases of visceral disease occurring each year. Most of the drugs commonly used to treat different forms of leishmaniasis are toxic and have unacceptable side effects. Moreover, cases of drug resistant leishmaniasis are on the rise. Due to non-existence of effective vaccine to date, improved immunoprophylactic approaches still remain desirable to combat leishmaniasis. Antileishmanial vaccines developed around the globe are discussed.

Dihydrobetulinic acid induces apoptosis in Leishmania donovani by targeting DNA topoisomerase I and II: implications in antileishmanial therapy.

Leishmaniasis is the second-most dreaded parasitic disease in the modern world, behind malaria. The lack of effective vaccines demand improved chemotherapy along with the development of lead compounds and newer targets. We report here that the pentacyclic triterpenoid, dihydrobetulinic acid (DHBA), is a novel lead compound for antileishmanial therapy. It acts by targeting DNA topoisomerases. DNA topoisomerase I and II activity was studied using relaxation and decatenation assays. Mechanistic studies were based on the decreased mobility of enzyme-bound DNA compared with free DNA and the differential mobility of nicked and supercoiled monomers in 1% agarose gel. Pulsed field gradient gel electrophoresis, confocal microscopy, and transmission electron microscopy were performed to assess cytotoxicity of the compound and ultrastructural damage of the parasite. Apoptosis was studied by the isolation of DNA from DHBA-treated parasites and subsequent electrophoresis in 1% agarose gel. DHBA inhibits growth of Leishmania donovani promastigotes and amastigotes with an IC50 of 2.6 and 4.1 microM respectively. The compound is a dual inhibitor of DNA topoisomerases that fails to induce DNA cleavage and acts by preventing the formation of enzyme-DNA binary complex, ultimately inducing apoptosis. Treatment of infected golden hamsters with the compound markedly reduces (> 92%) parasitic burden, both in spleen and liver. Interestingly, the 17-decarboxylated analogue, dihydrolupeol, does not inhibit DNA topoisomerase I and II, has no effect on parasitic growth, and also fails to induce apoptosis. DHBA is a potent antileishmanial agent that induces apoptosis by primarily targeting DNA topoisomerases. Therefore it is a strong candidate for use in designing new antileishmanial drugs.

Dendritic cell-based immunotherapy combined with antimony-based chemotherapy cures established murine visceral leishmaniasis.

Dendritic cells (DCs) have been proposed to play a critical role as adjuvants in vaccination and immunotherapy. In this study we evaluated the combined effect of soluble Leishmania donovani Ag (SLDA)-pulsed syngeneic bone marrow-derived DC-based immunotherapy and antimony-based chemotherapy for the treatment of established murine visceral leishmaniasis. Three weekly injections of SLDA-pulsed DCs into L. donovani-infected mice reduced liver and splenic parasite burden significantly, but could not clear parasite load from these organs completely. Strikingly, the conventional antileishmanial chemotherapy (sodium antimony gluconate) along with injections of SLDA-pulsed DCs resulted in complete clearance of parasites from both these organs. Repetitive in vitro stimulation of splenocytes from uninfected or L. donovani-infected mice with SLDA-pulsed DCs led to the emergence of CD4(+) T cells with characteristics of Th1 cells. Our data indicate that DC-based immunotherapy enhances the in vivo antileishmanial potential of antimony or vice versa.

Synthesis of a novel quinoline derivative, 2-(2-methylquinolin-4-ylamino)-N-phenylacetamide--a potential antileishmanial agent.

Some novel quinoline derivatives were prepared and tested for antileishmanial activity. 2-(2-Methylquinolin-4-ylamino)-N-phenylacetamide (2) was found to be significantly more active than the standard antileishmanial drug sodium antimony gluconate (SAG) in reducing the parasite load both in the spleen and liver at a much lower concentration in hamster models. The results suggest that the compound could be exploited as an antileishmanial drug.

Combination therapy with indolylquinoline derivative and sodium antimony gluconate cures established visceral leishmaniasis in hamsters.

2-(2"-Dichloroacetamidobenzyl)-3-(3'-indolylquinoline), designated indolylquinoline derivative A, reduced the splenic and the liver parasite burdens by >93.0% in Leishmania donovani-infected hamsters, whereas sodium antimony gluconate (SAG) reduced the burdens approximately 80.0%. Complete clearance of parasitemia from the livers and spleens was noticed when infected animals received indolylquinoline derivative A plus SAG, suggesting that indolylquinoline derivative A has potential as a new agent for sole or conjunctive therapy for leishmaniasis.