Synthesis and Biological Evaluation of Novel Multi-target-Directed Benzazepines Against Excitotoxicity

Excitotoxicty, a key pathogenic event is characteristic of the onset and development of neurodegeneration. The glutamatergic neurotransmission mediated through different glutamate receptor subtypes plays a pivotal role in the onset of excitotoxicity. The role of NMDA receptor (NMDAR), a glutamate receptor subtype, has been well established in the excitotoxicity pathogenesis. NMDAR overactivation triggers excessive calcium influx resulting in excitotoxic neuronal cell death. In the present study, a series of benzazepine derivatives, with the core structure of 3-methyltetrahydro-3H-benzazepin-2-one, were synthesised in our laboratory and their NMDAR antagonist activity was determined against NMDA-induced excitotoxicity using SH-SY5Y cells. In order to assess the multi-target-directed potential of the synthesised compounds, Aβ_1–42 aggregation inhibitory activity of the most potent benzazepines was evaluated using thioflavin T (ThT) and Congo red (CR) binding assays as Aβ also imparts toxicity, at least in part, through NMDAR overactivation. Furthermore, neuroprotective, free radical scavenging, anti-oxidant and anti-apoptotic activities of the two potential test compounds (7 and 14) were evaluated using primary rat hippocampal neuronal culture against Aβ_1–42-induced toxicity. Finally, in vivo neuroprotective potential of 7 and 14 was assessed using intracerebroventricular (ICV) rat model of Aβ_1–42-induced toxicity. All of the synthesised benzazepines have shown significant neuroprotection against NMDA-induced excitotoxicity. The most potent compound (14) showed relatively higher affinity for the glycine binding site as compared with the glutamate binding site of NMDAR in the molecular docking studies. 7 and 14 have been shown experimentally to abrogate Aβ_1–42 aggregation efficiently. Additionally, 7 and 14 showed significant neuroprotective, free radical scavenging, anti-oxidant and anti-apoptotic properties in different in vitro and in vivo experimental models. Finally, 7 and 14 attenuated Aβ_1–42-induced tau phosphorylation by abrogating activation of tau kinases, i.e. MAPK and GSK-3β. Thus, the results revealed multi-target-directed potential of some of the synthesised novel benzazepines against excitotoxicity.     

Designing B‐ and T‐cell multi‐epitope based subunit vaccine using immunoinformatics approach to control Zika virus infection

The Zika virus is a rapidly spreading Aedes mosquito‐borne sickness, which creates an unanticipated linkage birth deformity and neurological turmoil. This study represents the use of the combinatorial immunoinformatics approach to develop a multiepitope subunit vaccine using the structural and nonstructural proteins of the Zika virus. The designed subunit vaccine consists of cytotoxic T‐lymphocyte and helper T‐lymphocyte epitopes accompanied by suitable adjuvant and linkers. The presence of humoral immune response specific B‐cell epitopes was also confirmed by B‐cell epitope mapping among vaccine protein. Further, the vaccine protein was characterized for its allergenicity, antigenicity, and physiochemical parameters and found to be safe and immunogenic. Molecular docking and molecular dynamics studies of the vaccine protein with the toll‐like receptor‐3 were performed to ensure the binding affinity and stability of their complex. Finally, in silico cloning was performed for the effective expression of vaccine construct in the microbial system (Escherichia coli K12 strain). Aforementioned approaches result in the multiepitope subunit vaccine which may have the ability to induce cellular as well as humoral immune response. Moreover, this study needs the experimental validation to prove the immunogenic and protective behavior of the developed subunit vaccine.     

Evaluation of a diospyrin derivative as antileishmanial agent and potential modulator of ornithine decarboxylase of Leishmania donovani

World health organization has called for academic research and development of new chemotherapeutic strategies to overcome the emerging resistance and side effects exhibited by the drugs currently used against leishmaniasis. Diospyrin, a bis-naphthoquinone isolated from Diospyros montana Roxb., and its semi-synthetic derivatives, were reported for inhibitory activity against protozoan parasites including Leishmania. Presently, we have investigated the antileishmanial effect of a di-epoxide derivative of diospyrin (D17), both in vitro and in vivo. Further, the safety profile of D17 was established by testing its toxicity against normal macrophage cells (IC₅₀ ∼ 20.7 μM), and also against normal BALB/c mice in vivo. The compound showed enhanced activity (IC₅₀ ∼ 7.2 μM) as compared to diospyrin (IC₅₀ ∼ 12.6 μM) against Leishmania donovani promastigotes. Again, D17 was tested on L. donovani BHU1216 isolated from a sodium stibogluconate-unresponsive patient, and exhibited selective inhibition of the intracellular amastigotes (IC₅₀ ∼ 0.18 μM). Also, treatment of infected BALB/c mice with D17 at 2 mg/kg/day reduced the hepatic parasite load by about 38%. Subsequently, computational docking studies were undertaken on selected enzymes of trypanothione metabolism, viz. trypanothione reductase (TryR) and ornithine decarboxylase (ODC), followed by the enzyme kinetics, where D17 demonstrated non-competitive inhibition of the L. donovani ODC, but could not inhibit TryR.     

A high‐throughput capillary isoelectric focusing immunoassay for fingerprinting protein sialylation

The serum half‐life, biological activity, and solubility of many recombinant glycoproteins depend on their sialylation. Monitoring glycoprotein sialylation during cell culture manufacturing is, therefore, critical to ensure product efficacy and safety. Here a high‐throughput method for semi‐quantitative fingerprinting of glycoprotein sialylation using capillary isoelectric focusing immunoassay on NanoPro (Protein Simple) platform was developed. The method was specific, sensitive, precise, and robust. It could analyze 2 μL of crude cell culture samples without protein purification, and could automatically analyze from 8 samples in 4 h to 96 samples in 14 h without analyst supervision. Furthermore, its capability to detect various changes in sialylation fingerprints during cell culture manufacturing process was indispensable to ensure process robustness and consistency. Moreover, the changes in the sialylation fingerprints analyzed by this method showed strong correlations with intact mass analysis using liquid chromatography and mass spectrometry. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:235–241, 2016     

Thermodynamic effects of replacements of Pro residues in helix interiors of maltose-binding protein

Introduction of Pro residues into helix interiors results in protein destabilization. It is currently unclear if the converse substitution (i.e., replacement of Pro residues that naturally occur in helix interiors would be stabilizing). Maltose-binding protein is a large 370-amino acid protein that contains 21 Pro residues. Of these, three nonconserved residues (P48, P133, and P159) occur at helix interiors. Each of the residues was replaced with Ala and Ser. Stabilities were characterized by differential scanning calorimetry (DSC) as a function of pH and by isothermal urea denaturation studies as a function of temperature. The P48S and P48A mutants were found to be marginally more stable than the wild-type protein. In the pH range of 5-9, there is an average increase in T(m) values of P48A and P48S of 0.4 degrees C and 0.2 degrees C, respectively, relative to the wild-type protein. The other mutants are less stable than the wild type. Analysis of the effects of such Pro substitutions in MBP and in three other proteins studied to date suggests that substitutions are more likely to be stabilizing if the carbonyl group i-3 or i-4 to the mutation site is not hydrogen bonded in the wild-type protein.     

Verification of hypocholesterolemic effect of fermented milk on human subjects with different cholesterol levels

The possible hypocholesterolemic effect of acidophilus milk was evaluated on 27 human subjects having different levels of serum cholesterol, i.e. < 2.0 (group C1), 2.0-2.2 (C2), 2.2-2.5 (C3) and > 2.5 g/L (C4). The acidophilus milk was prepared by fermentation of low-fat milk with Lactobacillus acidophilus and was fed to each volunteer at the rate of 200 mL/d for 20 d. Blood samples from the volunteers were collected and analyzed for lipid profile twice prior to, during and after feeding, keeping a gap of 10 d between two collections. A significant decrease (p < 0.05) in average total cholesterol was found in the C2 and C3 groups, amounting to 21 and 12%, respectively. The average LDL cholesterol decreased in C2, C3 and C4 groups by 0.54, 0.26 and 0.46 g/L, respectively. In the C2 group, the LDL/HDL and total/HDL ratio was also reduced by 1.4 and 1.3, respectively. However, in the C1 group, the average total and LDL cholesterol level did not show any significant change but serum triacylglycerols and VLDL cholesterol showed a significant (p < 0.05) increase of 0.53 and 0.11 g/L, respectively. Regression analysis of the data revealed a square trend in most of the parameters over time period. Overall, the feeding had the best effect in the subjects with lipidemic status of borderline cholesterol level (2.0-2.2 g/L) group.     

Secreted meningeal chemokines,but not VEGFA,modulate the migratory properties of medulloblastoma cells

Leptomeningeal metastasis is a cause of morbidity and mortality in medulloblastoma, but the understanding of molecular mechanisms driving this process is nascent. In this study, we examined the secretory chemokine profile of medulloblastoma cells (DAOY) and a meningothelial cell line (BMEN1). Conditioned media (CM) of meningothelial cells increased adhesion, spreading and migration of medulloblastoma. VEGFA was identified at elevated levels in the CM from BMEN1 cells (as compared to DAOY CM); however, recombinant VEGFA alone was insufficient to enhance medulloblastoma cell migration. In addition, bevacizumab, the VEGFA scavenging monoclonal antibody, did not block the migratory phenotype induced by the CM. These results reveal that paracrine factors secreted by meningothelial cells can influence migration and adherence of medulloblastoma tumor cells, but VEGFA may not be a specific target for therapeutic intervention in this context.     

A New Player in the Spermiogenesis Pathway of Caenorhabditis elegans

Precise timing of sperm activation ensures the greatest likelihood of fertilization. Precision in Caenorhabditis elegans sperm activation is ensured by external signaling, which induces the spherical spermatid to reorganize and extend a pseudopod for motility. Spermatid activation, also called spermiogenesis, is prevented from occurring prematurely by the activity of SPE-6 and perhaps other proteins, termed “the brake model.” Here, we identify the spe-47 gene from the hc198 mutation that causes premature spermiogenesis. The mutation was isolated in a suppressor screen of spe-27(it132ts), which normally renders worms sterile, due to defective transduction of the activation signal. In a spe-27(+) background, spe-47(hc198) causes a temperature-sensitive reduction of fertility, and in addition to premature spermiogenesis, many mutant sperm fail to activate altogether. The hc198 mutation is semidominant, inducing a more severe loss of fertility than do null alleles generated by CRISPR-associated protein 9 (Cas9) technology. The hc198 mutation affects an major sperm protein (MSP) domain, altering a conserved amino acid residue in a β-strand that mediates MSP–MSP dimerization. Both N- and C-terminal SPE-47 reporters associate with the forming fibrous body (FB)-membranous organelle, a specialized sperm organelle that packages MSP and other components during spermatogenesis. Once the FB is fully formed, the SPE-47 reporters dissociate and disappear. SPE-47 reporter localization is not altered by either the hc198 mutation or a C-terminal truncation deleting the MSP domain. The disappearance of SPE-47 reporters prior to the formation of spermatids requires a reevaluation of the brake model for prevention of premature spermatid activation.