Molecular Docking Analysis of 120 Potential HPV Therapeutic Epitopes Using a New Analytical Method

International Journal of Peptide Research and Therapeutics - The accurate modelling and scoring of protein–peptide (Pr–Pe) complexes are determining factors in the drug discovery...     

In silico design and in vitro expression of novel multiepitope DNA constructs based on HIV-1 proteins and Hsp70 T-cell epitopes

Objectives

Epitope-driven vaccines carrying highly conserved and immunodominant epitopes have emerged as promising approaches to overcome human immunodeficiency virus-1 (HIV-1) infection.

Methods

Two multiepitope DNA constructs encoding T cell epitopes from HIV-1 Gag, Pol, Env, Nef and Rev proteins alone and/or linked to the immunogenic epitopes derived from heat shock protein 70 (Hsp70) as an immunostimulatory agent were designed. In silico analyses were applied including MHC-I and MHC-II binding, MHC-I immunogenicity and antigen processing, population coverage, conservancy, allergenicity, toxicity and hemotoxicity. The peptide-MHC-I/MHC-II molecular docking and cytokine production analyses were carried out for predicted epitopes. The selected highly immunogenic T-cell epitopes were then used to design two multiepitope fusion constructs. Next, prediction of the physicochemical and structural properties, B cell epitopes, and constructs-toll-like receptors (TLRs) molecular docking were performed for each construct. Finally, the eukaryotic expression plasmids harboring totally 12 cytotoxic T Lymphocyte (CTL) and 10 helper T lymphocytes (HTL) epitopes from HIV-1 proteins (i.e., pEGFP-N1-gag-pol-env-nef-rev), and linked to 2 CTL and 2 HTL epitopes from Hsp70 (i.e., pEGFP-N1-hsp70-gag-pol-env-nef-rev) were generated and transfected into HEK-293 T cells for evaluating the percentage of multiepitope peptides expression using flow cytometry and western blotting.

Results

The designed DNA constructs could be successfully expressed in mammalian cells. The expression rates of Gag-Pol-Env-Nef-Rev-GFP and Hsp70-Gag-Pol-Env-Nef-Rev-GFP were about 56–60% as the bands of?~?63 and?~?72 kDa confirmed in western blotting, respectively.

Conclusion

The combined in silico/in vitro methods indicated two multiepitope constructs can be produced and used as probable effective immunogens for HIV-1 vaccine development.

     

Utilization of the human gamma-satellite insulator for the enhancement of anti-PCSK9 monoclonal antibody expression in Chinese hamster ovary cells

Molecular Biology Reports - Monoclonal antibodies (mAbs) are widely employed as invaluable therapeutics for a vast number of human disorders. Several approaches have been introduced for the...     

Structure-based prediction of protein–protein interactions between GhWlim5 Domain1 and GhACTIN-1 proteins: a practical evidence with improved fibre strength

Cotton fibre quality is a multigenic trait. Genetic modification of different genes to achieve high quality fibre is difficult without knowing the mechanism lying behind genes interaction. Based on background knowledge an attempt to explore the potential structural interactions between Gossypium hirsutum Wlim5 domain1 and Gossypium hirsutum ACTIN-1 proteins was done in current study. Sequence features of the LIM domain1 of GhWlim5 protein were identified through multiple sequence alignment analysis, and a phylogenetic tree was built to identify evolutionary relationships between sequences. Conservation indicated the evolutionary importance of side chain residues and the presence of several aliphatic and/or bulky residues, which stabilize the protein core and facilitate packing of zinc fingers. The structures of GhWlim5 domain1 and GhACTIN-1 proteins were modelled and validated through computational methods. Validation of GhACTIN-1 and GhWlim5 domain1 structures indicated good structural quality with 99.7% and 100% of the favoured number of residues in allowed regions and Z-score, within the ranges of − 9.87 and − 4.17, respectively. Docking analysis indicated various possible modes of interaction between these two proteins with favourable binding affinities. Based on our strong binding interaction results between GhWlim5 domain1 and GhACTIN-1 proteins, we further investigated the role of over-expression of GhWlim5 by transformation in cotton plants under fibre specific promoter and transgenic plants displayed significant increases in fibre strength.

     

Design,Synthesis, Antibacterial Evaluation and Molecular Docking Studies of Some Newer Baenzothiazole Containing Aryl and Alkaryl Hydrazides

The alarming rise of bacterial resistance is occurring worldwide and endangering the efficacy of antibiotics. Therefore, development of new and efficient antibacterial agents remains paramount. In the present work, we designed and synthesized a series of N′-(1,3-benzothiazol-2-yl)-substituted aryl/aralkyl hydrazides C1 – C27 and evaluated them in vitro for their antibacterial activity. Among all tested compounds, C10 , C15 , and C24 showed potent activity against Staphylococcus aureus ATCC 43300 (MRSA). Minimum bactericidal concentration studies of synthesized compounds are performed against selected bacterial strains. Time kill kinetics showed that the compounds C10 and C15 possess bactericidal activity against MRSA ATCC 43300, while compound C24 possess bactericidal activity against S. aureus NCIM 5022. In the extra-precision docking, compounds C1 – C27 exhibited interactions mainly with the N-terminal and central domains of S. aureus GyrB catalytic pocket. Binding free energy (ΔG_bind) of compounds C1 – C27 /3U2K complexes were computed by MM-GBSA approach. Free energy components indicated Coulomb energy term as favorable for binding, while van der Waals and electrostatic solvation energy terms strongly disfavored the binding. ADMET properties of synthesized compounds C1 – C27 are also computed.     

Cloning and functional characterization of endo-β-1,4-glucanase gene from metagenomic library of vermicompost

In the vermicomposting of paper mill sludge, the activity of earthworms is very dependent on dietetic polysaccharides including cellulose as energy sources. Most of these polymers are degraded by the host microbiota and considered potentially important source for cellulolytic enzymes. In the present study, a metagenomic library was constructed from vermicompost (VC) prepared with paper mill sludge and dairy sludge (fresh sludge, FS) and functionally screened for cellulolytic activities. Eighteen cellulase expressing clones were isolated from about 89,000 fosmid clones libraries. A short fragment library was constructed from the most active positive clone (cMGL504) and one open reading frame (ORF) of 1,092 bp encoding an endo-β-1,4-glucanase was indentified which showed 88% similarity with Cellvibrio mixtus cellulase A gene. The endo-β-1,4-glucanase cmgl504 gene was overexpressed in Escherichia coli. The purified recombinant cmgl504 cellulase displayed activities at a broad range of temperature (25–55°C) and pH (5.5–8.5). The enzyme degraded carboxymethyl cellulose (CMC) with 15.4 U, while having low activity against avicel. No detectable activity was found for xylan and laminarin. The enzyme activity was stimulated by potassium chloride. The deduced protein and three-dimensional structure of metagenome-derived cellulase cmgl504 possessed all features, including general architecture, signature motifs, and N-terminal signal peptide, followed by the catalytic domain of cellulase belonging to glycosyl hydrolase family 5 (GHF5). The cellulases cloned in this work may play important roles in the degradation of celluloses in vermicomposting process and could be exploited for industrial application in future.     

Galinsosides A and B,bioactive flavanone glucosides from Galinsoga parviflora

Galinsosides A (1) and B (2), new flavanone glucosides together with two known flavanones, 7,3′,4′-trihydroxyflavanone (3) and 3,5,7,3′,4′-pentahydroxyflavanone (4) have been isolated from an ethyl acetate- soluble fraction of Galinsoga parviflora. Their structures were assigned on the basis of spectral studies. Compound 1 showed significant antioxidant and urease inhibitory activity while compound 2 was moderately active. On the other hand, 2 showed inhibitory potential against α-glucosidase.     

Development of Novel Prime-Boost Strategies Based on a Tri-Gene Fusion Recombinant L. tarentolae Vaccine against Experimental Murine Visceral Leishmaniasis

Visceral leishmaniasis (VL) is a vector-borne disease affecting humans and domestic animals that constitutes a serious public health problem in many countries. Although many antigens have been examined so far as protein- or DNA-based vaccines, none of them conferred complete long-term protection. The use of the lizard non-pathogenic to humans Leishmania (L.) tarentolae species as a live vaccine vector to deliver specific Leishmania antigens is a recent approach that needs to be explored further. In this study, we evaluated the effectiveness of live vaccination in protecting BALB/c mice against L. infantum infection using prime-boost regimens, namely Live/Live and DNA/Live. As a live vaccine, we used recombinant L. tarentolae expressing the L. donovani A2 antigen along with cysteine proteinases (CPA and CPB without its unusual C-terminal extension (CPB^-CTE)) as a tri-fusion gene. For DNA priming, the tri-fusion gene was encoded in pcDNA formulated with cationic solid lipid nanoparticles (cSLN) acting as an adjuvant. At different time points post-challenge, parasite burden and histopathological changes as well as humoral and cellular immune responses were assessed. Our results showed that immunization with both prime-boost A2-CPA-CPB^-CTE-recombinant L. tarentolae protects BALB/c mice against L. infantum challenge. This protective immunity is associated with a Th1-type immune response due to high levels of IFN-γ production prior and after challenge and with lower levels of IL-10 production after challenge, leading to a significantly higher IFN-γ/IL-10 ratio compared to the control groups. Moreover, this immunization elicited high IgG1 and IgG2a humoral immune responses. Protection in mice was also correlated with a high nitric oxide production and low parasite burden. Altogether, these results indicate the promise of the A2-CPA-CPB^-CTE-recombinant L. tarentolae as a safe live vaccine candidate against VL.     

Rutin as promising drug for the treatment of Parkinson’s disease: an assessment of MAO-B inhibitory potential by docking,molecular dynamics and DFT studies

ABSTRACT

Inhibitors of monoamine oxidase (MAO)-B have been used for many years in the therapy of Parkinson’s disease (PD). Owing to the safety concerns of the currently used agents, the discovery of novel scaffolds is of considerable interest. MAO-B inhibitory potential of rutin, a flavonoid derived from natural sources, has been established in experimental findings. Hence, the current study seeks to examine the interactions between rutin and crystal structure of human MAO-B enzyme. Molecular docking calculations, as well as molecular dynamics simulations, were employed to investigate the binding mode and the stability of the rutin/MAO-B complex. Energies of highest occupied/lowest unoccupied molecular orbitals were computed through DFT studies and used to calculate electron affinity, hardness, chemical potential, electronegativity, and electrophilicity index in order to investigate the capability of these parameters to influence the ligand–receptor interactions. It was found that rutin traverses both the entrance cavity and the substrate cavity, forcing the Ile-199 ‘gate’ to rotate into its open conformation. It results in the fusion of the two cavities of the MAO-B binding site and directly leads to better binding interactions. Results of the current study can be used for lead modification and development of novel drugs for the treatment of PD.     

Synthesis and evaluation of 7-chloro-4-(piperazin-1-yl)quinoline-sulfonamide as hybrid antiprotozoal agents

A new series of 4-aminochloroquinoline based sulfonamides were synthesized and evaluated for antiamoebic and antimalarial activities. Out of the eleven compounds evaluated (F1–F11), two of them (F3 and F10) showed good activity against Entamoeba histolytica (IC₅₀ <5 μM). Three of the compounds (F5, F7 and F8) also displayed antimalarial activity against the chloroquine-resistant (FCR-3) strain of Plasmodium falciparum with IC₅₀ values of 2 μM. Compound F7, whose crystal structure was also determined, inhibited β-haematin formation more potently than quinine. To further understand the action of hybrid molecules F7 and F8, molecular docking was carried out against the homology model of P. falciparum enzyme dihydropteroate synthase (PfDHPS). The complexes showed that the inhibitors place themselves nicely into the active site of the enzyme and exhibit interaction energy which is in accordance with our activity profile data. Application of Lipinski ‘rule of five’ on all the compounds (F1–F11) suggested high drug likeness of F7 and F8, similar to quinine.