Exploration of fluoroquinolone resistance in Streptococcus pyogenes: comparative structure analysis of wild‐type and mutant DNA gyrase

Quinolone resistance‐determining region is known to be the druggability site of the target protein that undergoes frequent mutation and thus renders quinolone resistance. In the present study, ligands were tested for their inhibitory activity against DNA gyrase of Streptococcus pyogenes involved in DNA replication. In silico mutational analysis on modelled gyrase A revealed that GLU85 had the most possible interactions with all the ligands used for the study. The amino acid residue GLU85 had also been predicted with an essential role of maintaining the three‐dimensional structure of the protein. When introduced with a mutation (GLU 85 LYS) on this particular residue, it had readily denatured the whole α‐helix (from 80 to 90 amino acids). This was confirmed through the molecular dynamics simulation and revealed that this single mutation can cause many functional and structural changes. Furthermore, LYS85 mutation has altered the original secondary structure of the protein, which in turn led to the steric hindrance during the ligand–receptor interaction. The results based on the G‐score revealed that ligands have reduced interaction with the mutant protein. The semisynthetic fluoroquinolone 6d, which is an exception, forms a strong interaction with the mutant protein and was experimentally verified using the antimicrobial test. Hence, the present study unravels the fact that mutation at the drug binding site is the major cause for different level of resistance by the S. pyogenes when exposed against the varying concentrations of the fluoroquinolones. Furthermore, a comparative assessment of quinolone derivative with the older generation fluoroquinolones will be of great impact for S. pyogenes–related infections. Copyright © 2013 John Wiley & Sons, Ltd.     

AFLP assessment of genetic diversity among Indian Mucuna accessions

Amplified fragment length polymorphism (AFLP) marker was used to assess diversity in germplasm collection of Mucuna species which has gained tremendous attention in the recent past due to its promising nutritional, agronomic and medicinal attributes. Twenty five accessions comprising five species, collected from seven states of India were evaluated with twelve AFLP primer combinations that generated a total of 1,612 fragments with an average of 134 fragments per primer combination. The values of polymorphic information content (PIC), marker index (MI) and the resolving power (Rp) demonstrated the utility of the primer combinations used in the present study for discriminating the Mucuna accessions. UPGMA and Principal coordinate analysis (PCoA) of the genotypic data revealed clustering of accessions as per phenetic and genetic relationships. The Jaccard’s similarity coefficient values suggested good variability among the M. pruriens accessions indicating their utility in breeding programs. Molecular diversity presented in this study combined with the datasets on other morphological/agronomic traits will be highly useful for selecting appropriate accessions for plant improvement through conventional as well as molecular breeding approaches and for evolving suitable conservation strategies.     

Inhibiting NF-κB-inducing kinase (NIK): Discovery,structure-based design,synthesis, structure–activity relationship,and co-crystal structures

The discovery, structure-based design, synthesis, and optimization of NIK inhibitors are described. Our work began with an HTS hit, imidazopyridinyl pyrimidinamine 1. We utilized homology modeling and conformational analysis to optimize the indole scaffold leading to the discovery of novel and potent conformationally constrained inhibitors such as compounds 25 and 28. Compounds 25 and 31 were co-crystallized with NIK kinase domain to provide structural insights.     

Spectrofluorometric analytical applications of cyclodextrins

Cyclodextrins (CDs) are a family of cyclic oligosaccharides composed of α‐(1,4)‐linked glucopyranose subunits. The most important feature of CDs is their ability to form inclusion complexes (host–guest complexes) with a very wide range of solid, liquid and gaseous compounds by a molecular complexation. During the last decade, a considerable number of research papers has been focused on the use of CDs to enhance fluorescence intensity of different analytes and to develop CD‐induced spectrofluorimetric method. In this review, the various spectrofluorimetric methods based on host–inclusion complex are presented. Copyright © 2013 John Wiley & Sons, Ltd.     

Biosynthesis of silver nanoparticles using Bacillus subtilis EWP-46 cell-free extract and evaluation of its antibacterial activity

This study highlights the ability of nitrate-reducing Bacillus subtilis EWP-46 cell-free extract used for preparation of silver nanoparticles (AgNPs) by reduction of silver ions into nano silver. The production of AgNPs was optimized with several parameters such as hydrogen ion concentration, temperature, silver ion (Ag⁺ ion) and time. The maximum AgNPs production was achieved at pH 10.0, temperature 60 °C, 1.0 mM Ag⁺ ion and 720 min. The UV–Vis spectrum showed surface plasmon resonance peak at 420 nm, energy-dispersive X-ray spectroscopy (SEM–EDX) spectra showed the presence of element silver in pure form. Atomic force microscopy (AFM) and transmission electron microscopy images illustrated the nanoparticle size, shape, and average particle size ranging from 10 to 20 nm. Fourier transform infrared spectroscopy provided the evidence for the presence of biomolecules responsible for the reduction of silver ion, and X-ray diffraction analysis confirmed that the obtained nanoparticles were in crystalline form. SDS-PAGE was performed to identify the proteins and its molecular mass in the purified nitrate reductase from the cell-free extract. In addition, the minimum inhibitory concentration and minimum bactericidal concentration of AgNPs were investigated against gram-negative (Pseudomonas fluorescens) and gram-positive (Staphylococcus aureus) bacteria.     

Ligand‐based pharmacophore modelling and screening of DNA minor groove binders targeting Staphylococcus aureus

The recognition of DNA by small molecules is of special importance in the design of new drugs. Many natural and synthetic compounds have the ability to interact with the minor groove of DNA. In the present study, identification of minor groove binding compounds was attained by the combined approach of pharmacophore modelling, virtual screening and molecular dynamics approach. Experimentally reported 32 minor groove binding compounds were used to develop the pharmacophore model. Based on the fitness score, best three pharmacophore hypotheses were selected and used as template for screening the compounds from drug bank database. This pharmacophore‐based screening provides many compounds with the same pharmacological properties. All these compounds were subjected to four phases of docking protocols with combined Glide‐quantum‐polarized ligand docking approach. Molecular dynamics results indicated that selected compounds are more active and showed good interaction in the binding site of DNA. Based on the scoring parameters and energy values, the best compounds were selected, and antibacterial activity of these compounds was identified using in vitro antimicrobial techniques. Copyright © 2014 John Wiley & Sons, Ltd.     

Expression of Leptospira membrane proteins Signal Peptidase (SP) and Leptospira Endostatin like A (Len A) in BL-21(DE3) is toxic to the host cells

Heterologous expression of Integral Membrane Proteins (IMPs) is reported to be toxic to the host system in many studies. Even though there are reports on various concerns like transformation efficiency, growth properties, protein toxicity, inefficient expression and protein degradation in IMP overexpression, no studies so far addressed these issues in a comprehensive way. In the present study, two transmembrane proteins of the pathogen Leptospira interrogans, namely Signal peptidase (SP), and Leptospira Endostatin like A (Len-A) were taken along with a cytosolic protein Hydrolase (HYD) to assess the differences in transformation efficiency, protein toxicity, and protein stability when over expressed in Escherichia coli (E. coli). Bioinformatics analysis to predict the transmembrane localization indicated that both SP and Len are targeted to the membrane. The three proteins were expressed in full length in the E. coli expression strain, BL 21 (DE3). Significant changes were observed for the strains transformed with IMP genes under the parameters analysed such as, the transformation efficiency, survival of colonies on IPTG-plate, culture growth kinetics and protein expression compared to the strain harbouring the cytosolic protein gene.