Maintenance of recombinant plasmid vectors in host bacteria relies on the presence of selection antibiotics in the growth media to suppress plasmid -free segregants. However, presence of antibiotic resistance genes and antibiotics themselves is not acceptable in several applications of biotechnology. Previously, we have shown that FabV-Triclosan selection system can be used to select high and medium copy number plasmid vectors in E. coli. Here, we have extended our previous work and demonstrated that expression vectors containing FabV can be used efficiently to express heterologous recombinant proteins in similar or better amounts in E. coli host when compared with expression vectors containing β-lactamase. Use of small amount of non-antibiotic Triclosan as selection agent in growth medium, enhanced plasmid stability, applicability in various culture media, and compatibility with other selection systems for multiple plasmid maintenance are noteworthy features of FabV-Triclosan selection system.
A novel and potent series of ene-amides featuring azetidines has been developed as FabI inhibitors active against drug resistant Gram-positive pathogens particularly staphylococcal organisms. Most of the compounds from the series possessed excellent biochemical inhibition of Staphylococcus aureus FabI enzyme and whole cell activity against clinically relevant MRSA, MSSA and MRSE organisms which are responsible for significant morbidity and mortality in community as well as hospital settings. The binding mode of one of the leads, AEA16, in Escherichia coli FabI enzyme was determined unambiguously using X-ray crystallography. The lead compounds displayed good metabolic stability in mice liver microsomes and pharmacokinetic profile in mice. The in vivo efficacy of lead AEA16 has been demonstrated in a lethal murine systemic infection model.
A series of novel 2-amino-4-(3-hydroxy-4-phenoxyphenyl)-6-(4-substituted phenyl) nicotinonitriles were synthesized and evaluated against HepG2, A-549 and Vero cell lines. Compounds 3b (IC50 16.74 ± 0.45 µM) and 3p (IC50 10.57 ± 0.54 µM) were found to be the most active compounds against A-549 cell line among the evaluated compounds. Further 3b- and 3p-induced apoptosis was characterized by AO/EB (acridine orange/ethidium bromide) nuclear staining method and also by DNA fragmentation study. A decrease in cell viability and initiation of apoptosis was clearly evident through the morphological changes in the A-549 cells treated with 3b and 3p when stained with this method. Fragmentation of DNA into nucleosomes was observed which further confirmed the cell apoptosis in cells treated with compound 3b. Flow cytometry studies confirmed the cell cycle arrest at G2/M phase in A549 cells treated with compound 3b. Further in silico studies performed supported the in vitro anticancer activity of these compounds as depicted by dock score and binding energy values.