Satish Kumar Awasthi

Bactericidal antisense effects of peptide-PNA conjugates

Antisense peptide nucleic acids (PNAs) can specifically inhibit Escherichia coli gene expression and growth and hold promise as anti-infective agents and as tools for microbial functional genomics. Here we demonstrate that chemical modification improves the potency of standard PNAs. We show that 9- to 12-mer PNAs, especially when attached to the cell wall/membrane-active peptide KFFKFFKFFK, provide improvements in antisense potency in E. coli amounting to two orders of magnitude while retaining target specificity. Peptide–PNA conjugates targeted to ribosomal RNA (rRNA) and to messenger RNA (mRNA) encoding the essential fatty acid biosynthesis protein Acp prevented cell growth. The anti-acpP PNA at 2 μM concentration cured HeLa cell cultures noninvasively infected with E. coli K12 without any apparent toxicity to the human cells. These results indicate that peptides can be used to carry antisense PNA agents into bacteria. Such peptide–PNA conjugates open exciting possibilities for anti-infective drug development and provide new tools for microbial genetics.

Cell-Dependent Differential Cellular Uptake of PNA, Peptides, and PNA-Peptide Conjugates

Peptide nucleic acid (PNA) oligomers were conjugated to cell-penetrating peptides: pAnt, a 17-residue fragment of the Drosophila protein Antennapedia, and pTat, a 14-amino acid fragment of HIV protein Tat. A 14-mer PNA was attached to the peptide by disulfide linkage or by maleimide coupling. The uptake of (directly or indirectly, via biotin) fluorescein-labeled peptides, PNAs, or PNA-peptide conjugates was studied by fluorescence microscopy, confocal laser scanning microscopy, and fluorometry in five cell types. In SK-BR-3, HeLa, and IMR-90 cells, the PNA-peptide conjugates and a T1, backbone-modified PNA were readily taken up (2 microM). The PNA was almost exclusively confined to vesicular compartments in the cytosol. However, the IMR-90 cells also showed a weak diffuse staining of the cytoplasm. In the U937 cells, we observed a very weak and exclusively vesicular staining with the PNA-peptide conjugates and the T(lys)-modified PNA. No evident uptake of the unmodified PNA was seen. In H9 cells, both peptides and the PNA-peptide conjugates quickly associated with the membrane, followed by a weak intracellular staining. A cytotoxic effect resulting in artificial staining of the cells was observed with fluoresceinated peptides and PNA-peptide conjugates at concentrations above 5-10 microM, depending on cell type and incubation time. We conclude that uptake of PNAs in many cell types can be achieved either by conjugating to certain peptides or simply by charging the PNA backbone using lysine PNA units. The uptake is time, temperature, and concentration dependent and mainly endocytotic. Our results also show that proper controls for cytotoxicity should always be carried out to avoid misinterpretation of visual data.

Antifilarial Activity of 1,3-Diarylpropen-1-One: Effect on Glutathione-S-Transferase, a Phase II Detoxification Enzyme

Chalcone derivatives were evaluated for their antifilarial activity on Setaria cervi using glutathione-S-transferase (GST) as a drug target. The compounds 1-(4-benzotriazol-1-yl-phenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (5), and 3-(4-methoxyphenyl)-1-(4-pyrrolidin-1-yl-phenyl) prop-2-en-1-one (7) showed a significant suppression (P < 0.01) in GST activity of adult female parasite extract at 3 microM concentration in vitro. However, GST activity was detected along with depletion in GSH level. Except Compounds 1 and 2, all exhibited a significant effect on the motility and viability of adult parasites. Compounds 3-(4-chlorophenyl)-1-(4-piperidin-1-yl-phenyl)prop-2-en-1-one (3), 1-(4-benzotriazol-1-yl-phenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (5), and 3-(4-methoxyphenyl)-1-(4-pyrrolidin-1-yl-phenyl) prop-2-en-1-one (7) exhibited major irreversible effects on viability and resulted in parasite death and also inhibited the GST activity by 84-100% in vitro. We report for the first time the antifilarial activity of chalcones on GST of adult parasites. This study also strengthens our previous findings where GST is reported as a potential drug target for antifilarials.

Antimalarial Activity of Newly Synthesized Chalcone Derivatives In Vitro

Twenty‐seven novel chalcone derivatives were synthesized using Claisen‐Schmidt condensation and their antimalarial activity against asexual blood stages of Plasmodium falciparum was determined. Antiplasmodial IC50 (half‐maximal inhibitory concentration) activity of a compound against malaria parasites in vitro provides a good first screen for identifying the antimalarial potential of the compound. The most active compound was 1‐(4‐benzimidazol‐1‐yl‐phenyl)‐3‐(2, 4‐dimethoxy‐phenyl)‐propen‐1‐one with IC50 of 1.1 μg/mL, while that of the natural phytochemical, licochalcone A is 1.43 μg/mL. The presence of methoxy groups at position 2 and 4 in chalcone derivatives appeared to be favorable for antimalarial activity as compared to other methoxy‐substituted chalcones. Furthermore, 3, 4, 5‐trimethoxy groups on chalcone derivative probably cause steric hindrance in binding to the active site of cysteine protease enzyme, explaining the relative lower inhibitory activity.

Antimalarial pharmacodynamics of chalcone derivatives in combination with artemisinin against Plasmodium falciparum in vitro

Use of artemisinin based combination therapies (ACTs) is increasing in treatment of malaria. Their extensive and indiscriminate deployment will ultimately lead to selection of resistance. Thus, alternate ACTs are needed. We reported in vitro antimalarial potential of chalcone derivatives. A few potent chalcones were selected for their antimalarial interaction in combination with artemisinin in vitro. Combinations evaluated show synergistic or additive interactions. Chalcones act on broad range of asexual stages of the parasite. The synergistic combinations decrease hemozoin formation in parasitized erythrocytes. These combinations do not affect new permeation pathways induced in the host cells. This is the first report showing antiplasmodial interactions between artemisinin and synthetic chalcone azole derivatives. Thus, chalcones and artemisinin combinations open the possibility of novel ACTs.

Inhibition of Mycobacterium smegmatis Gene Expression and Growth Using Antisense Peptide Nucleic Acids

Antisense agents that inhibit genes at the mRNA level are attractive tools for genome-wide studies and drug target validation. The approach may be particularly well suited to studies of bacteria that are difficult to manipulate with standard genetic tools. Antisense peptide nucleic acids (PNA) with attached carrier peptides can inhibit gene expression in Escherichia coli and Staphylococcus aureus. Here we asked whether peptide-PNAs could mediate antisense effects in Mycobacterium smegmatis. We first targeted the gfp reporter gene and observed dose- and sequence-dependent inhibition at low micromolar concentrations. Sequence alterations within both the PNA and target mRNA sequences eliminated inhibition, strongly supporting an antisense mechanism of inhibition. Also, antisense PNAs with various attached peptides showed improved anti-gfp effects. Two peptide-PNAs targeted to the essential gene inhA were growth inhibitory and caused cell morphology changes that resemble that of InhA-depleted cells. Therefore, antisense peptide-PNAs can efficiently and specifically inhibit both reporter and endogenous essential genes in mycobacteria.

Diversification in the synthesis of antimalarial trioxane and tetraoxane analogs

Artemisinin and its semi synthetic analogs have established themselves as effective antimalarial drugs. Since artemisinin is naturally isolated, in short supply and is very expensive to synthesize, extensive research on the discovery of peroxidic antimalarial compounds has intrigued scientists to explore structurally simple yet diverse cyclic peroxides. Consequently, trioxanes and tetraoxanes have been identified as promising candidates for the development of the next generation of antimalarial drugs. To combine the latest synthetic routes and methodologies employed in their synthesis along with earlier reports (dating back to 1899), we present an integrated overview on the diversified synthesis of trioxanes, tetraoxanes and their hybrids.

Introducing nanocrystalline CeO2 as heterogeneous environmental friendly catalyst for the aerobic oxidation of para-xylene to terephthalic acid in water

CeO2 nanoparticles exposed in (100) and (111) surfaces have been synthesized and explored as a heterogeneous catalyst for the first time in the oxidation of para-xylene to terephthalic acid. The synthesis and catalysis reaction was environmental friendly, where water was used as the solvent. Ceria nanoparticles were synthesized with controlled size of 15 nm and high surface area of 268 m2 g−1 magnitude. These particles were exploited as a novel heterogeneous catalyst for aqueous phase oxidation of para-xylene to bypass all the hazardous steps involve in the manufacture of industrially important terephthalic acid. The result shows the formation of 30–40% terephthalic acid under mild reaction condition, i.e. at 70 °C in water, by avoiding the corrosive bromide promoter and acetic acid solvent. The recyclability studies reveal that the recovered ceria catalyst retained its activity in para-xylene conversion without the change in the fluorite crystal structure, crystallite size and morphology of CeO2. At last, a radical mechanism for this particular catalytic activity of the catalysis reaction has been proposed based on the high surface area and the corresponding available exposed active (100) and (111) surfaces.

Synthetic application of gold nanoparticles and auric chloride for the synthesis of 5-substituted 1H-tetrazoles

An effective one-pot, convenient gold catalyzed synthesis of 5-substituted 1H-tetrazoles has been discussed. The study demonstrated the comparative overview for utilization of gold(III) and gold nano-particles (spheres) as a catalyst. Detailed understandings of the mechanism, surface area effect (in reference to nanoparticles) of gold in the activation of nitriles for nucleophilic (3 + 2) cycloaddition of sodium azide to give the product have also been studied.

2-(4-Chloro-phen-yl)chromen-4-one.

The title compound, C15H9ClO2, is a synthetic flavonoid obtained by the cyclization of 3-(4-chlorophenyl)-1-(2-hydroxyphenyl)prop-2-en-1-one. The 4-chlorophenyl ring is twisted at an angle of 11.54° with respect to the chromen-4-one skeleton. In the crystal, pairs of molecules are interconnected by weak Cl⋯Cl interactions [3.3089 (10) Å] forming dimmers which are further peripherally connected through intermolecular C—H⋯O hydrogen bonds.