Structure-based design of selective phosphodiesterase 4B inhibitors based on ginger phenolic compounds

Phosphodiesterase 4 (PDE4) has been established as a drug target for inflammatory diseases of respiratory tract like asthma and chronic obstructive pulmonary disease. The selective inhibitors of PDE4B, a subtype of PDE4, are devoid of adverse effects like nausea and vomiting commonly associated with non-selective PDE4B inhibitors. This makes the development of PDE4B subtype selective inhibitors a desirable research goal. Thus, in the present study, molecular docking, molecular dynamic simulations and binding free energy were performed to explore potential selective PDE4B inhibitors based on ginger phenolic compounds. The results of docking studies indicate that some of the ginger phenolic compounds demonstrate higher selective PDE4B inhibition than existing selective PDE4B inhibitors. Additionally, 6-gingerol showed the highest PDE4B inhibitory activity as well as selectivity. The comparison of binding mode of PDE4B/6-gingerol and PDE4D/6-gingerol complexes revealed that 6-gingerol formed additional hydrogen bond and hydrophobic interactions with active site and control region 3 (CR3) residues in PDE4B, which were primarily responsible for its PDE4B selectivity. The results of binding free energy demonstrated that electrostatic energy is the primary factor in elucidating the mechanism of PDE4B inhibition by 6-gingerol. Dynamic cross-correlation studies also supported the results of docking and molecular dynamics simulation. Finally, a small library of molecules were designed based on the identified structural features, majority of designed molecules showed higher PDE4B selectivity than 6-gingerol. These results provide important structural features for designing new selective PDE4B inhibitors as anti-inflammatory drugs and promising candidates for synthesis and pre-clinical pharmacological investigations.     

First report of leaf spot disease caused by Phoma eupyrena Sacc. on Aloe vera from Madhya Pradesh,India

A leaf spot disease caused by Phoma eupyrena Sacc. on Aloe vera was reported from Madhya Pradesh, India. Disease symptoms were observed as irregular to elongated, sunken lesion on both leaf surfaces which later turns creamish brown with maroon margin. The fungal colonies were whitish or light pale to dark grey colour. The pycnidia were glabrous and solitary, papillate, and indistinctly uniostiolate averages 100–260 μm in diameter. Conidia were ellipsoidal, 4.5–7.5 × 2.5–3.75 μm in size, with two large guttules. To the best of our knowledge, this is the first report of P. eupyrena causing leaf spot disease on A. vera in India.     

Probiotic Lactobacillus reuteri promotes TNF-induced apoptosis in human myeloid leukemia-derived cells by modulation of NF-kappaB and MAPK signalling

The molecular mechanisms of pro-apoptotic effects of human-derived Lactobacillus reuteri ATCC PTA 6475 were investigated in this study. L. reuteri secretes factors that potentiate apoptosis in myeloid leukemia-derived cells induced by tumour necrosis factor (TNF), as indicated by intracellular esterase activity, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labelling assays and poly (ADP-ribose) polymerase cleavage. L. reuteri downregulated nuclear factor-κB (NF-κB)-dependent gene products that mediate cell proliferation (Cox-2, cyclin D1) and cell survival (Bcl-2, Bcl-xL). L. reuteri suppressed TNF-induced NF-κB activation, including NF-κB-dependent reporter gene expression in a dose-and time-dependent manner. L. reuteri stabilized degradation of IκBα and inhibited nuclear translocation of p65 (RelA). Although phosphorylation of IκBα was not affected, subsequent polyubiquitination necessary for regulated IκBα degradation was abrogated by L. reuteri . In addition, L. reuteri promoted apoptosis by enhancing mitogen-activated protein kinase (MAPK) activities including c-Jun N-terminal kinase and p38 MAPK. In contrast, L. reuteri suppressed extracellular signal-regulated kinases 1/2 in TNF-activated myeloid cells. L. reuteri may regulate cell proliferation by promoting apoptosis of activated immune cells via inhibition of IκBα ubiquitination and enhancing pro-apoptotic MAPK signalling. An improved understanding of L. reuteri- mediated effects on apoptotic signalling pathways may facilitate development of future probiotics-based regimens for prevention of colorectal cancer and inflammatory bowel disease.     

Thiophene containing triarylmethanes as antitubercular agents

A new series of thiophene containing triarylmethane derivatives were synthesized from the Friedel-Crafts alkylation of diarylcarbinols followed by incorporation of amino alkyl chains. These were evaluated against Mycobacterium tuberculosis H37R(v) and showed the activity in the range of 3.12-12.5 microg/mL in vitro.     

The Hin recombinase assembles a tetrameric protein swivel that exchanges DNA strands

Most site-specific recombinases can be grouped into two structurally and mechanistically different classes. Whereas recombination by tyrosine recombinases proceeds with little movements by the proteins, serine recombinases exchange DNA strands by a mechanism requiring large quaternary rearrangements. Here we use site-directed crosslinking to investigate the conformational changes that accompany the formation of the synaptic complex and the exchange of DNA strands by the Hin serine recombinase. Efficient crosslinking between residues corresponding to the ‘D-helix’ region provides the first experimental evidence for interactions between synapsed subunits within this region and distinguishes between different tetrameric conformers that have been observed in crystal structures of related serine recombinases. Crosslinking profiles between cysteines introduced over the 35 residue E-helix region that constitutes most of the proposed rotating interface both support the long helical structure of the region and provide strong experimental support for a subunit rotation mechanism that mediates DNA exchange.     

A Nonsense Mutation in a Cinnamyl Alcohol Dehydrogenase Gene Is Responsible for the Sorghum brown midrib6 Phenotype

brown midrib6 (bmr6) affects phenylpropanoid metabolism, resulting in reduced lignin concentrations and altered lignin composition in sorghum (Sorghum bicolor). Recently, bmr6 plants were shown to have limited cinnamyl alcohol dehydrogenase activity (CAD; EC 1.1.1.195), the enzyme that catalyzes the conversion of hydroxycinnamoyl aldehydes (monolignals) to monolignols. A candidate gene approach was taken to identify Bmr6. Two CAD genes (Sb02g024190 and Sb04g005950) were identified in the sorghum genome based on similarity to known CAD genes and through DNA sequencing a nonsense mutation was discovered in Sb04g005950 that results in a truncated protein lacking the NADPH-binding and C-terminal catalytic domains. Immunoblotting confirmed that the Bmr6 protein was absent in protein extracts from bmr6 plants. Phylogenetic analysis indicated that Bmr6 is a member of an evolutionarily conserved group of CAD proteins, which function in lignin biosynthesis. In addition, Bmr6 is distinct from the other CAD-like proteins in sorghum, including SbCAD4 (Sb02g024190). Although both Bmr6 and SbCAD4 are expressed in sorghum internodes, an examination of enzymatic activity of recombinant Bmr6 and SbCAD4 showed that Bmr6 had 1 to 2 orders of magnitude greater activity for monolignol substrates. Modeling of Bmr6 and SbCAD4 protein structures showed differences in the amino acid composition of the active site that could explain the difference in enzyme activity. These differences include His-57, which is unique to Bmr6 and other grass CADs. In summary, Bmr6 encodes the major CAD protein involved in lignin synthesis in sorghum, and the bmr6 mutant is a null allele.Plant cell walls constitute a vast reserve of fixed carbon. Cellulose and lignin are the first and second most abundant polymers on the planet, respectively (Jung and Ni, 1998). The world community has started to look to biomass as substrates for plant-based biologically sustainable fuels, which would mitigate carbon dioxide emission and reduce petroleum dependence (Sarath et al., 2008; Schmer et al., 2008). In the current generation of biofuels, ethanol is being synthesized via the fermentation of grain starch or sugarcane juice. For the next generation of biofuels, research is being directed toward the conversion of lignocellulosic biomass into biofuels (Chang, 2007). As bioenergy technologies progress, the conversion of biomass to biofuels could involve a range of chemical, biochemical, and fermentation processes to produce biofuels; alternate biofuels, such as butanol or dimethylfuran, are also on the horizon (Ezeji et al., 2007; Roman-Leshkov et al., 2007). Most liquid biofuel production processes will likely rely on the conversion of the cell wall polysaccharides cellulose and hemicellulose into monomeric sugars.Plant cell walls consist of a complex polysaccharide moiety composed of cellulose microfibrils, composed of β-1,4-linked Glc polymers (Carpita and McCann, 2000). Connecting the cellulose microfibrils to each other is a hemicellulose network, whose structure and composition are species dependent, and which is mainly composed of glucuronoarabinoxylans in grasses (Carpita and McCann, 2000). Lignin, a nonlinear heterogeneous polymer derived from aromatic precursors, cross-links these polysaccharides, rigidifying and reinforcing the cell wall structure (Carpita and McCann, 2000). The addition of lignin polymers to the polysaccharide matrix creates a barrier that is chemically and microbially resistant.Lignin can block the liberation of sugars from the cell wall polysaccharide moieties, release compounds that can inhibit microbes used for fermenting sugars to fuels, and adhere to hydrolytic enzymes. Understanding lignin synthesis, structure, and function to increase cell wall digestibility has long been a goal for forage improvement and paper processing (Mackay et al., 1997; Jung and Ni, 1998). Recently, manipulating lignin has also become an important target for bioenergy feedstock improvement (Chen and Dixon, 2007; Li et al., 2008).Lignin is derived from the phenylpropanoid pathway and contains primarily three types of phenolic subunits: p-hydroxyphenyl, guaiacyl, and syringyl units (Dixon et al., 2001). The phenolic aldehyde precursors are reduced into their corresponding alcohols (monolignols) and subsequently transported to the cell wall (Fig. 1), where laccases and peroxidases catalyze lignin polymerization through the formation of monolignol radicals (Boerjan et al., 2003). Therefore, most research efforts to manipulate lignin have focused on biosynthesis of the monolignols. Most of the enzymes involved in monolignol synthesis have been cloned and characterized in Arabidopsis (Arabidopsis thaliana) and other dicot species, using both mutagenic and transgenic approaches to study the impact of these gene products on dicot cell walls (Anterola and Lewis, 2002). However, there are significant differences in the architecture, polysaccharide composition, and phenylpropanoid composition of grass cell walls compared with those of dicots (Carpita and McCann, 2000; Vogel and Jung, 2001). For example, grasses contain significant amounts of p-coumaric acid and ferulic acid that are cross-linked to cell wall polysaccharides through ester and ether linkages in addition to their presence in lignin (Grabber et al., 1991; Boerjan et al., 2003). Because many of the proposed dedicated bioenergy crops are grasses, there is a need to identify and understand the function of the gene products involved in lignin biosynthesis in these species (Vermerris et al., 2007; Li et al., 2008; Sarath et al., 2008).Open in a separate windowFigure 1.The CAD enzyme and its role in the monolignol biosynthetic pathway. A, CAD catalyzes the conversion of cinnamyl aldehydes to alcohols using NADPH as its cofactor. p-Coumaryl aldehyde and alcohol, R₁ and R₂ = H; caffeoyl aldehyde and alcohol, R₁ and R₂ = OH; coniferyl aldehyde and alcohol, R₁ = H and R₂ = OCH₃; sinapyl aldehyde and alcohol, R₁ and R₂ = OCH₃. B, A simplified model of the lignin biosynthetic pathway where CAD catalyzes the final step in monolignol biosynthesis.The brown midrib phenotype has been useful for identifying mutants affecting lignin synthesis in grasses because it is a visible phenotype. Spontaneous brown midrib mutants were first discovered in maize (Zea mays; Jorgenson, 1931) and were subsequently generated in sorghum (Sorghum bicolor) using diethyl sulfate mutagenesis (Porter et al., 1978). Brown midrib mutants in maize, sorghum, and pearl millet (Pennisetum glaucum) have increased forage digestibility for livestock (Cherney et al., 1990; Akin et al., 1993; Jung et al., 1998; Oliver et al., 2004). In maize and sorghum, there are at least four brown midrib loci in their respective genomes (Jorgenson, 1931; Porter et al., 1978; Gupta, 1995). The genes encoding bm3 in maize and bmr12 in sorghum are the only loci cloned to date, and both encode highly similar caffeic acid O-methyl transferases (Vignols et al., 1995; Bout and Vermerris, 2003). A second brown midrib locus associated with reduced cinnamyl alcohol dehydrogenase (CAD) activity has been identified both in maize (bm1; Halpin et al., 1998) and sorghum (bmr6; Bucholtz et al., 1980; Pillonel et al., 1991). CAD is a member of the alcohol dehydrogenase superfamily of proteins that catalyzes the conversion of the hydroxycinnamoyl aldehydes into alcohols prior to their incorporation into lignin polymers (Fig. 1). Reduced CAD activity results in increased digestibility on dry weight basis, altered cell wall architecture, reduced lignin level, and the incorporation of phenolic aldehydes into lignin in sorghum and maize (Pillonel et al., 1991; Provan et al., 1997; Halpin et al., 1998; Marita et al., 2003; Shi et al., 2006; Palmer et al., 2008). The reduced CAD activity in bm1 has been genetically mapped to a region of the maize genome that contained a CAD gene, ZmCAD2 (Halpin et al., 1998), but a mutation was not identified. However, it has recently been shown that bm1 down-regulated the expression of several lignin biosynthetic genes, suggesting its gene product may be a regulatory protein (Shi et al., 2006; Guillaumie et al., 2007).To identify the mutation responsible for the bmr6 phenotype and to characterize how bmr6 impacts the lignin biosynthetic pathway, a candidate gene approach was taken. Here, we describe the cloning and characterization of Bmr6 and a related protein, SbCAD4. The identification and characterization of Bmr6 has revealed the major monolignol CAD protein in the grasses, which is likely to aid the development of new strategies to increase conversion of sorghum and other grass feedstocks to biofuels.     

Peganine hydrochloride dihydrate an orally active antileishmanial agent

Protozoic infections caused by genus Leishmania pose an enormous public health threat in developing countries, compounded by the toxicity and resistance to current therapies. Under the aegis of our ongoing program on drug discovery and development on antileishmanial agents from plants, we carried out bioassay guided fractionation on Peganum harmala seeds which resulted in the isolation of 1 as an antileishmanial agent. 2D-NMR spectral data and single crystal X-ray crystallography data indicated 1 as peganine hydrochloride in dihydrated form. The compound 1 exhibited in-vitro activity against both extracellular promastigotes as well as intracellular amastigotes residing within murine macrophages in Leishmania donovani. Furthermore, 1 also exhibited in-vivo activity, 79.6 (±8.07)% against established VL in hamsters at a dose of 100 mg/kg b.wt.