Platinum (II) and palladium (II) complexes with (N,N') and (C,N,N') ligands derived from pyrazole as anticancer and antimalarial agents: synthesis, characterization and in vitro activities

The study of the reactivity of three 1-(2-dimethylaminoethyl)-1H-pyrazole derivatives of general formula [1-(CH2)2NMe2}-3,5-R2-pzol] {where pzol represents pyrazole and Rdouble bond; length as m-dashH (1a), Me (1b) or Ph (1c)} with [MCl2(DMSO)2] (Mdouble bond; length as m-dashPt or Pd) under different experimental conditions allowed us to isolate and characterize cis-[M{κ2-N,N′-{[1-(CH2)2NMe2}-3,5-R2-pzol])}Cl2] {MMdouble bond; length as m-dashPtPt (2a-2c) or Pd (3a-3c)} and two cyclometallated complexes [M{κ3-C,N,N′-{[1-(CH2)2NMe2}-3-(C5H4)-5-Ph-pzol])}Cl] {Mdouble bond; length as m-dashPt(II) (4c) or Pd(II) (5c)}. Compounds 4c and 5c arise from the orthometallation of the 3-phenyl ring of ligand 1c. Complex 2a has been further characterized by X-ray crystallography. Ligands and complexes were evaluated for their in vitro antimalarial against Plasmodium falciparum and cytotoxic activities against lung (A549) and breast (MDA MB231 and MCF7) cancer cellular lines. Complexes 2a-2c and 5c exhibited only moderate antimalarial activities against two P. falciparum strains (3D7 and W2). Interestingly, cytotoxicity assays revealed that the platinacycle 4c exhibits a higher toxicity than cisplatin in the three human cell lines and that the complex 2a presents a remarkable cytotoxicity and selectivity in lung (IC50 = 3 μM) versus breast cancer cell lines (IC50 > 20 μM). Thus, complexes 2c and 4c appear to be promising leads, creating a novel family of anticancer agents. Electrophoretic DNA migration studies in presence of the synthesized compounds have been performed, in order to get further insights into their mechanism of action

Thiacetazone, an Antitubercular Drug that Inhibits Cyclopropanation of Cell Wall Mycolic Acids in Mycobacteria

Background. Mycolic acids are a complex mixture of branched, long-chain fatty acids, representing key components of the highly hydrophobic mycobacterial cell wall. Pathogenic mycobacteria carry mycolic acid sub-types that contain cyclopropane rings. Double bonds at specific sites on mycolic acid precursors are modified by the action of cyclopropane mycolic acid synthases (CMASs). The latter belong to a family of S-adenosyl-methionine-dependent methyl transferases, of which several have been well studied in Mycobacterium tuberculosis, namely, MmaA1 through A4, PcaA and CmaA2. Cyclopropanated mycolic acids are key factors participating in cell envelope permeability, host immunomodulation and persistence of M. tuberculosis. While several antitubercular agents inhibit mycolic acid synthesis, to date, the CMASs have not been shown to be drug targets. Methodology/Principle Findings. We have employed various complementary approaches to show that the antitubercular drug, thiacetazone (TAC), and its chemical analogues, inhibit mycolic acid cyclopropanation. Dramatic changes in the content and ratio of mycolic acids in the vaccine strainMycobacterium bovis BCG, as well as in the related pathogenic speciesMycobacterium marinum were observed after treatment with the drugs. Combination of thin layer chromatography, mass spectrometry and Nuclear Magnetic Resonance (NMR) analyses of mycolic acids purified fromdrug-treated mycobacteria showed a significant loss of cyclopropanation in both the a- and oxygenated mycolate sub-types. Additionally, High-Resolution Magic Angle Spinning (HR-MAS) NMR analyses on whole cells was used to detect cell wall-associated mycolates and to quantify the cyclopropanation status of the cell envelope. Further, overexpression of cmaA2, mmaA2 or pcaA in mycobacteria partially reversed the effects of TAC and its analogue on mycolic acid cyclopropanation, suggesting that the drugs act directly on CMASs. Conclusions/Significance. This is a first report on them echanism of action of TAC, demonstrating the CMASs as its cellular targets in mycobacteria. The implications of this study may be important for the design of alternative strategies for tuberculosis treatment

Polysaccharide structural variability in mycobacteria: identification and characterization of phosphorylated mannan and arabinomannan.

Arabinomannan (AMannan) and mannan (Mannan) are major polysaccharides antigens of the mycobacterial capsule. They are highly related to the lipoarabinomannan (LAM) and lipomannan (LM) lipoglycans of the cell wall, known to participate to the immunopathogenesis of mycobacterial infections. Here we present the identification of two related polysaccharides from Mycobacterium kansasii that co-purified with AMannan and Mannan. Structural analysis using GC, MALDI-MS and NMR clearly established these molecules as non-acylated phosphorylated AMannan and Mannan designated P-AMannan and P-Mannan, respectively. These glycoconjugates represent a new source of polysaccharide structural variability in mycobacteria and constitute unique tools for structure-activity relationship studies in order to investigate the role of fatty acids in the biological functions of LAM and LM. The potential participation of these polysaccharides in influencing the outcome of the infection is also discussed

Mycobacterial lipomannan induces MAP kinase phosphatase-1 expression in macrophages.

Mycobacterial lipomannan (LM) and lipoarabinomannan (LAM) regulate macrophage activation by interacting with Toll-like receptors (TLRs). The intracellular signalling pathways elicited by these complex molecules are poorly defined. We have demonstrated that LM purified from various mycobacterial species, but not LAM from Mycobacterium kansasii or Mycobacterium bovis BCG, induced expression of the MAP kinase phosphatase 1 (MKP-1) in macrophages. Anti-TLR2 antibodies, as well as specific ERK and p38 MAPK inhibitors, decreased MKP-1 transcription in LM-stimulated cells. These findings suggest that the binding of LM to TLR2 triggers MAPK activation, followed by an up-regulation of MKP-1 expression, which in turn may act as a negative regulator of MAPK activation

Binding of NADP + triggers an open-to-closed transition in a mycobacterial FabG β-ketoacyl-ACP reductase

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Structural patterns of rhamnogalacturonans modulating Hsp-27 expression in cultured human keratinocytes

International audiencePolysaccharide extracts were obtained from chestnut bran (Castanea sativa), grape marc (Vitis vinifera) and apple marc (Malus spp.) and fractionated by size exclusion chromatography after endopolygalacturonase degradation. Compositional and linkage analyses by GC and GC-MS showed the characteristic rhamnogalacturonan structure with specific arabinan (apple marc) and type II arabinogalactan (chestnut bran, grape marc) side chains. Type II arabinogalactan rhamnogalacturonan from chestnut bran significantly stimulated the in vitro differentiation of human keratinocytes, giving evidence of a tight structure-function relationship. This molecule comprises short and ramified 3- and 3,6-β-D-galactan and 5- and 3,5-α-L-arabinan side chains, but also contains significant amounts of t-Xyl and 4-Xyl with a characteristic 2:1 ratio. Enzymatic hydrolysis of this polysaccharide produced fragments of lower molecular weight with unchanged xylose content which conserved the same ability to stimulate human keratinocyte differentiation. It could be then speculated that dimeric xylosyl-xylose and/or longer oligomeric xylose side chains attached to a galacturonan and closely associated to hairy rhamno-galacturonan domains are essential patterns that could determine the biological activity of pectins

Involvement of ST6Gal I‐mediated α2,6 sialylation in myoblast proliferation and differentiation

Myogenesis is a physiological process which involves the proliferation of myoblasts and their differentiation into multinucleated myotubes, which constitute the future muscle fibers. Commitment of myoblasts to differentiation is regulated by the balance between the myogenic factors Pax7 and MyoD. The formation of myotubes requires the presence of glycans, especially N‐glycans, on the cell surface. We examined here the involvement of α2,6 sialylation during murine myoblastic C2C12 cell differentiation by generating a st6gal1‐knockdown C2C12 cell line; these cells exhibit reduced proliferative potential and precocious differentiation due to the low expression of Pax7. The earlier fusion of st6gal1‐knockdown cells leads to a high fusion index and a drop in reserve cells (Pax7+/MyoD−). In st6gal1‐knockdown cells, the Notch pathway is inactivated; consequently, Pax7 expression is virtually abolished, leading to impairment of the proliferation rate. All these results indicate that the decrease in α2,6 sialylation of N‐glycans favors the differentiation of most cells and provokes a significant loss of reserve cells

Mucus composition and bacterial communities associated with the tissue and skeleton of three scleractinian corals maintained under culture conditions

International audienceCorals live in close association with bacterial communities, but the nature of the relationship is still poorly understood. In this study, three scleractinian coral species, Galaxea fascicularis, Pavona cactus and Turbinaria reniformis were incubated under different laboratory conditions, and the composition of the bacterial community associated with their tissue or skeleton was compared between species or between species and seawater using denaturing gradient gel electrophoresis (DGGE). The amount of dissolved organic carbon (DOC) excreted and the mucus glycoconjugate composition were also determined for each species. The aim of the study was to assess if the bacterial community composition was species-specific or linked either to the seawater composition, or to the quality and quantity of carbon released by each coral. Results obtained showed that DOC release was significantly different (P < 0.0001) for the three species, with the highest excretion rate for G. fascicularis. Also, the mucus of G. fascicularis and P. cactus mainly contained galactose and glucose whereas the mucus of T. reniformis contained more glucose and xylose. Cluster analyses of microbial community composition showed that the bacterial community was species-specific in the coral tissue but not in the skeleton, in all conditions. It remained specific when corals were incubated in the same or in different aquaria, and under different seawater renewal rates. Since DOC release rates and bacterial composition were both different according to the coral species considered, a link might be suggested between the two parameters. Sequencing of DGGE bands indicated that some bacterial phylotypes were consistently retrieved in all samples of a given species

Mycobacterium marinum lipooligosaccharides are unique caryophyllose-containing cell wall glycolipids that inhibit tumor necrosis factor-alpha secretion in macrophages.

International audienceEarlier studies have reported a role for lipooligosaccharides (LOSs) in sliding motility, biofilm formation, and infection of host macrophages in Mycobacterium marinum. Although a LOS biosynthetic gene cluster has recently been identified in this species, many structural features of the different LOSs (LOS-I-IV) are still unknown. This clearly hampers assessing the contribution of each LOS in mycobacterial virulence as well as structure-function-based studies of these important cell wall-associated glycolipids. In this study, we have identified an M. marinum isolate, M. marinum 7 (Mma7), which failed to produce LOS-IV but instead accumulated large amounts of LOS-III. Local genomic comparison of the LOS biosynthetic cluster established the presence of a highly disorganized region in Mma7 compared with the standard M strain, characterized by multiple genetic lesions that are likely to be responsible for the defect in LOS-IV production in Mma7. Our results indicate that the glycosyltransferase LosA alone is not sufficient to ensure LOS-IV biosynthesis. The availability of different M. marinum strains allowed us to determine the precise structure of individual LOSs through the combination of mass spectrometric and NMR techniques. In particular, we established the presence of two related 4-C-branched monosaccharides within LOS-II to IV sequences, of which one was never identified before. In addition, we provided evidence that LOSs are capable of inhibiting the secretion of tumor necrosis factor-alpha in lipopolysaccharide-stimulated human macrophages. This unexpected finding suggests that these cell wall-associated glycolipids represent key effectors capable of interfering with the establishment of a pro-inflammatory response