Adriane R. Todeschini

Synthesis and evaluation of analgesic, antiinflammatory and antiplatelet properties of new 2-pyridylarylhydrazone derivatives†

Abstract This work describes recent results from our research program aiming at the synthesis and pharmacological evaluation of new compounds acting as antiinflammatory, analgesic and platelet antiaggregatory. In this paper the synthesis and the pharmacological profile as analgesic, antiinflammatory and anti-platelet of new functionalized 2-pyridylarylhydrazone derivatives 5a–r are discussed. This class of N-heterocyclic derivatives represents a new series of prototype candidates with analgesic and antiinflammatory properties possessing also an important anti-aggregating activity. The pharmacological results herein disclosed suggest that the antiinflammatory and analgesic activities of these new pyridynehydrazone derivatives observed in the carrageenan pleurisy model and acetic acid writhing test, respectively, is probably due to an interference on the arachidonic acid (AA) metabolism. The most important antiinflammatory derivative 2-(2-formylfurane)pyridylhydrazone 5p presented a 79% inhibition of pleurisy at a dose of 80.1 μmol/kg. We also described the results concerning the mechanism of action of this series of N-heterocyclic derivatives in platelet aggregation which suggest a Ca2+ participation, probably by a complexation scavenger mechanism. Compound 2-(2-formylfurane)pyridylhydrazone 5p was able to complex Ca2+ in in vitro experiments at 100 μM concentration, indicating that this series of compounds can act as Ca2+ scavenger depending on the nature of the aryl moiety present at the imine subunit.

Ganglioside GM2-Tetraspanin CD82 Complex Inhibits Met and Its Cross-talk with Integrins, Providing a Basis for Control of Cell Motility through Glycosynapse

Glycosphingolipids (GSLs) at the cell surface membrane are associated or complexed with signal transducers (Src family kinases and small G-proteins), tetraspanins, growth factor receptors, and integrins. Such organizational framework, defining GSL-modulated or -dependent cell adhesion, motility, and growth, is termed “glycosynapse” (Hakomori, S., and Handa, K. (2002) FEBS Lett. 531, 88–92; Hakomori, S. (2004) Ann. Braz. Acad. Sci. 76, 553–572). We describe here the functional organization of the glycosynaptic microdomain, and the mechanisms for control of cell motility and invasiveness, in normal bladder epithelial HCV29 cells versus highly invasive bladder cancer YTS1 cells, both derived from transitional epithelia. (i) Ganglioside GM2, but not GM3 or globoside, interacted specifically with tetraspanin CD82, and such a complex inhibited hepatocyte growth factor (HGF)-induced activation of Met tyrosine kinase in a dose-dependent manner. (ii) Depletion of GM2 in HCV29 cells by treatment with d-threo-1-phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (P4), or reduction of CD82 expression by RNA interference, significantly enhanced HGF-induced Met tyrosine kinase and cell motility. (iii) In contrast, YTS1 cells, lacking CD82, displayed HGF-independent activation of Met tyrosine kinase and high cell motility. Transfection of the CD82 gene to YTS1 inhibited HGF dose-dependent Met tyrosine kinase activity and cell motility, due to formation of the GM2-CD82 complex. (iv) Adhesion of YTS1 or YTS1/CD82 cells to laminin-5-coated plates, as compared with noncoated plates, strongly enhanced Met activation, and the degree of activation was further increased in association with GSL depletion by P4. Laminin-5-dependent Met activation was minimal in HCV29 cells. These findings indicate that GSL, particularly GM2, forms a complex with CD82, and that such complex interacts with Met and thereby inhibits HGF-induced Met tyrosine kinase activity, as well as integrin to Met cross-talk.

Costimulation of Host T Lymphocytes by a Trypanosomal trans-Sialidase: Involvement of CD43 Signaling

Trans-sialidase is a membrane-bound and shed sialidase from Trypanosoma cruzi, the protozoan parasite responsible for Chagas disease. We investigated the role of soluble trans-sialidase on host CD4+ T cell activation. Trans-sialidase activated naive CD4+ T cells in vivo. Both enzymatically active and inactive recombinant trans-sialidases costimulated CD4+ T cell activation in vitro. Costimulation resulted in increased mitogen-activated protein kinase activation, proliferation, and cytokine synthesis. Furthermore, active and inactive trans-sialidases blocked activation-induced cell death in CD4+ T cells from T. cruzi-infected mice. By flow cytometry, inactive trans-sialidase bound the highly sialylated surface Ag CD43 on host CD4+ T cells. Both costimulatory and antiapoptotic effects of trans-sialidases required CD43 signaling. These results suggest that trans-sialidase family proteins are involved in exacerbated host T lymphocyte responses observed in T. cruzi infection.

Glycoinositolphospholipid from Trypanosoma cruzi: Structure, Biosynthesis and Immunobiology

The pathogenic protozoan parasite Trypanosoma cruzi expresses on its surface an unusual family of glycoinositolphospholipids (GIPLs) closely related to glycosylphosphatidylinositol (GPI) anchors. Different parasite isolates express distinct GIPLs which fall into two series, depending on the substitution of the third mannosyl residue in the conserved glycan sequence Man4-(AEP)-GlcN-InsPO4 by ethanolamine phosphate or beta-galactofuranose. Although the exact role of these molecules in the cell biology and pathogenicity of T. cruzi remains unknown, the lipid and glycan moieties impart distinct responses to host T and B lymphocytes and phagocytes, overall favouring an immune response permissive to the parasite. The biosynsthesis of GIPLs follows a pathway similar to that observed for GPI anchors. However, a more detailed understanding might enable the development of specific inhibitors of parasite-specific enzymes and lead to novel drugs to ameliorate Chagas disease.

Enzymatically Inactive trans-Sialidase from Trypanosoma cruzi Binds Sialyl and β-Galactopyranosyl Residues in a Sequential Ordered Mechanism

Host/parasite interaction mediated by carbohydrate/lectin recognition results in the attachment to and invasion of host cells and immunoregulation, enabling parasite replication and establishment of infection. Trypanosoma cruzi, the protozoan responsible for Chagas disease, expresses on its surface a family of enzymatically active and inactive trans-sialidases. The parasite uses the active trans-sialidase for glycoprotein sialylation in an unusual trans-glycosylation reaction. Inactive trans-sialidase is a sialic acid-binding lectin that costimulates host T cells through leucosialin (CD43) engagement. The co-mitogenic effect of trans-sialidase can be selectively abrogated by N-acetyllactosamine, suggesting the presence of an additional carbohydrate binding domain for galactosides, in addition to that for sialic acid. Here we investigated the interaction of inactive trans-sialidase in the presence of β-galactosides. By using NMR spectroscopy, we demonstrate that inactive trans-sialidase has a β-galactoside recognition site formed following a conformational switch induced by sialoside binding. Thus prior positioning of a sialyl residue is required for the β-galactoside interaction. When an appropriate sialic acid-containing molecule is available, both sialoside and β-galactoside are simultaneously accommodated in the inactive trans-sialidase binding pocket. This is the first report of a lectin recognizing two distinct ligands by a sequential ordered mechanism. This uncommon binding behavior may play an important role in several biological aspects of T. cruzi/host cell interaction and could shed more light into the catalytic mechanism of the sialic acid transfer reaction of enzymatically active trans-sialidase.

Biosynthetic Machinery Involved in Aberrant Glycosylation: Promising Targets for Developing of Drugs Against Cancer

Cancer cells depend on altered metabolism and nutrient uptake to generate and keep the malignant phenotype. The hexosamine biosynthetic pathway is a branch of glucose metabolism that produces UDP-GlcNAc and its derivatives, UDP-GalNAc and CMP-Neu5Ac and donor substrates used in the production of glycoproteins and glycolipids. Growing evidence demonstrates that alteration of the pool of activated substrates might lead to different glycosylation and cell signaling. It is already well established that aberrant glycosylation can modulate tumor growth and malignant transformation in different cancer types. Therefore, biosynthetic machinery involved in the assembly of aberrant glycans are becoming prominent targets for anti-tumor drugs. This review describes three classes of glycosylation, O-GlcNAcylation, N-linked, and mucin type O-linked glycosylation, involved in tumor progression, their biosynthesis and highlights the available inhibitors as potential anti-tumor drugs.

Trypanosoma cruzi Subverts Host Cell Sialylation and May Compromise Antigen-specific CD8+ T Cell Responses

Upon activation, cytotoxic CD8+ T lymphocytes are desialylated exposing β-galactose residues in a physiological change that enhances their effector activity and that can be monitored on the basis of increased binding of the lectin peanut agglutinin. Herein, we investigated the impact of sialylation mediated by trans-sialidase, a specific and unique Trypanosoma transglycosylase for sialic acid, on CD8+ T cell response of mice infected with T. cruzi. Our data demonstrate that T. cruzi uses its trans-sialidase enzyme to resialylate the CD8+ T cell surface, thereby dampening antigen-specific CD8+ T cell response that might favor its own persistence in the mammalian host. Binding of the monoclonal antibody S7, which recognizes sialic acid-containing epitopes on the 115-kDa isoform of CD43, was augmented on CD8+ T cells from ST3Gal-I-deficient infected mice, indicating that CD43 is one sialic acid acceptor for trans-sialidase activity on the CD8+ T cell surface. The cytotoxic activity of antigen-experienced CD8+ T cells against the immunodominant trans-sialidase synthetic peptide IYNVGQVSI was decreased following active trans-sialidase- mediated resialylation in vitro and in vivo. Inhibition of the parasites native trans-sialidase activity during infection strongly decreased CD8+ T cell sialylation, reverting it to the glycosylation status expected in the absence of parasite manipulation increasing mouse survival. Taken together, these results demonstrate, for the first time, that T. cruzi subverts sialylation to attenuate CD8+ T cell interactions with peptide-major histocompatibility complex class I complexes. CD8+ T cell resialylation may represent a sophisticated strategy to ensure lifetime host parasitism.

Evidences for the involvement of cell surface glycans in stem cell pluripotency and differentiation

Induced pluripotent stem (iPS) cells are somatic cells that have been reprogrammed to a pluripotent state via the introduction of defined transcription factors. Although iPS is a potentially valuable resource for regenerative medicine and drug development, several issues regarding their pluripotency, differentiation propensity and potential for tumorigenesis remain to be elucidated. Analysis of cell surface glycans has arisen as an interesting tool for the characterization of iPS. An appropriate characterization of glycan surface molecules of human embryonic stem (hES) cells and iPS cells might generate crucial data to highlight their role in the acquisition and maintenance of pluripotency. In this study, we characterized the surface glycans of iPS generated from menstrual blood-derived mesenchymal cells (iPS-MBMC). We demonstrated that, upon spontaneous differentiation, iPS-MBMC present high amounts of terminal β-galactopyranoside residues, pointing to an important role of terminal-linked sialic acids in pluripotency maintenance. The removal of sialic acids by neuraminidase induces iPS-MBMC and hES cells differentiation, prompting an ectoderm commitment. Exposed β-galactopyranose residues might be recognized by carbohydrate-binding molecules found on the cell surface, which could modulate intercellular or intracellular interactions. Together, our results point for the first time to the involvement of the presence of terminal sialic acid in the maintenance of embryonic stem cell pluripotency and, therefore, the modulation of sialic acid biosynthesis emerges as a mechanism that may govern stem cell differentiation.

Structural elucidation of the repeat unit in highly branched acidic exopolysaccharides produced by nitrogen fixing Burkholderia

Burkholderia kururiensis, strain M130, an endophytic diazotrophic bacterium isolated from rice roots, produces acetylated acidic exopolysaccharides which can be separated by anion exchange chromatography. These were characterized by nuclear magnetic resonance spectroscopy, methylation analysis and Smith degradation. The exopolysaccharides eluted with 0.5 M NaCl were produced when the bacterium was grown in a medium containing mannitol as the sole carbon source, and showed to be a mixture of two different polymers, composed of hepta or octasaccharide repeat units, consistent with following structure: [structure: see text]. The ability of diazotrophic Burkholderia to produce two exopolysaccharides that differ by the presence of a terminal glucosyl residue provides insight into polysaccharide function with potentially significant biological consequences in the endophytic-host plant interaction.

The Major Surface Carbohydrates of the Echinococcus granulosus Cyst: Mucin-Type O-Glycans Decorated by Novel Galactose-Based Structures †

The cestodes constitute important but understudied human and veterinary parasites. Their surfaces are rich in carbohydrates, on which very little structural information is available. The tissue-dwelling larva (hydatid cyst) of the cestode Echinococcus granulosus is outwardly protected by a massive layer of carbohydrate-rich extracellular matrix, termed the laminated layer. The monosaccharide composition of this layer suggests that its major carbohydrate components are exclusively mucin-type O-glycans. We have purified these glycans after their release from the crude laminated layer and obtained by MS and NMR the complete structure of 10 of the most abundant components. The structures, between two and six residues in length, encompass a limited number of biosynthetic motifs. The mucin cores 1 and 2 are either nondecorated or elongated by a chain of Galpbeta1-3 residues. This chain can be capped by a single Galpalpha1-4 residue, such capping becoming more dominant with increasing chain size. In addition, the core 2 N-acetylglucosamine residue is in cases substituted with the disaccharide Galpalpha1-4Galpbeta1-4, giving rise to the blood P(1)-antigen motif. Larger, also related, glycans exist, reaching at least 18 residues in size. The glycans described are related but larger than those previously described from an Echinococcus multilocularis mucin [Hulsmeier, A. J., et al. (2002) J. Biol. Chem. 277, 5742-5748]. Our results reveal that the E. granulosus cyst exposes to the host only a few different major carbohydrate motifs. These motifs are composed essentially of galactose units and include the elongation by (Galpbeta1-3)(n) and the capping by Galpalpha1-4, novel in animal mucin-type O-glycans.