Novel potent liposome agonists of triggering receptor expressed on myeloid cells 2 phenocopy antibody treatment in cells.

The receptor Triggering Receptor Expressed on Myeloid cells 2 (TREM2) is associated with several neurodegenerative diseases including Alzheimer's Disease and TREM2 stimulation represents a novel therapeutic opportunity. TREM2 can be activated by antibodies targeting the stalk region, most likely through receptor dimerization. Endogenous ligands of TREM2 are suggested to be negatively charged apoptotic bodies, mimicked by phosphatidylserine incorporated in liposomes and other polyanionic molecules likely binding to TREM2 IgV fold. However, there has been much discrepancy in the literature on the nature of phospholipids (PLs) that can activate TREM2 and on the stability of the corresponding liposomes over time. We describe optimized liposomes as robust agonists selective for TREM2 over TREM1 in cellular system. The detailed structure/activity relationship studies of lipid polar heads indicate that negatively charged lipid heads are required for activity and we identified the shortest maximally active PL sidechain. Optimized liposomes are active on both TREM2 common variant and TREM2 R47H mutant. Activity and selectivity were further confirmed in different native TREM2 expressing cell types including on integrated cellular responses such as stimulation of phagocytic activity. Such tool agonists will be useful in further studies of TREM2 biology in cellular systems alongside antibodies, and in the design of small molecule synthetic TREM2 agonists.

Poly(ethylene glycol acrylate)-functionalized hydrogels for heparan sulfate oligosaccharide recognition.

Heparan sulfates are complex polysaccharides belonging to the family of glycosaminoglycans that participate to the regulation of cell behavior and tissue homeostasis. The biological activities conferred to heparan sulfates are largely dependent on the content and positioning of the sulfate groups along their saccharidic units. At present, identification of particular sulfation patterns in biologically relevant heparan sulfate sequences remains challenging. Although several approaches for structure analysis exist, the complexity of heparan sulfates makes new and original approaches still required. Here, we used molecular imprinting technologies to prepare a library of polyethylene glycol acrylate functionalized hydrogels with the aim to investigate their applicability as specific recognizing systems for fondaparinux, a synthetic pentasaccharide analog to the antithrombin binding site of heparin. Adequate choice of the hydrogel composition and controlling rebinding conditions were important determinants for improving the sulfated oligosaccharide recognition specificity and selectivity. Our results suggest that molecular imprinting approaches could be a possibility for the specific recognition of biologically active sequences in heparan sulfates.

NMR and DFT analysis of trisaccharide from heparin repeating sequence.

NMR and density functional theory (DFT) have afforded detailed information on the molecular geometry and spin-spin coupling constants of a trisaccharide from the heparin repeating-sequence. The fully optimized molecular structures of two trisaccharide conformations (differing from each other in the form of the central iduronic acid residue) were obtained using the B3LYP/6-311+G(d,p) level of theory in the presence of solvent, the latter included as either explicit water molecules or via a continuum solvent model. NMR spin-spin coupling constants were also computed using various basis sets and functionals and then compared with measured experimental values. Optimized structures for both conformers showed differences in geometry at the glycosidic linkages and in the formation of intramolecular hydrogen bonds. Three-bond proton-proton coupling constants ((3)JH-C-C-H), based on fully optimized geometry computed using the B3LYP/6-311+G(d,p)/UFF level of theory and hydrated with 57 water molecules, showed that the best agreement with experiment was obtained with the 6-311+G(d,p) basis set and a weighted average of 55:45 ((1)C4:(2)S0) of the IdoA2S forms. Other basis sets, DGDZVP and TZVP, also gave acceptable data for most coupling constants, with DGDZVP outperforming the TZVP. Detailed analysis of Fermi-contact contributions to (3)JH-C-C-H showed that important contributions arise from oxygen at both glycosidic linkages, as well as from oxygen atoms on the neighboring monosaccharide units. Their contribution to the Fermi term cannot be neglected and must be taken into account for a correct description of coupling constants. The analysis also showed that the magnitude of paramagnetic (PSO) and diamagnetic (DSO) spin-orbit contributions is comparable to the magnitude of the Fermi-contact contribution in some coupling constants in the IdoA2S residue. Calculations of the localized molecular orbital contributions to the DSO terms from separate conformational residues showed that the contribution from adjacent residues is not negligible and can be important for the spin-spin coupling constants between protons located close to the geometrical center of the molecule. These contributions should be taken into account when interpreting DSO terms in spin-spin coupling constants especially in large molecules.

Characterization of glycosaminoglycan (GAG) sulfatases from the human gut symbiont Bacteroides thetaiotaomicron reveals the first GAG-specific bacterial endosulfatase.

Despite the importance of the microbiota in human physiology, the molecular bases that govern the interactions between these commensal bacteria and their host remain poorly understood. We recently reported that sulfatases play a key role in the adaptation of a major human commensal bacterium, Bacteroides thetaiotaomicron, to its host (Benjdia, A., Martens, E. C., Gordon, J. I., and Berteau, O. (2011) J. Biol. Chem. 286, 25973-25982). We hypothesized that sulfatases are instrumental for this bacterium, and related Bacteroides species, to metabolize highly sulfated glycans (i.e. mucins and glycosaminoglycans (GAGs)) and to colonize the intestinal mucosal layer. Based on our previous study, we investigated 10 sulfatase genes induced in the presence of host glycans. Biochemical characterization of these potential sulfatases allowed the identification of GAG-specific sulfatases selective for the type of saccharide residue and the attachment position of the sulfate group. Although some GAG-specific bacterial sulfatase activities have been described in the literature, we report here for the first time the identity and the biochemical characterization of four GAG-specific sulfatases. Furthermore, contrary to the current paradigm, we discovered that B. thetaiotaomicron possesses an authentic GAG endosulfatase that is active at the polymer level. This type of sulfatase is the first one to be identified in a bacterium. Our study thus demonstrates that bacteria have evolved more sophisticated and diverse GAG sulfatases than anticipated and establishes how B. thetaiotaomicron, and other major human commensal bacteria, can metabolize and potentially tailor complex host glycans.

Synthetic oligosaccharides as active pharmaceutical ingredients: lessons learned from the full synthesis of one heparin derivative on a large scale.

Covering: up to November 2013. Heparin and heparan sulfate are natural polysaccharides with strong structural variations, which are responsible for their numerous specific biological properties. One key target of heparin, among others, is antithrombin, a serine protease inhibitor that, upon activation, mainly targets anticoagulation factors IIa and Xa. It is well documented that inhibition of the latter is due to a specific pentasaccharidic sequence, its synthetic analog being the registered drug fondaparinux. The replacement of hydroxyls by methoxy groups, N-sulfates by O-sulfonates and the modulation of the sulfation pattern gave rise to both idraparinux and its neutralizable form, idrabiotaparinux, two pentasaccharides with a significantly increased half-life compared to fondaparinux. Although numerous efforts have been devoted to improving the chemoenzymatic preparation of heparin fragments, enzymes are usually selective for their natural substrates, which limits the generation of some specific non-natural structures. Up to now, total synthesis has proved to be a valuable approach for the preparation of tailor-made and pure saccharides in the milligram to gram scale. This highlight will focus on the synthesis and the technical challenges associated with the development and the production of complex carbohydrates which will be exemplified with idrabiotaparinux. Particular attention will be paid to the process improvements needed in order to implement the production in a pilot plant, achieving batch generation on a multi-kilogram scale with a purity higher than 99.5%, and with no unknown impurity over 0.1%.

Influence of heparin mimetics on assembly of the FGF.FGFR4 signaling complex.

Fibroblast growth factor (FGF) signaling regulates mammalian development and metabolism, and its dysregulation is implicated in many inherited and acquired diseases, including cancer. Heparan sulfate glycosaminoglycans (HSGAGs) are essential for FGF signaling as they promote FGF.FGF receptor (FGFR) binding and dimerization. Using novel organic synthesis protocols to prepare homogeneously sulfated heparin mimetics (HM), including hexasaccharide (HM(6)), octasaccharide (HM(8)), and decasaccharide (HM(10)), we tested the ability of these HM to support FGF1 and FGF2 signaling through FGFR4. Biological assays show that both HM(8) and HM(10) are significantly more potent than HM(6) in promoting FGF2-mediated FGFR4 signaling. In contrast, all three HM have comparable activity in promoting FGF1.FGFR4 signaling. To understand the molecular basis for these differential activities in FGF1/2.FGFR4 signaling, we used NMR spectroscopy, isothermal titration calorimetry, and size-exclusion chromatography to characterize binding interactions of FGF1/2 with the isolated Ig-domain 2 (D2) of FGFR4 in the presence of HM, and binary interactions of FGFs and D2 with HM. Our data confirm the existence of both a secondary FGF1.FGFR4 interaction site and a direct FGFR4.FGFR4 interaction site thus supporting the formation of the symmetric mode of FGF.FGFR dimerization in solution. Moreover, our results show that the observed higher activity of HM(8) relative to HM(6) in stimulating FGF2.FGFR4 signaling correlates with the higher affinity of HM(8) to bind and dimerize FGF2. Notably FGF2.HM(8) exhibits pronounced positive binding cooperativity. Based on our findings we propose a refined symmetric FGF.FGFR dimerization model, which incorporates the differential ability of HM to dimerize FGFs.