Adhesive interactions of leukocytes and endothelial cells initiate leukocyte migration to inflamed tissue and are important for immune surveillance. Acute and chronic inflammatory diseases show a dysregulated immune response and result in a massive efflux of leukocytes that contributes to further tissue damage. Therefore, targeting leukocyte trafficking may provide a potent form of antiinflammatory therapy. Leukocyte migration is initiated by interactions of the cell adhesion molecules E-, L-, and P-selectin and their corresponding carbohydrate ligands. Compounds that efficiently address these interactions are therefore of high therapeutic interest. Based on this rationale we investigated synthetic dendritic polyglycerol sulfates (dPGS) as macromolecular inhibitors that operate via a multivalent binding mechanism mimicking naturally occurring ligands. dPGS inhibited both leukocytic L-selectin and endothelial P-selectin with high efficacy. Size and degree of sulfation of the polymer core determined selectin binding affinity. Administration of dPGS in a contact dermatitis mouse model dampened leukocyte extravasation as effectively as glucocorticoids did and edema formation was significantly reduced. In addition, dPGS interacted with the complement factors C3 and C5 as was shown in vitro and reduced C5a levels in a mouse model of complement activation. Thus, dPGS represent an innovative class of a fully synthetic polymer therapeutics that may be used for the treatment of inflammatory diseases.anti-inflammatory drug | complement inhibition | multiple target binding | multivalent selectin inhibitor | synthetic polymer

A versatile route for the synthesis of highly functionalized, polyanionic macromolecules based on dendritic polyglycerol was applied by means of the Huisgen-Sharpless-Meldal 1,3-dipolar cycloaddition ("click-reaction") of polyglycerolazide precursors and alkyne-functionalized anions such as sulfonates, carboxylates, phosphonates, and bisphosphonates. In addition, the corresponding polyglycerol phosphate has been synthesized via direct hydroxyl interconversion of polyglycerol to the corresponding phosphate with a degree of functionalization >80% by analogy to the synthesis of previously reported polyglycerol sulfates (dPGS). On the basis of the finding that dPGS exhibits high affinity for L- and P-selectin, the potential of these novel polyanionic, multivalent macromolecules of varying anionic nature as L-selectin inhibitors has been evaluated in vitro by means of a competitive concentration dependent binding assay. Affinity of all polyanions toward L-selectin was demonstrated with distinct IC(50) values ranging from the low nanomolar to the high micromolar range. The efficiency of L-selectin inhibition increases in the order carboxylate < phosphate < phosphonate ≈ sulfonate < bisphosphonate < sulfate. Additional DLS and ζ-potential measurements of these polyanions were performed to correlate their binding affinity toward L-selectin with their anionic nature. However, a direct correlation of effective charge and particle size with the determined IC(50) values turned out to require further in-depth studies on the microstructure of the polyanions but clearly indicate an exceptional position of dPGS among the studied dendritic polyelectrolytes.

␣-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors undergo constitutive and ligand-induced internalization that requires dynamin and the clathrin adaptor complex AP-2. We report here that an atypical basic motif within the cytoplasmic tails of AMPA-type glutamate receptors directly associates with 2-adaptin by a mechanism similar to the recognition of the presynaptic vesicle protein synaptotagmin 1 by AP-2. A synaptotagmin 1-derived AP-2 binding peptide competes the interaction of the AMPA receptor subunit GluR2 with AP-2 and increases the number of surface active glutamate receptors in living neurons. Moreover, fusion of the GluR2-derived tail peptide with a synaptotagmin 1 truncation mutant restores clathrin/AP-2-dependent internalization of the chimeric reporter protein. These data suggest that common mechanisms regulate AP-2-dependent internalization of pre-and postsynaptic membrane proteins.endocytosis ͉ postsynaptic ͉ sorting signal ͉ synaptic plasticity F ast neurotransmission at excitatory synapses is mediated by heterotetrameric ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors composed of combinations of four subunits (GluR1-4). AMPA receptors interact with different factors including the transmembrane protein stargazin (1), PDZ proteins GRIP1/ABP, SAP97, and PICK1, and NSF (2). Accumulating evidence suggests that rapid changes in functional postsynaptic AMPA receptor numbers are important means of controlling synaptic efficacy (2-5). AMPA receptors undergo constitutive and regulated clathrin-and dynamin-dependent endocytosis via distinct AMPA-or NMDAinduced signaling cascades (reviewed in refs. 2-5). How exactly AMPA receptor cargo is targeted for clathrin-mediated internalization remains an open question. One possibility is that AMPA receptors are recognized by endocytic adaptor proteins such as the clathrin adaptor complex AP-2, a major endocytic protein interaction hub (6-8). NMDA-induced AMPA receptor internalization can be blocked by overexpression of a GluR2 cytoplasmic tail (CT) peptide (pep2r) or by mutating the putative AP-2 binding motif within the GluR2 CT. Infusion of hippocampal CA1 neurons with the putative AP-2-blocking peptide prevents induction of long-term depression (LTD), suggesting that the association of GluR2 with AP-2 may be an important determinant for NMDA-induced LTD (7). Whether AP-2 directly binds to GluR2 CTs and via which of its four subunits is unknown.Here we have identified the molecular determinants responsible for binding of AP-2 to the CTs of AMPA-type glutamate receptors. We demonstrate that the 2 subunit of AP-2 interacts directly and with nanomolar affinity with a basic motif found in CTs of GluR1-3 and the presynaptic vesicle protein synaptotagmin 1. Our data thus suggest that common mechanisms regulate AP-2-dependent internalization of pre-and postsynaptic membrane proteins.

Hyperbranched polyglycerols (HPGs) are ideal scaffolds for the multivalent presentation of saccharides, due to their biocompatible, carbohydrate-like properties; here, we report the conjugation of galactosesugar moieties to HPG, and the multivalent effect of these constructs on selectin binding.