Species-specific and conserved epitopes on mouse and human E-selectin important for leukocyte adhesion.

Selectins are C-type, cell surface lectins that are key players in leukocyte adhesion to the blood vessel wall endothelium. We describe here epitopes for a series of novel monoclonal antibodies (moAbs), UZ4-UZ7, directed against mouse E-selectin. All four antibodies specifically bind to mouse E-selectin, but not to P- or L-selectin, and all inhibit the adhesion of granulocytes, peripheral blood lymphocytes, and promyelocytic HL-60 cells to cytokine-activated mouse endothelium. Three moAbs, UZ5, UZ7, and UZ6, specifically inhibit mouse E-selectin-mediated adhesion by binding to epitopes in domains CR1 or CR2. moAb UZ4 inhibits leukocyte adhesion to both human and murine endothelium activated with IL-1 or other proinflammatory stimuli. UZ4 is the first described moAb that detects an epitope in the lectin domain which is conserved in both murine and human E-selectin (CXKKKL), but is not present in the other members of the selectin family, P- and L-selectin. Interestingly, UZ5, UZ6, and UZ7 more efficiently interfere with lymphocyte than with granulocyte adhesion to cytokine-activated endothelium, while UZ4 completely blocks adhesion of PMN, lymphocytes, and HL-60 and U937 cell lines. The data suggest that E-selectin-ligand engagement differs between lymphocytes and PMN, and that these differences may be accentuated by the CR1 and CR2 domains in the E-selectin cell adhesion molecule.

Statins selectively inhibit leukocyte function antigen-1 by binding to a novel regulatory integrin site.

The beta2 integrin leukocyte function antigen-1 (LFA-1) has an important role in the pathophysiology of inflammatory and autoimmune diseases. Here we report that statin compounds commonly used for the treatment of hypercholesterolemia selectively blocked LFA-1-mediated adhesion and costimulation of lymphocytes. This effect was unrelated to the statins' inhibition of 3-hydroxy-3-methylglutaryl coenzyme-A reductase; instead it occurred via binding to a novel allosteric site within LFA-1. Subsequent optimization of the statins for LFA-1 binding resulted in potent, selective and orally active LFA-1 inhibitors that suppress the inflammatory response in a murine model of peritonitis. Targeting of the statin-binding site of LFA-1 could be used to treat diseases such as psoriasis, rheumatoid arthritis, ischemia/reperfusion injury and transplant rejection.

A homogeneous fluorometric assay for measuring cell adhesion to immobilized ligand using V-well microtiter plates.

We have developed a homogeneous high-capacity assay format for measuring integrin- and selectin-dependent cell binding to immobilized ligand using V-well microtiter plates. 2',7'-Bis(2-carboxyethyl)-5-(and-6)-carboxylfluorescence, acetoxymethylester-labeled cells are added to ligand-coated V-shaped microtiter wells. Bound cells are separated from free cells using centrifugal force to produce shear stress. Nonadherent cells accumulate in the nadir of the well and are measured using a fluorescence plate reader. Antibody or low-molecular-weight inhibitors of either the ligand or the cell surface receptor result in less cell binding, more cells in the pellet, and increased signal. The optimization and validation of the very late antigen-4/vascular cell adhesion molecule-1 assay is described in detail. We demonstrate that this assay can be rapidly adapted to measure other integrin- and selectin-mediated interactions. This assay format has several advantages over conventional assays. The centrifugal process is biologically relevant and eliminates the washing steps to remove nonadherent cells that can cause well-to-well and plate-to-plate variation. Because the assay is robust with a high signal-to-noise ratio and low variability, it is ideally suited for studying multiple parameters of cell adhesion and for high capacity screening.

Structural basis for LFA-1 inhibition upon lovastatin binding to the CD11a I-domain.

The lymphocyte function-associated antigen (LFA-1) belongs to the family of beta2-integrins and plays an important role in T-cell activation and leukocyte migration to sites of inflammation. We report here that lovastatin, a drug clinically used for lowering cholesterol levels, inhibits the interaction of human LFA-1 with its counter-receptor intercellular adhesion molecule-1. Using nuclear magnetic resonance spectroscopy and X-ray crystallography we show that the inhibitor binds to a highly conserved domain of the LFA-1 alpha-chain called the I-domain. The first three-dimensional structure of an integrin inhibitor bound to its receptor reveals atomic details for a hitherto unknown mode of LFA-1 inhibition. It also sheds light into possible mechanisms of LFA-1 mediated signalling and will support the design of novel anti-adhesive and immunosuppressive drugs.

Assembly of binding loops on aromatic templates as VCAM-1 mimetics.

The design and synthesis of cyclic mimetics of VCAM-1 protein that reproduce the integrin-binding domain are presented. The unprotected peptide precursor 37-43, Thr-Gln-Ile-Asp-Ser-Pro-Leu, was grafted onto functional templates of type naphthalene, biphenyl and benzyl through the chemoselective formation of C- and N-terminal oximes resulting in a mixture of four isomeric forms due to syn-anti isomerism of the oxime bonds. Some isomers could be monitored by HPLC and identified by NMR. The molecule containing a naphthalene-derived template was found to inhibit the VCAM-1/VLA-4 interaction more efficiently than previously reported for sulfur-bridged cyclic peptides containing similar sequences. The finding confirms the importance of incorporating conformational constraints between the terminal ends of the peptide loop 37-43 in the design of synthetic inhibitors of the VCAM-1/integrin interaction.

Selectin/glycoconjugate binding assays for the identification and optimization of selectin antagonists.

In this study we describe ELISA-type P- and L-selectin binding assays for the analysis of selectin antagonists. A biotinylated polyacrylamide-type glycoconjugate containing sialyl Lewis A (sLe(a)-polymer) is utilized as a synthetic ligand for both selectins analogous to the E-selectin assay we have developed recently. Following precomplexation of sLe(a)-polymer with streptavidin-peroxidase, the complex is added to microtiter plates coated with the recombinant selectins. Binding of sLe(a)-polymer to the immobilized selectins is measured by the peroxidase reaction. SLe(a)-polymer was found to bind to P- and L-selectin in a cation-dependent manner. The interaction of the polymer was blocked by neutralizing anti-P- and anti-L-selectin antibody, respectively. The reference compounds heparin and fucoidan inhibited in both assays. Sialyl Lewis X (sLe(x)) blocked binding to L-selectin by 46% at 3 mM, whereas no inhibition was observed in the P-selectin assay up to 3 mM. Control polymers containing sialic acid or beta-d-glucose instead of sLe(a) weakly bound or failed to bind to the selectins. Both assays are rapid to perform and of low variability. The P-selectin assay was successfully employed to identify and optimize novel carbohydrate-based P-selectin antagonists. The P-, L-, and E-selectin assays were used to determine the fine selectivity of several sLe(x)-related selectin antagonists. These studies together suggest that sLe(a)-polymer-based selectin assays are well suited for primary screening and the characterization of selectin antagonists.

Design, synthesis and biological evaluation of aryl-substituted sialyl Lewis X mimetics prepared via cross-metathesis of C-fucopeptides.

Several aryl substituted C-fucopeptides have been developed as sialyl Lewis X mimetics. Although the compounds have a much simpler structure compared to SLe(x), up to 3-times higher binding affinity toward E-selectin and > 1000 times toward P-selectin was observed. Furthermore, a convenient strategy for generating a number of analogues from a SLe(x) mimetic template at a very late stage of the synthesis was introduced, using a ruthenium catalyzed cross olefin metathesis under benchtop conditions.

Synthesis of sialyl Lewis x mimetics as selectin inhibitors by enzymatic aldol condensation reactions.

Several D-mannosyl phosphate/phosphonate derivatives have been enzymatically prepared as sialyl Lewis x tetrasaccharide mimics, which showed strong-to-moderate inhibition against E-, P-, and L-selectins. The synthesis of these mimics is very straightforward; mannosyl aldehyde derivatives are condensed with dihydroxyacetone phosphate (DHAP) in the presence of a DHAP-dependent aldolase to provide mannosyl phosphates.

Synthesis of sialyl Lewis X mimetics using the Ugi four-component reaction.

Application of the Ugi four-component condensation to rapidly synthesize a library of glycopeptide mimics of the tetrasaccharide SLe(x) as inhibitors of E- and P-selectin, and to study the effect of varied functionality in mimics on the inhibition is described.

Synthesis of novel 6-amido-6-deoxy-l-galactose derivatives as sialyl Lewis X mimetics.

The synthesis and biological potency of several sialyl Lewis X (SLe(x)) mimetics is described. These mimics incorporate all of the critical functional groups present in SLe(x) necessary for binding to E-selectin. L-Galactose is used to mimic the naturally occurring L-fucose residue in SLe(x) due to the identical arrangement of the 2-, 3-, and 4-hydroxyl groups. Several synthetically and enzymatically prepared amino acids were used to mimic the D-galactose residue. Because of the variability incorporated in the synthesis of these amino acids the spatial requirements necessary for efficient binding were investigated. A carboxylate bearing side chain was introduced as a sialic acid mimic and the chain length was varied to maximize biological activity. By investigating the optimal arrangement of these two factors mimics were produced which were up twofold more active than SLe(x).

Design and synthesis of C-linked fucosides as inhibitors of E-selectin.

Two series of C-linked fucosides as mimetics for the tetrasaccharide sialyl Lewis X have been synthesized and tested as inhibitors of E-Selectin. The fucopeptides have been prepared from three key intermediates, including alpha-C-allyl fucose, natural and unnatural amino acids bearing hydroxyl groups and an alpha, omega-diacid moiety for the imitation of the essential three parts of SLex, i.e., the Fuc, Gal, and NeuAc. The nature and distance of the linkage of the fucose moiety to the amino acids as well as the distance between the amino acids and the terminal carboxylic acid group turned out to be crucial for the biological activity. In addition the necessity of both OH groups (4- and 6-OH) in the Gal part could be confirmed. Conformational NMR study of the most active mimetic supports the structure-activity relationship. A second series of mimetics was prepared, where Fuc and Gal moieties were purely C-linked. In the synthesis of beta-C-allyl galactose an intramolecular 1,2-hydride shift led to an interesting side product. However, the substituted glycosidic oxygens led to a substantial loss of conformational constrain, which could not be compensated and resulted in low activity.

An E-selectin binding assay based on a polyacrylamide-type glycoconjugate.

Here we show that biotinylated polyacrylamide-type glycoconjugates which contain sialyl Lewis X (sLex-polymer) or sialyl Lewis A (sLea-polymer) are ligands for E-selectin. sLea-polymer bound E-selectin with higher affinity than sLex-polymer. Based on this property we used the sLea-polymer to establish a sensitive cell-free binding assay for the characterization of E-selectin antagonists. The assay involves complexation of the biotinylated sLea-polymer with streptavidin-peroxidase. This complex is incubated with E-selectin mouse Ckappa fusion protein immobilized onto microtiter plates. Bound complex is detected by the peroxidase reaction. sLea-polymer bound in a Ca2+-dependent manner consistent with the function of E-selectin as a C-type lectin. Control glycoconjugates with sialic acid (alpha-Neu5Ac), Lewis A (Lea), or beta-D-glucose residues instead of sLea failed to interact with the E-selectin. Neutralizing anti-E-selectin antibodies blocked completely binding to E-selectin. This demonstrates specificity of the assay system. sLex blocked binding of the sLea-polymer to E-selectin by 50% at a concentration of 550 microM (IC50). The assay was used to characterize sLea-polymers with differing sLea content as multivalent inhibitors of E-selectin binding. The inhibitory activity of these polymeric forms of sLea increased with their sLea content up to IC50s in the low micromolar range. The binding assay described is sensitive, rapid, and simple and of low variability. Therefore it should be advantageous for the identification and characterization of novel E-selectin antagonists.

Functional serotonin 5-HT1D receptors and 5-HT1D beta receptor mRNA expression in human umbilical vein endothelial cells.

Pharmacological evidence has suggested the presence of 5-hydroxytryptamine (5-HT, serotonin), 5-HT(1D) receptors on endothelial cells but these receptors have never been identified unambiguously on this type of cells. We now report that human umbilical vein endothelial cells (HUVEC) express 5-HT(1D) receptors coupled to inhibition of cyclic AMP formation. 5-HT and 5-HT(1D) receptor agonists 5-carboxamidotryptamine (5-CT) and sumatriptan were approximately equipotent at inhibiting forskolin-stimulated cyclic AMP accumulation in HUVEC (mean pEC50 7.6-8.2, maximal effect 30% inhibition). The 5-HT(1A) receptor antagonist, 8-OH-DPAT was clearly less potent (pEC50 6.2) and less efficacious. The selective 5-HT(1D) receptor agonist, GR127935 (1 nM) markedly inhibited the effect of 5-HT (apparent pK(B) 10.8). Reverse transcription-polymerase chain reaction analysis showed the mRNA for 5-HT(1D beta) receptors to be expressed in HUVEC. These results demonstrate the presence of functional 5-HT(1D) receptors and the expression of 5-HT(1D beta) receptor mRNA in HUVEC. They support the involvement of 5-HT(1D beta) receptors in endothelial-mediated responses to 5-HT.