Selectin Antagonists Research Articles

ChemInform Abstract: Drug Design, Synthesis, and Evaluation of a Non‐sugar‐based Selectin Antagonist.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

ChemInform Abstract: Design and Synthesis of (E)‐Selectin Antagonists

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Drug design, synthesis, and evaluation of a non-sugar-based selectin antagonist

We have designed a series of simple rigid compounds ( 2) having a phenyl ring attached to three essential groups necessary for selectin binding, i.e., a fucose unit, a carboxylic acid, and the hydrophobic part. In this series of compound 2, 2a exhibited strong inhibitory activity in in vitro P-selectin mediated cell adhesion assay. The novel type of compound 2a would be a potential lead compound for selectin antagonist.

Solid-phase synthesis of hydroxyproline-based cyclic hexapeptides

Cyclic peptides are excellent tools to investigate the functional and spatial requirements for ligands to bind to a given target. In this paper we report the synthesis of a library of cyclic hexapeptides, designed to be Selectin antagonists. Based on molecular modelling calculations, these peptides contain a hydroxyproline building block that serves also as the point of attachment to the solid phase. A modified THP linker has been prepared to bind the hydroxy group of this amino acid to aminomethyl SynPhase™ Lanterns. Amino acids of the d- and l-series are used and their influence onto the cyclisation step is also investigated.

Studies on selectin blocker. 9. SARs of non-sugar selectin blocker against E-, P-, L-selectin bindings

As a part of study of selectin blockers, we have already reported that a non-sugar selectin antagonist ( 3) was successfully discovered using a computational screening (Hiramatsu, Y.; Tsukida, T.; Nakai, Y.; Inoue, Y.; Kondo, H. J. Med. Chem. 2000, 43, 1476). To investigate the SARs of compound 3 against E-, P-, and L-selectins, we synthesized the derivatives of compound 3 and evaluated their inhibitory activities toward selectin bindings. The structural diversity of compound 3 contained the following: (1) a modification of the spacer unit ( 4– 7), (2) a modification of the tail unit ( 8– 11), (3) a modification of the head unit ( 12– 18). As a result, it was found that a non-sugar based selectin blocker ( 3) could be a potential lead compound for E-, P-, and L-selectin blockers and some of the derivatives showed broad and/or selective inhibitory activities toward the E-, P-, and L-selectins. In addition, it was found that the experimental evidence well supported that the computational screening using 3D-pharmacophore model could be useful methodology to find out a new lead for the several type of selectin blockers, which included a broad and/or a selective inhibitor.

Development of potent non-carbohydrate imidazole-based small molecule selectin inhibitors with antiinflammatory activity.

A novel series of non-carbohydrate imidazole-based selectin inhibitors has been discovered via high-throughput screening using a P-selectin ELISA-based assay system. The initial lead 1 had an IC(50) of 17 microM in the P-selectin ELISA; this potency was significantly improved via an extensive SAR exploration. One of the current lead compounds (29) has an IC(50) of 300 nM in a P-selectin ELISA; it also has good activity in P- and E-selectin cell adhesion assays and shows efficacy in vivo. These compounds represent a novel series of sLe(X) mimetics with antiinflammatory activity. Their unique profile supports our interest in their further evaluation as drug candidates for the treatment of inflammation. Herein we describe the syntheses, optimization, and SAR of this series of novel potent selectin antagonists.

Design and Synthesis of E-Selectin Antagonists

Selectin-mediated recruitment of leukocytes plays a crucial role in a number of diseases and pathological situations, for example in inflammation, reperfusion injury, rheumatoid arthritis and respiratory diseases. Substantial research efforts are directed toward development of carbohydrate-derived drugs that interfere with the inflammatory response by blocking the selectin binding site. This article describes two approaches for the improvement of the inhibitory potency of the lead structure sialyl Lewisx (sLex). One approach is based on the preorganization of mimics in their bioactive conformation to reduce entropic costs. For the conformational analysis of mimics, molecular modeling based tools were developed. They allow the rational design of selectin antagonists with simplified structures, but increased inhibitory potency. Alternatively, additional carbohydrate/lectin contacts can be identified for the improvement of enthalpic contributions. Following this approach, an additional hydrophobic interaction of the antagonists with E-selectin leads to a 60-fold improvement of E-selectin affinity. Antagonists have been synthesized using chemical or chemoenzymatic methods. Finally, a flow chart for the biological evaluation of the antagonists is presented.

P-Selectin Antagonism Causes Dose-dependent Venous Thrombosis Inhibition

Inhibition of P-selectin by antibody or selectin antagonist decreases inflammation and thrombosis. This study evaluates the dose-response relationship using a selectin receptor antagonist. Eight male baboons (Papio anubis) underwent inferior vena caval thrombosis using a 6 h balloon occlusion model. Three animals received 500 microg/kg P-selectin antagonist (rPSGL-Ig) and five 1 mg/kg rPSGL-Ig with or without a non-anticoagulant dose of Dalteparin. These animals were compared to our published results in this model with 4 saline controls and 8 animals that received 4 mg/kg rPSGL-Ig. A statistically significant dose-response relationship existed between rPSGL-Ig dose and thrombosis (p < 0.01), and between rPSGL-Ig dose and spontaneous recanalization (p<0.05). Inflammatory assessment revealed decreased gadolinium enhancement in all rPSGL-Ig groups compared to previously reported control, despite no significant differences in inflammatory cell extravasation. No dose of rPSGL-Ig caused anticoagulation. Selectin antagonism results in a dose-dependent decrease in thrombosis and increase in spontaneous recanalization.

TNF-alpha increases entry of macromolecules into luminal endothelial cell glycocalyx.

The endothelial luminal glycocalyx has been largely ignored as a target in vascular pathophysiology even though it occupies a key location. As a model of the inflammatory response, we tested the hypothesis that tumor necrosis factor-alpha (TNF-alpha) can alter the properties of the endothelial apical glycocalyx. In the intact hamster cremaster microcirculation, fluorescein isothiocyanate (FITC)-labeled Dextrans 70, 580, and 2,000 kDa are excluded from a region extending from the endothelial surface almost 0.5 micrometer into the lumen. This exclusion zone defines the boundaries of the glycocalyx. Red blood cells (RBC) under normal flow conditions are excluded from a region extending even farther into the lumen. The cremaster microcirculation was pretreated with topical or intrascrotal applications of TNF-alpha. After infusion of FITC-dextran, FITC-albumin, or FITC-immunoglubulin G (IgG) via a femoral cannula, microvessels were observed with bright-field and fluorescence microscopy to obtain estimates of the anatomic diameters and the widths of fluorescent tracer columns and of the RBC columns (means +/- SE). After 2 h of intrascrotal TNF-alpha exposure, there was a significant increase in access of FITC-Dextrans 70 and 580 to the space bounded by the apical glycocalyx in arterioles, capillaries, and venules, but no significant change in access of FITC-Dextran 2,000. The effects of TNF-alpha could be observed as early as 20 min after the onset of topical application. TNF-alpha treatment also significantly increased the penetration rate of FITC-Dextran 40, FITC-albumin, and FITC-IgG into the glycocalyx and caused a significant increase in the intraluminal volume occupied by flowing RBC. White blood cell adhesion increased during TNF-alpha application, and we used the selectin antagonist fucoidan to attenuate leukocyte adhesion during TNF-alpha stimulation. This did not inhibit the TNF-alpha-mediated increase in permeation of the glycocalyx. These results show that proinflammatory cytokines can cause disruption of the endothelial apical glycocalyx, leading to an increased macromolecular permeation in the absence of an increase in leukocyte recruitment.

A Solid-Phase Synthesis for β-Turn Mimetics of Sialyl Lewis X

A solid-phase synthesis of heterocyclic β-turn mimetics of sialyl Lewis X, which is a natural carbohydrate ligand of selectins, was established. This synthetic method could be very useful for drug discovery of selectin antagonists using combinatorial chemistry techniques.

Effects of selectin–sialyl Lewisx blockade on mesenteric microvascular permeability associated with cardiopulmonary bypass

Objectives: Cardiopulmonary bypass is associated with an inflammatory response that is associated with a neutrophil-mediated microvascular barrier injury. We studied the effects of blocking neutrophil-endothelial tethering on microvascular permeability and edema formation during cardiopulmonary bypass. Using a selectin antagonist that prevents interactions with their ligands, we hypothesized that there would be less neutrophil infiltration into the tissue and a reduction in microvascular permeability and edema formation. Methods: A canine mesenteric lymphatic fistula was created to measure Starling forces and to determine microvascular permeability. Normothermic, atrial-femoral cardiopulmonary bypass was initiated (70-90 mL · kg –1 · min –1). Intestinal tissue water was determined with microgravimetry. Ileal tissue myeloperoxidase was measured as an index of neutrophil tissue infiltration. One experimental group received the selectin antagonist TBC 1269 before the initiation of bypass, and the control group received saline solution. Results: There was a modest increase in microvascular permeability in both groups, as evidenced by significantly increased transvascular protein clearance and a trend toward a decrease in reflection coefficient. There were no differences in the experimental group compared with the control group. Ileal tissue myeloperoxidase levels were lower in the experimental group than in the control group. Conclusions: The selectin antagonist TBC 1269 reduces neutrophil infiltration into the ileum without altering ileal microvascular permeability or edema associated with cardiopulmonary bypass. (J Thorac Cardiovasc Surg 2000;119:1255-61)

Pharmacology of selectin inhibitors in ischemia/reperfusion states.

Recently, the selectin family of glycoprotein adhesion molecules (P-selectin, E-selectin, and L-selectin) has been implicated in the pathogenesis of a number of inflammatory disease states. The selectins modulate the early adhesive interactions between circulating neutrophils and the endothelium. Both P-selectin and E-selectin can be expressed on the surface of endothelial cells following stimulation by a number of inflammatory mediators. In contrast, L-selectin is constitutively expressed on the surface of neutrophils at very high levels. In addition, neutrophils also express ligands for the endothelial selectins, including the carbohydrate sialyl Lewis(x) and the high-affinity ligand P-selectin glycoprotein ligand 1, which facilitate neutrophil-endothelial interactions. Selectins have been extensively investigated in ischemia/reperfusion injury states. The study of selectin involvement in ischemia/reperfusion injury has been facilitated by the development of highly specific selectin antagonists, including monoclonal antibodies, carbohydrates, small molecule inhibitors, and soluble forms of P-selectin glycoprotein ligand 1. This article reviews the results of current studies of selectin antagonists in experimental models of ischemia/reperfusion injury.

Synthesis of the C-Glycoside Analogue of a Novel Sialyl Lewis X Mimetic

Sialyl Lewis X (sLex) mimetics that can function as selectin antagonists have received considerable attention in connection with the development of novel antiinflammatory therapies. An interesting structure that emerged from the studies of the Wong group is the 1,1-Gal-Man disaccharide 2, reported to bind E-selectin 5 times more strongly than sLex. The C-glycoside derivative 3 is of interest both as a conformational probe for selectin binding and as a hydrolytically stable analogue. Herein we illustrate a novel methodology for beta-C-galacto-disaccharides in the synthesis of 3. The protocol has as a key step a novel oxocarbenium ion-enol ether cyclization to give a C1-substituted galactal.

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 (sLea-polymer) is utilized as a synthetic ligand for both selectins analogous to the E-selectin assay we have developed recently. Following precomplexation of sLea-polymer with streptavidin–peroxidase, the complex is added to microtiter plates coated with the recombinant selectins. Binding of sLea-polymer to the immobilized selectins is measured by the peroxidase reaction. SLea-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 (sLex) 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 β-d-glucose instead of sLea 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 sLex-related selectin antagonists. These studies together suggest that sLea-polymer-based selectin assays are well suited for primary screening and the characterization of selectin antagonists.

ChemInform Abstract: Design and Investigation of Highly Practical Glycosylation on the Development of Selectin Antagonist

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Oligonucleotide inhibitors of P-selectin-dependent neutrophil-platelet adhesion.

P-selectin, an inducible cell adhesion molecule, mediates rolling of neutrophils on activated vascular endothelium. Because rolling is an early event of the inflammatory response, therapeutic applications of selectin antagonists have been of broad interest. There are, however, no truly satisfactory therapeutic candidates among known inhibitors. Consequently, we have used Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technology, a process based on oligonucleotide combinatorial chemistry and in vitro selection, to develop aptamer antagonists of P-selectin. Equilibrium dissociation constants for aptamer/P-selectin binding range from 16 to 710 pM, a 10(5)-10(6)-fold improvement compared with the minimal carbohydrate ligand, sialyl Lewis X (sLeX). Aptamer binding is divalent cation dependent and, unlike sLeX, is specific for P-selectin. The selectivity for human P-selectin relative to human E-selectin or human L-selectin is 10(4)-10(5). In vitro, aptamers bind with subnanomolar affinities to P-selectin expressed on thrombin-activated platelets, inhibit the binding of P-selectin-IgG chimera to sLeX and to neutrophils, and block the binding activated platelets to neutrophils in flow cytometry and in hydrodynamic assays. Extrapolating from their in vitro characteristics, these novel P-selectin-specific antagonists may be suitable candidates for therapeutic development.

Anaphylaxis-induced mesenteric vascular permeability, granulocyte adhesion, and platelet aggregates in rat.

This study investigates the response of small venules to IgE-dependent, antigen-mediated mast cell activation. Intravital microscopy was utilized to visualize 25- to 40-micron mesenteric venules, mast cell degranulation (on-line detection), vascular permeability changes (albumin leakage), leukocyte adhesion, and the formation of platelet aggregates in rats sensitized with 10 microg of intraperitoneal egg albumin (EA) in saline- or sham-sensitized (saline alone) rats. Sensitized rats challenged with EA (1 mg/ml superfusing mesentery), but not sensitized rats challenged with BSA or sham-sensitized rats challenged with EA, exhibited mast cell degranulation with significant time-dependent increases in vascular permeability (inhibited by diphenhydramine, salbutamol, and indomethacin), leukocyte adhesion (inhibited by Web-2086), and the formation of cellular aggregates (platelet), which were associated with intermittent obstruction of venular flow. Anti-platelet antibody, but not anti-neutrophil antibody or fucoidin (selectin antagonist), prevented platelet aggregate formation. Compound 48/80-induced mast cell degranulation caused similar changes in permeability (via different mediators) and leukocyte adhesion but did not induce platelet aggregation. EA-induced platelet aggregation was not inhibited by any of the mediators tested, and platelets isolated from sensitized rats failed to aggregate in response to direct EA challenge, suggesting release of an unidentified inflammatory mediator as the factor initiating platelet aggregation.

Large scale synthesis of linker-modified sialyl LewisX, LewisX and N-acetyllactosamine

The synthesis of sialyl-LewisX (1b), LewisX (2) and N-acetyllactosamine (3), each being attached to the 1β-O-(6-amino)hexyl handle, were scaled up to gram amounts to obtain sufficient material for thorough pharmaceutical evaluations and for derivatisations aiming at more potent selectin antagonists. The disaccharide 3 was synthesised from inexpensive lactose to provide a versatile building block, either to be used for alternative approaches to the Lewis type oligosaccharides, or to prepare polyvalent LacNAc templates to be further elaborated by glycosyltransferase reactions. All syntheses were directed to reasonable large scale procedures, especially by minimising the number of steps and the use of heavy metal salts in glycosylations.

Synthesis of novel Sialyl-Lewis X glycomimetics as selectin antagonists

A series of low molecular weight sialyl-Lewis X analogs based on either rigid or flexible replacements for the carbohydrates were designed as orally available anti-inflammatory drugs, synthesized and tested in cell-based adhesion assays. The flexible glycomimetic 7a lacking any glycoside or peptide linkage was prepared in 4 steps and 41% overall yield from methyl 3,5-dihydroxybenzoate and the fucose derivative 18 and exhibited about equal binding affinity to E- and P-selectin compared to the parent tetrasaccharide.

Scintillation Proximity Assay for E-, P-, and L-Selectin Utilizing Polyacrylamide-Based Neoglycoconjugates as Ligands

In this study, a novel scintillation proximity assay (SPA) that uses radiolabeled soluble neoglycoconjugates as synthetic alternatives to the natural E-, P-, and L-selectin counterligands was developed. The neoglycoconjugates contained sialyl LewisXor sialyl LewisAattached via a three-carbon spacer to a poly[N-(hydroxyethyl)acrylamide] backbone, thus presenting the carbohydrates in a multivalent form. Selectin–ZZ fusion proteins were immobilized on anti-rabbit IgG-coated SPA beads via a rabbit IgG bridge. The neoglycoconjugate ligands bound to all three bead-immobilized selectins, with the highest binding levels apparent with E-selectin. Saturation binding studies with E-selectin revealed a complex interaction indicative of two or more binding affinities. The response to carbohydrate inhibitors was comparable in E-selectin assays that used either the neoglycoconjugates or the tritium-labeled HL60 cells as selectin counterligands. The incorporation of tyrosine sulfate groups into the backbone of the neoglycoconjugate resulted in enhanced binding avidity to both P- and L-selectin, indicating that the sulfate-containing neoglycoconjugates are viable synthetic mimics of the natural P- and L-selectin counterligands. The use of these radiolabeled neoglycoconjugates in conjunction with SPA results in a format ideally suited for the high-throughput screening for selectin antagonists. Furthermore, this approach can potentially be used to measure other low-avidity lectin–carbohydrate interactions.