Michael I. Bird

Isolation and characterisation of potential respiratory syncytial virus receptor(s) on epithelial cells.

Respiratory syncytial virus (RSV) infection causes severe lower respiratory diseases in infancy, early childhood and the elderly. RSV infections respond poorly to current therapies. Therefore, we initiated a search for novel drug targets by investigating the characteristics and identity of RSV adhesion receptors on mammalian cells. Soluble human lectins, complex polysaccharides and a low molecular selectin antagonist, TBC1269, were used to characterise and isolate the RSV receptor on a human epithelial cell line (Hep2 cells). The binding characteristics of the RSV receptor on Hep2 cells were similar to those reported for L-selectin. The carbohydrate-based selectin antagonists, fucoidan and TBC 1269, inhibit RSV infection both in vitro and in a mouse model of infection. Furthermore, we have isolated annexin II as a potential RSV receptor on Hep2 cells. The expression of annexin II was increased after RSV infection. Recombinant annexin II binds to RSV G-protein, heparin and plasminogen and the binding is inhibited by a selectin antagonist, TBC1269. These findings indicate that inhibitors of annexin II could have potential in treating RSV infection.

P-SELECTIN BINDS TO BACTERIAL LIPOPOLYSACCHARIDE

Multiple organ failure associated with disseminated intravascular coagulation is a frequent complication in septic shock patients. Accumulation of platelets and neutrophils in the organs contributes to the manifestation of lipopolysaccharide (LPS)‐induced organ failure. Although a direct interaction between LPS and platelets is well documented, the nature of the surface receptor for LPS on platelets is unknown. In this article we show that P‐selectin is a receptor for LPS. The binding of LPS to P‐selectin is independent of Ca2+ , and is blocked by antibodies to P‐selectin, lipid A and fucoidan. Platelets pre‐treated with thrombin showed fourfold higher binding of fluorescein isothiocyanate (FITC)‐conjugated LPS compared to untreated platelets and the binding of FITC‐conjugated LPS to platelets was blocked in the presence of anti‐P‐selectin antibodies. It is likely that the binding of LPS via P‐selectin on activated platelets or epithelium could have a significant role in the pathophysiology of organ failure in septic shock.

Monomeric and Multimeric Blockers of Selectins: Comparison of in vitro and in vivo Activity

The potency of the oligosaccharides SiaLex, SiaLea, HSO3Lex, and HSO3Lea, their conjugates with polyacrylamide (PAA, 40 kD), and other monomeric and polymeric selectin inhibitors has been compared with that of the polysaccharide fucoidan. The following assay systems were used: 1) a 96-well assay based either on the use of recombinant E-, P-, and L- selectins or an analogous assay with natural P-selectin isolated from human platelets; 2) a platelet-based P-selectin cell assay; and 3) a rat model of peritoneal inflammation. IC50 values for the neoglycoconjugate SiaLea-PAA were 6, 40, and 85 µM for recombinant E-, P-, and L-selectins, respectively; all monomeric inhibitors were about two orders of magnitude weaker. PAA-conjugates, containing as a ligand tyrosine-O-sulfate (sTyr) in addition to one of the sialylated oligosaccharides, were the most potent synthetic blockers in vitro. Compared with fucoidan, the most potent known P- and L-selectin blocker, the bi-ligand glycoconjugate HSO3Lea-PAA-sTyr displayed similar inhibitory activity in vitro towards L-selectin and about ten times lower activity towards P-selectin. All of the tested synthetic polymers displayed a similar ability to inhibit neutrophil extravasation in the peritonitis model (in vivo) at 10 mg/kg. The data provide evidence that monomeric SiaLex is considerably more effective as a selectin blocker in vivo than in vitro, whereas the opposite is true for fucoidan and the bi-ligand neoglycoconjugate HSO3Lea-PAA-sTyr.

Assembly of P-Selectin Ligands on a Polymeric Template

High-affinity receptor-ligand interactions frequently involve molecular interactions at two distinct sites. A derivatized polyacrylic-based polymer was synthesized to allow substitution with multiple ligands (e.g., L(1) and L(2)) on the backbone. Two-site P-selectin-ligand interactions were first studied with SiaLe(x) (L(1)) and tyrosine sulfate (L(2)) covalently incorporated onto the flexible polymer. In competition assays, a marked synergistic inhibitory effect was observed when the polymer presented both L(1) and L(2) as opposed to either ligand alone. In a second approach, the SiaLe(X) ligand was reduced in complexity so that L(1) was fixed as Le(x) or Le(a), and alternative L(2) groups (to mimic sialic acid) were investigated. Certain combinations of L(1) and L(2) were better antagonists of P-selectin than SiaLe(x) itself. These approaches offer the potential of facilitating the discovery of novel inhibitors of receptors or enzymes.