Selectin inhibition modulates NF-kappa B and AP-1 signaling after liver ischemia/reperfusion.

The infiltration of neutrophils after ischemia and reperfusion (I/R) is facilitated by the expression of adhesion molecules on the surface of both leukocytes and endothelial cells. Adhesion molecules of the selectin family are of particular importance at the onset of neutrophil mediated injury, as demonstrated by the occurrence of many cellular interactions with the final extravasation of inflammatory leukocytes at the site of I/R damage. Previous studies demonstrated a prevention of neutrophil extravasation and protection of ischemic damage when a small anti-selectin molecule was used. In this study, we tested a new small anti-selectin compound (OC-229) in a murine model of partial hepatic I/R. The aim of this study was to determine the effect of OC-229 on liver function and histology after I/R and to evaluate its role in the modulation of the inflammatory molecular signaling pathways of NF-kappa B and AP-1 under the same experimental condition. Mice subjected to 90 min of partial (70-80%) hepatic ischemia and 3 h of reperfusion were divided into three groups (n = 9/group): sham, ischemic control, and treated group, which received 25 mg/kg of the anti-selectin small molecule OC-229. These groups were studied when the treatment was given at the time of reperfusion (no pretreatment was given). The parameters measured at 3 h of reperfusion included liver function tests (ALT and AST), liver histology, and liver tissue electrophoretic mobility shift assay (EMSA) for NF-kappa B and AP-1. It was demonstrated that the multiselectin inhibitor OC-229 offered significant protection for the ischemic liver when given at 25 mg/kg at the time of reperfusion. ALT and AST serum levels significantly decreased when the ischemic control and the group receiving OC-229 were compared (p = .01). Treated animals demonstrated better histological findings as well. The EMSA showed dissociation of NF-kappa B and AP-1 activity in the liver nuclear extracts after selectin inhibition treatment. A reduction in the activity of AP-1 and an increment in NF-kappa B activation was seen. In this work, we obtained evidence that the small-molecule selectin inhibitor OC-229 offered functional and histological protection of the ischemic liver when given at 25 mg/kg at the time for reperfusion. There was dissociation in the activation signals of NF-kappa B and AP-1. Increase in NF-kappa B and reduction of the activation of AP-1 were noted at 3 h of reperfusion.

Selectin antagonists : therapeutic potential in asthma and COPD.

Asthma and COPD are chronic inflammatory conditions that affect hundreds of millions of patients worldwide. New therapeutics are desperately needed, especially those that target the underlying causes and prevent disease progression. Although asthma and COPD have distinct etiologies, both are associated with reduced airflow caused by excess infiltration of inflammatory cells into healthy lung tissues. As selectin-mediated adhesion of leukocytes to the vascular endothelium is a key early event in the initiation of the inflammatory response, selectin inhibition is thought to be a good target for therapeutic intervention. Three known selectins are expressed in distinct subsets of cells: P-selectin is presented on the surface of activated platelets and endothelial cells, L-selectin is constitutively expressed on leukocytes, and E-selectin synthesis is upregulated in activated endothelial cells. They mediate cell-cell adhesion in the shear flow of the bloodstream via specialized interactions with clusters of oligosaccharides presented on cell surface glycopeptide ligands. The role of selectin-ligand interactions in the inflammatory response has been demonstrated in various animal models, prompting considerable attention from the pharmaceutical industry.Drug discovery efforts have yielded many different classes of selectin inhibitors, including soluble protein ligands, antibodies, oligosaccharides and small molecules. Although many selectin inhibitors have shown activity in preclinical models, clinical progress of selectin-directed therapies has been slow. Early approaches employed carbohydrate-based inhibitors to mimic the natural ligand sialyl Lewis X; however, these compounds proved challenging to develop. Cytel's CY 1503, a complex oligosaccharide, progressed to phase II/III trials for reperfusion injury, but further development was halted when it failed to demonstrate clinical efficacy. Two protein-based selectin inhibitors have reached phase II development. These included Wyeth's recombinant soluble P-selectin ligand, TSI (PSGL-1), which was discontinued after disappointing results in myocardial infarction trials and Protein Design Labs' humanized anti-L-selectin monoclonal antibody, which is currently in development for trauma. Bimosiamose, discovered by Encysive Pharmaceutical and presently being developed by Revotar Biopharmaceuticals, is an 863 g/mol molecular weight dimer with minimal carbohydrate content and is, to date, the leading selectin inhibitor in clinical development. This compound has shown promise in a phase IIa 'proof of concept' trial in patients with asthma, reducing airway recruitment of eosinophils after intravenous administration. Further clinical development of an inhaled formulation is underway. Despite a significant need for new therapeutics, selectin inhibitors have not yet been explored for the treatment of COPD. Bimosiamose represents an important proof of principle, and hopefully continued success will spark renewed interest in selectin-directed therapeutics for respiratory diseases.

Inhibition of fructose-1,6-bisphosphatase by a new class of allosteric effectors.

A highly constrained pseudo-tetrapeptide (OC252-324) further defines a new allosteric binding site located near the center of fructose-1,6-bisphosphatase. In a crystal structure, pairs of inhibitory molecules bind to opposite faces of the enzyme tetramer. Each ligand molecule is in contact with three of four subunits of the tetramer, hydrogen bonding with the side chain of Asp187 and the backbone carbonyl of residue 71, and electrostatically interacting with the backbone carbonyl of residue 51. The ligated complex adopts a quaternary structure between the canonical R- and T-states of fructose-1,6-bisphosphatase, and yet a dynamic loop essential for catalysis (residues 52-72) is in a conformation identical to that of the T-state enzyme. Inhibition by the pseudo-tetrapeptide is cooperative (Hill coefficient of 2), synergistic with both AMP and fructose 2,6-bisphosphate, noncompetitive with respect to Mg2+, and uncompetitive with respect to fructose 1,6-bisphosphate. The ligand dramatically lowers the concentration at which substrate inhibition dominates the kinetics of fructose-1,6-bisphosphatase. Elevated substrate concentrations employed in kinetic screens may have facilitated the discovery of this uncompetitive inhibitor. Moreover, the inhibitor could mimic an unknown natural effector of fructose-1,6-bisphosphatase, as it interacts strongly with a conserved residue of undetermined functional significance.