Stimulation of liver-directed cholesterol flux in mice by novel N-acetylgalactosamine-terminated glycolipids with high affinity for the asialoglycoprotein receptor.

OBJECTIVE: Interventions that promote liver-directed cholesterol flux can suppress atherosclerosis, as demonstrated for scavenger receptor-BI overexpression in hypercholesterolemic mice. In analogy, we speculate that increasing lipoprotein flux to the liver via the asialoglycoprotein receptor (ASGPr) may be of therapeutic value in hypercholesterolemia. METHODS AND RESULTS: A bifunctional glycolipid (LCO-Tyr-GalNAc3) with a high-nanomolar affinity for the ASGPr (inhibition constant 2.1+/-0.2 nmol/L) was synthesized that showed rapid association with lipoproteins on incubation with serum. Prior incubation of LCO-Tyr-GalNAc3 with radiolabeled low-density lipoprotein or high-density lipoprotein (0.5 microg/microg of protein) resulted in a dramatic induction of the liver uptake of these lipoproteins when injected intravenously into mice (70+/-3% and 78+/-1%, respectively, of the injected dose at 10 minutes of low-density lipoprotein and high-density lipoprotein), as mediated by the ASGPr on hepatocytes. Intravenously injected LCO-Tyr-GalNAc3 quantitatively incorporated into serum lipoproteins and evoked a strong and persistent (> or =48 hour) cholesterol-lowering effect in normolipidemic mice (37+/-2% at 6 hours) and hyperlipidemic apoE(-/-) mice (32+/-2% at 6 hours). The glycolipid was also effective on subcutaneous administration. CONCLUSIONS: LCO-Tyr-GalNAc3 is very effective in promoting cholesterol uptake by hepatocytes and, thus, may be a promising alternative for the treatment of those hyperlipidemic patients who do not respond sufficiently to conventional cholesterol-lowering therapies.

Design and synthesis of novel N-acetylgalactosamine-terminated glycolipids for targeting of lipoproteins to the hepatic asialoglycoprotein receptor.

A novel glycolipid has been prepared that contains a cluster glycoside with an unusually high affinity for the asialoglycoprotein receptor (ASGPr) and a bile acid moiety that mediates stable incorporation into lipidic particles. The glycolipid spontaneously associated with low-density lipoproteins (LDL) and high-density lipoproteins (HDL) within human and murine plasma, and loading of lipoproteins with this glycolipid resulted in an efficient dose-dependent recognition and uptake of LDL and HDL by the liver (and not by spleen) upon intravenous injection into wild-type mice. Preinjection with asialoorosomucoid largely inhibited the uptake, establishing that both HDL and LDL were selectively recognized and processed by the ASGPr on liver parenchymal cells. Finally, repeated intravenous administration of the glycolipid to hyperlipidemic LDL receptor-deficient mice evoked an efficient and persistent cholesterol-lowering effect. These results indicate that the glycolipid may be a promising alternative for the treatment of hyperlipidemic patients who do not respond sufficiently to current cholesterol-lowering therapies.

P-selectin as a candidate target in atherosclerosis.

P-selectin is of critical importance in early atherogenesis by initiating leukocyte rolling at the site of endothelial injury. In order to validate P-selectin as a candidate target for the development of anti-atherogenic strategies, we wanted to obtain quantitative information on P-selectin expression, and identify novel peptide-based lead structures that interact with P-selectin. P-selectin mRNA expression in the aortic arch and in other tissues of apoE-deficient (apoE-/-) mice was determined by real-time PCR technology. P-selectin mRNA expression of apoE-/- mice increased steadily with age to levels 14-fold higher than that of control animals. The onset and level of P-selectin expression correlated well with the extent of lesion development, and was more specific for atherosclerotic tissue as compared with other adhesion molecules. Phage display technology was used to obtain novel P-selectin antagonists. Phage display selections resulted in the isolation of a highly P-selectin-specific phage clone. Synthetic peptide-equivalents of this clone displaced the binding of the parent phage and antagonized the binding of a sialyl Lewis(x) analogue to P-selectin. In conclusion, P-selectin expression correlates with early and advanced atherosclerotic lesion development. P-selectin ligands, like the lead structure we have developed here, can therefore be considered as promising tools to identify, target or antagonize P-selectin function within the chronically inflamed arterial wall.

Rational optimization of a short human P-selectin-binding peptide leads to nanomolar affinity antagonists.

P-selectin plays an important role in the development of various diseases, including atherosclerosis and thrombosis. In our laboratory we recently identified a number of specific human P-selectin-binding peptides containing a Glu-Trp-Val-Asp-Val consensus motif, displaying a low micromolar affinity for P-selectin (IC(50) = 2 microm). In search of more potent antagonists for P-selectin, we have optimized the EWVDV pentapeptide core motif via a two-step combinatorial chemistry approach. A dedicated library of peptide derivatives was generated by introducing seven substituents at the N and C termini of the motif. In particular, pentapeptides with gallic acid or 1,3,5-benzenetricarboxylic acid substituents at the N terminus proved to be considerably more potent inhibitors of P-selectin binding than the parental peptide. After removal of the N-terminal glutamic acid from the core sequence, which appeared to be replaceable by a carboxamide function without loss of affinity, a second library was synthesized to map the chemical moieties within the gallic acid or 1,3,5-benzenetricarboxyl acid groups responsible for the enhanced P-selectin binding. Moreover, by varying the length and rigidity of the connective spacer, we have further optimized the spatial orientation of the N-terminal substituent. The combined use of phage display and subsequent combinatorial chemistry led to the design of a number of gallic acid- containing peptides with low nanomolar affinity for P-selectin both under static and dynamic conditions (IC(50) = 15.4 nm). These small synthetic antagonists, which are equally as potent as the natural ligand P-selectin glycoprotein ligand-1, are promising leads in anti-atherothrombotic therapy.