Design and synthesis of high affinity ligands for the asialoglycoprotein receptor (ASGP-R)

Abstract

The asialoglycoprotein receptor (ASGP-R) is a carbohydrate-binding protein from the C-type lectin family that is expressed exclusively and in high numbers on mammalian hepatocytes. The human ASGP-R is a transmembrane protein, consisting of two homologous subunits (H1 and H2), that recognizes and binds desialylated glycoproteins with terminal galactose or N-acetylgalactosamine residues. The binding process is followed by receptor-mediated endocytosis of the receptor-ligand complex by the parent hepatocyte. The ASGP-R is then recycled back to the surface, whereas the ligand is ferried to the lysosomes for enzymatic degradation. Due to its location and efficient ligand uptake, the ASGPR has for a long time been a validated target for liver-specific drug delivery. Furthermore, there is substantial evidence that the ASGP-R is involved in hepatitis B and C virus entry into the liver cells. The focus of this thesis was to design and synthesize various high affinity ligands for the ASGP-R that could be used as (1) drug carriers for liver-specific drug delivery, (2) small molecular weight inhibitors of hepatitis B/C entry, (3) a spinlabeled GalNAc-based molecular probe for second binding site screening by NMR, and (4) a set of trivalent compounds for investigating the local concentration effect on ligand affinity towards the ASGP-R by surface plasmon resonance (BIACORE). The trivalent drug carrier for liver-specific drug delivery was shown to bind with high affinity and selectivity to the ASGP-R, and is now awaiting the next step, namely, its conjugation to a therapeutic agent and in vivo testing. The TEMPO spin-labeled GalNAc derivative was successfully used as a first-site ligand for second-site screening by NMR, in which imidazole was identified as a potential second-site ligand. Therefore, after the removal of the TEMPO spin label the first-site ligand will be used in further studies, involving “in situ click chemistry”, in order to find the appropriate linker for joining the first- and secondsite ligands. The four trivalent compounds synthesized for investigating the local concentration effect had an identical molecular mass and scaffold, but differed in the ratio of D-galactose to D-glucose moieties per molecule. Since the affinity of glucose towards the ASGP-R is > 20 mM, and that of galactose is 2.2 mM, the affinity was expected to increase with increasing number of galactose moieties. However, the compound bearing two galactose and one glucose residue unexpectedly showed an affinity greater than that for a compound with three galactose residues. The phenomenon is yet to be explained and verified by further experiments. Nevertheless, the results presented in this work did confirm that the statistical local concentration effect has a weaker influence on multivalency than the chelate effect.