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

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.