Design, synthesis and evaluation of monovalent ligands for the asialoglycoprotein receptor (ASGP-R)

Stokmaier, Daniela; Khorev, Oleg; Cutting, Brian; Born, Rita; Ricklin, Daniel; Ernst, Thomas O.G.; Böni, Fabienne; Schwingruber, Kathrin; Gentner, Martin; Wittwer, Matthias; Spreafico, Morena; Vedani, Angelo; Rabbani, Said; Schwardt, Oliver; Ernst, Beat

doi:10.1016/j.bmc.2009.08.049

Cited by 40 publications

(39 citation statements)

References 55 publications

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“…3D). This value is somewhat lower than that obtained recently from SPR experiments [20], with the difference probably due to the effects of immobilization of ASGP-R CRD on the SPR sensor chips. We also determined the dissociation constants for Gal binding to ASGP-R CRD at various Ca 2+ concentrations.…”

Section: Ligand Binding To Asgp-r Crdcontrasting

confidence: 71%

NMR study of ligand release from asialoglycoprotein receptor under solution conditions in early endosomes

et al. 2012

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Asialoglycoprotein receptor (ASGP-R) is an endocytic C-type lectin receptor in hepatocytes that clears plasma glycoconjugates containing a terminal galactose or N-acetylgalactosamine. The carbohydrate recognition domain (CRD) of ASGP-R has three Ca 2+ binding sites (sites 1, 2 and 3), with Ca 2+ at site 2 being directly involved in ligand binding. Following endocytosis, the ligands are released from ASGP-R in endosomes to allow receptor recycling to the cell membrane. Although dissociation of the receptorligand complex is mediated by the acidic environment within the mature endosomes, many of these complexes also dissociate in the early time of endocytosis, where pH is approximately neutral. To investigate the mechanism of ligand release from ASGP-R in early endosomes, we examined the binding mode of Ca 2+ and ligands to ASGP-R CRD by NMR. We demonstrate that sites 1 and 2 of ASGP-R are high affinity Ca 2+ binding sites, site 3 is low affinity, and that Ca 2+ ions bind to sites 1 and 2 cooperatively. The pH and Ca 2+ concentration dependences of Ca 2+ binding states indicated that early endosome conditions favor apo-ASGP-R CRD, allowing ligand release. Our results elucidated that the cooperative binding mode of Ca 2+ makes it possible for ASGP-R to be more sensitive to Ca 2+ concentrations in early endosomes, and plays an important role in the efficient release of ligand from ASGP-R. In our proposed mechanism, ASGP-R can rapidly release Ca 2+ and its ligand even at nearly neutral pH. Sequence comparisons of endocytic C-type lectin receptors suggest that this mechanism is common in their family.

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Section: Ligand Binding To Asgp-r Crdcontrasting

confidence: 71%

NMR study of ligand release from asialoglycoprotein receptor under solution conditions in early endosomes

et al. 2012

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“…Stokmaier et al revealed that the binding affinity of galactose to ASGPR elevated 100–1000 fold from mono- to triantennary galactose structures, probably due to clustering effects [22]. The dissociation constant of monosaccharide with ASGPR was 10 –4 M, whereas those of triantennary and tetraantennary compounds with ASGPR were 5 × 10 −9 M and 9 × 10 −9 M, respectively [22]. Previously, we synthesized the mono-Lac-β-CD and revealed the reduction in accumulation of intracellular cholesterol in NPC-like HepG2 cells [14].…”

Section: Resultsmentioning

confidence: 99%

Synthesis of multi-lactose-appended β-cyclodextrin and its cholesterol-lowering effects in Niemann–Pick type C disease-like HepG2 cells

Motoyama

Nishiyama

Maeda

et al. 2017

Beilstein J. Org. Chem.

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Niemann–Pick type C (NPC) disease, characterized by intracellular accumulation of unesterified cholesterol and other lipids owing to defects in two proteins NPC1 and NPC2, causes neurodegeneration and other fatal neurovisceral symptoms. Currently, treatment of NPC involves the use of 2-hydroxypropyl-β-cyclodextrin (HP-β-CD). HP-β-CD is effective in the treatment of hepatosplenomegaly in NPC disease, albeit at a very high dose. One of the methods to reduce the required dose of HP-β-CD for treatment of NPC is to actively targeting hepatocytes with β-cyclodextrin (β-CD). The aim of the present study was to synthesize a novel multi-lactose-appended β-CD (multi-Lac-β-CD) and to evaluate its cholesterol-lowering effect in U18666A-treated HepG2 (NPC-like HepG2) cells. Further, the study aimed at delivering β-CD to hepatocytes via cholesterol-accumulated HepG2 cells, and indicated that the newly synthesized multi-Lac-β-CD had an average degree of substitution of lactose (DSL) of 5.6. This newly synthesized multi-Lac-β-CD was found to significantly decrease the concentration of intracellular cholesterol with negligible cytotoxicity as compared to HP-β-CD. An increased internalization of TRITC-multi-Lac-β-CD (DSL 5.6) as compared to TRITC-HP-β-CD was observed in NPC-like HepG2 cells. Further, the dissociation constant of peanut lectin with multi-Lac-β-CD (DSL5.6) was found to be extremely low (2.5 × 10−8 M). These results indicate that multi-Lac-β-CD (DSL5.6) diminished intracellular cholesterol levels in NPC-like HepG2 cells via asialoglycoprotein receptor (ASGPR)-mediated endocytosis.

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“…Measurements were performed by using flow cytometry in HepG2 cells. [a] Dissociation constant for the binding of the labeled glycoconjugates with the human ASGPR, calculated by using a simple competitive inhibition model with the IC 50 value measured with GalNAc and the inhibition constant with GalNAc ( K i =150×10 −6 ) . [b] Incomplete saturation or inhibition at the concentration range used.…”

Section: Figurementioning

confidence: 99%

ASGPR‐Mediated Uptake of Multivalent Glycoconjugates for Drug Delivery in Hepatocytes

et al. 2016

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Liver cells are an essential target for drug delivery in many diseases. The hepatocytes express the asialoglycoprotein receptor (ASGPR), which promotes specific uptake by means of N-acetylgalactosamine (GalNAc) recognition. In this work, we designed two different chemical architectures to treat Wilson's disease by intracellular copper chelation. Two glycoconjugates functionalized with three or four GalNAc units each were shown to enter hepatic cells and chelate copper. Here, we studied two series of compounds derived from these glycoconjugates to find key parameters for the targeting of human hepatocytes. Efficient cellular uptake was demonstrated by flow cytometry using HepG2 human heptic cells that express the human oligomeric ASGPR. Dissociation constants in the nanomolar range showed efficient multivalent interactions with the receptor. Both architectures were therefore concluded to be able to compete with endogeneous asialoglycoproteins and serve as good vehicles for drug delivery in hepatocytes.

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Design, synthesis and evaluation of monovalent ligands for the asialoglycoprotein receptor (ASGP-R)

Cited by 40 publications

References 55 publications

NMR study of ligand release from asialoglycoprotein receptor under solution conditions in early endosomes

NMR study of ligand release from asialoglycoprotein receptor under solution conditions in early endosomes

Synthesis of multi-lactose-appended β-cyclodextrin and its cholesterol-lowering effects in Niemann–Pick type C disease-like HepG2 cells

ASGPR‐Mediated Uptake of Multivalent Glycoconjugates for Drug Delivery in Hepatocytes

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