Synthesis and Evaluation of New Trivalent Ligands for Hepatocyte Targeting via the Asialoglycoprotein Receptor

Abstract

Since the asialoglycoprotein receptor (also known as the "Ashwell−Morell receptor" or ASGPR) was discovered as the first cellular mammalian lectin, numerous drug delivery systems have been developed and several gene delivery systems associated with multivalent ligands for liver disease targeting are undergoing clinical trials. The success of these systems has facilitated the further study of new ligands with comparable or higher affinity and less synthetic complexity. Herein, we designed two novel trivalent ligands based on the esterification of tris(hydroxymethyl) aminomethane (TRIS) followed by the azide−alkyne Huisgen cycloaddition with azido N-acetyl-D-galactosamine. The presented triazolyl glycoconjugates exhibited good binding to ASGPR, which was predicted using in silico molecular docking and assessed by a surface plasmon resonance (SPR) technique. Moreover, we demonstrated the low level of in vitro cytotoxicity, as well as the optimal spatial geometry and the required amphiphilic balance, for new, easily accessible ligands. The conjugate of a new ligand with Cy5 dye exhibited selective penetration into HepG2 cells in contrast to the ASGPR-negative PC3 cell line.

New Small-Molecule Glycoconjugates of Docetaxel and GalNAc for Targeted Delivery to Hepatocellular Carcinoma

“…Therefore, the contribution of this ASGPR-specific mechanism is highly significant in the case of hepatocytes and allows faster internalization of conjugate 9 in line with the literature data reporting that effective cellular uptake of ASGPR-targeted compounds occurs even within the first 10 min of treatment followed by further slow accumulation up to 30–40 min . The specificity of GalNAc-mediated cellular uptake was proved in parallel experiments in the presence of high concentrations of competing monovalent (GalNAc) or trivalent (GalNAc) 3 ligands (10 mM, 10 6 to 10 4 molar excess), which efficiently inhibit internalization of compound 9 (Figure ) but not compound 8 in HepG2 (compare Figure S7a,b, middle and bottom panels, Supporting Information). Such high inhibiting concentrations of GalNAc were reported previously .…”

“…Representative fluorescence (top) and bright-field (bottom) microscopy images of HepG2 cells treated with 1 μM of compound 9 either alone or in the presence of 10 mM of ASGPR-blocking ligands: monovalent GalNAc or trivalent (GalNAc) 3 . (For details see Supporting Information.…”

Development of Asialoglycoprotein-Mediated Hepatocyte-Targeting Antitumor Prodrugs Triggered by Glutathione

“…In other words, the hepatocyte-targeting capacity followed the W-1-5 (monoga-lactose) < W-2-9 (digalactose) < W-3-8 (trigalactose) order, obeying the rules of the galactose-recognition cluster effect toward ASGPR. Interestingly, it was reported that the galactose-modified bis-fluorophore conjugate, 24 the galactosamine-modified betulin conjugate, 25 and the trivalent galactosamine-modified Cy5 dye 26 showed a similar hepatocytetargeted tendency, also confirming the reliability of our results.…”

Discovery of Bivalent GalNAc-Conjugated Betulin as a Potent ASGPR-Directed Agent against Hepatocellular Carcinoma

“…Initially, two azide derivatives of N-acetyl-D-galactosamine 1 and 2 and also N-acetyl-D-glucosamine (GlcNAc) azide 3 were synthesized (Figure 1). We used β-azido glycosylation (FeCl 3 / TMSN 3 ) 32 or β-O-glycosylation (TMSOTf/2-azidoethanol) 33 reactions of β-D-galactosamine pentaacetate according to the previously published procedures, followed by alcoholysis of the O-acetyl groups by MeONa/MeOH. Esters 4−6 containing a residual 5-hexynoic acid moiety as a linker (Scheme 1) were synthesized in the second step.…”