Development of Triantennary N-Acetylgalactosamine Conjugates as Degraders for Extracellular Proteins

Abstract

Targeted protein degradation (TPD) technology has drawn significant attention from researchers in both academia and industry. It is rapidly evolved as a new therapeutic modality and also a useful chemical tool in selectively depleting various protein targets. As most efforts focus on cytosolic proteins using PROteolysis TArgeting Chimera (PROTAC), LYsosome TArgeting Chimera (LYTAC) recently emerged as a promising technology to deliver extracellular protein targets to lysosome for degradation through the cation-independent mannose-6-phosphate receptor (CI-M6PR). In this study, we exploited the potential of the asialoglycoprotein receptor (ASGPR), a lysosomal targeting receptor specifically expressed on liver cells, for the degradation of extracellular proteins including membrane proteins. The ligand of ASGPR, triantennary N-acetylgalactosamine (tri-GalNAc), was conjugated to biotin, antibodies, or fragments of antibodies to generate a new class of degraders. We demonstrated that the extracellular protein targets could be successfully internalized and delivered into lysosome for degradation in liver cell lines specifically by these degraders. This work will add a new dimension to TPD with cell type specificity.

Figure 1

Comparison of the application of tri-GalNAc in targeted protein degradation and drug delivery. Small molecule- and antibody-based tri-GalNAc degraders noncovalently capture the protein targets and transport the targets to lysosome for degradation via the interaction with ASGPR. Oligonucleotides covalently linked to tri-GalNAc enable their internalization into the cell through ASGPR. After trafficking to lysosome, a small amount of the oligonucleotides can escape from the endosome or lysosome to block or induce degradation of RNA.

Figure 2

Tri-GalNAc-biotin mediates ASGPR-dependent cellular uptake of NA-650 specifically in liver cells and transports NA-650 to lysosome for degradation. (A) Chemical structures of tri-GalNAc-biotin (compound 1), tri-GalNAc-CO₂H (compound 2), and tri-GalNAc-NHS ester (compound 3). (B) Cellular uptake of NA-650 in HepG2 cells treated with NA-650 alone (500 nM) or NA-650 (500 nM) and compound 1 (2 μM) or 2 (2 μM). (C) Inhibition of the internalization of NA-650 (500 nM) mediated by 1 (2 μM) in HepG2 cells by compound 2. (D, E) Comparison of the internalization of NA-650 (500 nM) mediated by 1 (2 μM) among HepG2, Huh7, and A549 cells incubated with NA-650 and compounds 1 or 2 for 16 (D) or 6 h (E). Data presented as mean ± SD, n = 3. **p < 0.01, ***p < 0.001, ****p < 0.0001. (F) Knockdown of ASGPR by siRNA. (G) Uptake of NA-650 (500 nM) in the presence of 1 (2 μM) within 4 h in HepG2 cells treated with control or ASGPR siRNA. (H) Confocal microscopy images of HepG2 cells treated with NA-650 (500 nM) and compound 1 (2 μM) for 18 h. Legend: internalized NA-650 (red); lysosome stained by Lysotracker (green); nuclei stained by Hoechst 33342 (blue); merged area (yellow). White arrows indicate the colocalization of NA-650 and the lysosome; scale bar: 20 μm. (I) In gel fluorescence analysis of NA-650 (500 nM) internalization and degradation in HepG2 cells by compound 1 (2 μM) in the presence or absence of leupeptin (0.1 mg/mL).

Figure 3

Tri-GalNAc labeled full length antibody goat anti-mouse IgG (Ab-GN) delivers target protein mouse anti-biotin IgG-647 into the cells. (A) Goat anti-mouse full length antibody labeling with various amounts of tri-GalNAc: UL = unlabeled; 3x = 3 mol equiv; 12x = 12 mol equiv; 25x = 25 mol equiv. N = the number of tri-GalNAc labeled on the antibody. (B) Uptake of mouse anti-biotin IgG-647 (50 nM) in the HepG2 cells treated with or without Ab-GN (25 nM) for 6 h. (C) Mouse anti-biotin IgG-647 (50 nM) uptake mediated by Ab-GN (25 nM) with or without 1 h premix before treatment for 6 h. The uptake of anti-biotin IgG-647 (50 nM) and Ab-647-GN (25 nM) were measured for comparison.

Figure 4

Uptake of mouse IgG-647 mediated by tri-GalNAc-labeled antibodies and compound 1. (A) Antibodies labeled with tri-GalNAc (25 mol equiv). (B) Comparison of the 6 h uptake of mouse anti-rabbit IgG-647 (50 nM) mediated by 25 nM of the goat anti-mouse IgG and goat anti-mouse IgG Fab with or without tri-GalNAc (GN) labeling. (C) Cellular uptake of mouse anti-biotin IgG-647 (P1, 50 nM), premixed mouse anti-biotin IgG-647 (50 nM)/goat anti-mouse IgG Fab (200 nM) complex (P2), and premixed mouse anti-biotin IgG-647 (50 nM)/goat anti-mouse IgG (200 nM) complex (P3) in the presence of compounds 1 (200 nM) or 2 (200 nM) for 6 h.

Figure 5

Tri-GalNAc-antibody mediates the uptake and degradation of mouse anti-biotin IgG-647 and EGFR in liver cells. (A) Internalization of mouse anti-biotin IgG-647 in cells incubated with mouse anti-biotin IgG-647 (50 nM) and 25 nM of goat anti-mouse IgG Fab with or without tri-GalNAc (GN) labeling for 6 h. (B) Mouse anti-biotin IgG-647 (50 nM) endocytosis and degradation in HepG2 cells in the presence or absence of leupeptin (0.1 mg/mL) for 6 h. (C) Ctx labeling with tri-GalNAc (25 mol equiv). (D) EGFR degradation in the presence of 30 nM Ctx-GN in HepG2 and Huh7 cells after 48 h treatment.