Human ASGR1 Stable Cell Line - HEK293T

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates plasma low-density lipoprotein cholesterol (LDL-C) levels by promoting degradation of the hepatic low-density lipoprotein receptor (LDLR). Current treatment approaches use antibodies that block PCSK9 binding to the LDLR to reduce circulating LDL-C concentrations or use siRNA to reduce PCSK9 synthesis and thus reduce circulating LDL-C levels. Previous studies have described several small molecules that are similar to therapeutic antibodies and can interfere with PCSK9 binding to the LDLR. Here, researchers report an alternative approach to reducing circulating PCSK9 levels, using heterobifunctional molecules that simultaneously bind to PCSK9 and the asialoglycoprotein receptor (ASGPR) to accelerate PCSK9 clearance and degradation. Various formats, including bispecific antibodies, antibody-small molecule conjugates, and heterobifunctional small molecules, have been demonstrated to bind in vitro and accelerate PCSK9 clearance in vivo. These molecules demonstrate a novel approach to inhibit PCSK9 by targeting plasma protein degradation (TPPD) and demonstrate the feasibility of heterobifunctional small molecule ligands to accelerate the clearance and degradation of pathogenic proteins in circulation.

Here, the researchers performed cellular studies to gain a deeper understanding of the mechanisms of PCSK9 clearance. HEK293 cells were chosen for these experiments because both ASGPR and LDLR levels are low, allowing for the overexpression of ASGR1 to control ASGPR levels and prevent PCSK9 uptake through the LDLR. While this better represents the normal physiological state, it complicates the interpretation of the experiments because compound 15 both prevents PCSK9 from binding to the LDLR and promotes its uptake through the ASGPR. ASGR1 overexpressing HEK293 cells were used to demonstrate that both ASGR1 and the heterobifunctional ligand (15) are required to accelerate PCSK9 clearance above that seen in parental HEK cells (Figure 1D). These results are quantified in Figure 1E, along with additional conditions that replicate the in vivo results: heterobifunctional ligand 15 is required to accelerate PCSK9 uptake, as neither ASGR1 ligand 1 nor PCSK9 ligand 11 increases uptake. Because excess ASGR1 ligand 1 or PCSK9 ligand 11 inhibits accelerated PCSK9 uptake, simultaneous binding of both ASGR1 and PCSK9 is required to accelerate uptake. The studies also demonstrated that inhibition of receptor-mediated endocytosis using PitStop2 or inhibition of lysosomal degradation using bafilomycin inhibits endogenous and heterobifunctional ligand-mediated PCSK9 uptake, albeit to different extents.

Figure 1. (D) Images of HEK293 cells (overexpressing or not overexpressing ASGR1) incubated with 50 nM PCSK9 (red) in the presence or absence of heterobifunctional ligand 15; (E) Quantitative analysis of images in panel D, where open bars represent HEK and solid bars represent ASGR1 overexpressing HEK. (Bagdanoff J T, et al., 2023)