Synthetic Site-Specific Antibody-Ligand Conjugates Promote Asialoglycoprotein Receptor-Mediated Degradation of Extracellular Human PCSK9.
Synthetic Site-Specific Antibody-Ligand Conjugates Promote Asialoglycoprotein Receptor-Mediated Degradation of Extracellular Human PCSK9.
复制标题
合成位点特异性抗体-配体缀合物促进去唾液酸糖蛋白受体介导的细胞外人 PCSK9 降解。
DOI：
10.1021/acschembio.3c00229
复制
发表时间：
2023
影响因子：
4
通讯作者：
Wang,Lai-Xi
中科院分区：
文献类型：
--
作者：
Donahue,ThomasC;Ou,Chong;Yang,Qiang;Flinko,Robin;Zhang,Xiao;Zong,Guanghui;Lewis,GeorgeK;Wang,Lai-Xi
使用细胞特异性溶酶体靶向受体的靶向降解正在成为消除疾病相关蛋白的新治疗策略。肝脏特异性人去唾液酸糖蛋白受体（ASGPR）是一种特别有吸引力的溶酶体靶向受体，用于靶向蛋白降解（TPD）。然而，不同聚糖配体用于ASGPR介导的溶酶体递送的效率仍有待进一步表征。在这项研究中，我们应用化学酶促Fc聚糖重塑方法来构建一系列位点特异性抗体-配体缀合物，这些缀合物携带天然的双触角和三触角N-聚糖以及合成的三-GalNAc配体。选择阿利司那（一种抗PCSK 9（前蛋白转化酶枯草杆菌蛋白酶/kexin 9型）抗体）和西妥昔单抗（一种抗EGFR抗体）分别证明ASGPR介导的细胞外和膜相关蛋白降解。发现缀合物中聚糖配体的性质和间隔基的长度对于受体结合和受体介导的PCSK 9降解至关重要，其阻断低密度脂蛋白受体（LDLR）功能并不利地影响低密度脂蛋白胆固醇的清除。有趣的是，抗体-三-GalNAc缀合物显示出其与ASGPR结合的明显钩状效应，而携带天然N-聚糖的抗体缀合物则没有。如基于细胞的测定中所示，抗体-三触角N-聚糖缀合物和抗体-三-GalNAc缀合物均可显著降低细胞外PCSK 9。然而，tri-GalNAc缀合物在受体介导的PCSK 9降解中显示出明显的钩状效应，而携带天然N-聚糖的抗体缀合物则没有。西妥昔单抗-三-GalNAc缀合物也显示出对膜相关蛋白、表皮生长因子受体（EGFR）的降解的类似钩状效应。这些结果表明，这两种类型的配体可能涉及一个不同的模式的受体结合和目标降解过程中的相互作用。有趣的是，与单独的抗体相比，还发现alirocumab-tri-GalNAc缀合物上调LDLR水平。这项研究展示了针对PCSK 9的靶向降解策略在降低低密度脂蛋白胆固醇（心脏病和中风的风险因素）方面的潜力。
Targeted degradation using cell-specific lysosome targeting receptors is emerging as a new therapeutic strategy for the elimination of disease-associated proteins. The liver-specific human asialoglycoprotein receptor (ASGPR) is a particularly attractive lysosome targeting receptor leveraged for targeted protein degradation (TPD). However, the efficiency of different glycan ligands for ASGPR-mediated lysosomal delivery remains to be further characterized. In this study, we applied a chemoenzymatic Fc glycan remodeling method to construct an array of site-specific antibody–ligand conjugates carrying natural bi- and tri-antennaryN-glycans as well as synthetic tri-GalNAc ligands. Alirocumab, an anti-PCSK9 (proprotein convertase subtilisin/kexin type 9) antibody, and cetuximab (an anti-EGFR antibody) were chosen to demonstrate the ASGPR-mediated degradation of extracellular and membrane-associated proteins, respectively. It was found that the nature of the glycan ligands and the length of the spacer in the conjugates are critical for the receptor binding and the receptor-mediated degradation of PCSK9, which blocks low-density lipoprotein receptor (LDLR) function and adversely affects clearance of low-density lipoprotein cholesterol. Interestingly, the antibody–tri-GalNAc conjugates showed a clear hook effect for its binding to ASGPR, while antibody conjugates carrying the natural N-glycans did not. Both the antibody–tri-antennary N-glycan conjugate and the antibody–tri-GalNAc conjugate could significantly decrease extracellular PCSK9, as shown in the cell-based assays. However, the tri-GalNAc conjugate showed a clear hook effect in the receptor-mediated degradation of PCSK9, while the antibody conjugate carrying the natural N-glycans did not. The cetuximab–tri-GalNAc conjugates also showed a similar hook effect on degradation of the membrane-associated protein, epidermal growth factor receptor (EGFR). These results suggest that the two types of ligands may involve a distinct mode of interactions in the receptor binding and target-degradation processes. Interestingly, the alirocumab–tri-GalNAc conjugate was also found to upregulate LDLR levels in comparison with the antibody alone. This study showcases the potential of the targeted degradation strategy against PCSK9 for reducing low-density lipoprotein cholesterol, a risk factor for heart disease and stroke.