Efficient Liver Targeting by Polyvalent Display of a Compact Ligand for the Asialoglycoprotein Receptor

A compact and stable bicyclic bridged ketal was developed as a ligand for the asialoglycoprotein receptor (ASGPR). This compound showed excellent ligand efficiency, and the molecular details of binding were revealed by the first X-ray crystal structures of ligand-bound ASGPR. This analogue was used to make potent di- and trivalent binders of ASGPR. Extensive characterization of the function of these compounds showed rapid ASGPR-dependent cellular uptake in vitro and high levels of liver/plasma selectivity in vivo. Assessment of the biodistribution in rodents of a prototypical Alexa647-labeled trivalent conjugate showed selective hepatocyte targeting with no detectable distribution in nonparenchymal cells. This molecule also exhibited increased ASGPR-directed hepatocellular uptake and prolonged retention compared to a similar GalNAc derived trimer conjugate. Selective release in the liver of a passively permeable small-molecule cargo was achieved by retro-Diels–Alder cleavage of an oxanorbornadiene linkage, presumably upon encountering intracellular thiol. Therefore, the multicomponent construct described here represents a highly efficient delivery vehicle to hepatocytes.本研究开发了一种紧凑稳定的双环桥连缩酮作为去唾液酸糖蛋白受体（asialoglycoprotein receptor, ASGPR）的配体。该化合物展现出优异的配体效率，其结合的分子细节通过首个配体结合型ASGPR的X射线晶体结构得以揭示。此类似物被用于制备强效的ASGPR二价与三价结合剂。对这些化合物功能的全面表征显示，它们在体外可快速实现ASGPR依赖的细胞摄取，并在体内展现出高水平的肝/血浆选择性。对典型Alexa647标记的三价偶联物在啮齿类动物体内的生物分布评估结果表明，其可选择性靶向肝细胞，且未在非实质细胞中检测到分布。与类似的N-乙酰半乳糖胺（GalNAc）衍生三聚体偶联物相比，该分子还表现出更强的ASGPR导向肝细胞摄取能力与更长的滞留时间。通过氧降冰片二烯连接子的逆狄尔斯-阿尔德裂解，可在肝脏中选择性释放被动渗透性小分子载荷，推测该过程在遭遇细胞内硫醇时触发。因此，本文所报道的多组分构建体可作为一种高效的肝细胞递送载体。