Enzyme‐Assisted Synthesis and In Vitro Characterization of Bifunctional PCSK9 Inhibitors

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a well-established target for lowering cholesterol and is abundantly present in the extracellular space. Inhibitors of PCSK9 have achieved marked success in the clinic, but an alternative strategy for therapeutic modulation is emerging through the degradation of PCSK9. This novel strategy has been enabled by the identification of cell surface receptors such as the asialoglycoprotein receptor (ASGPR), which mediates the lysosomal degradation of extracellular ligands. Given the importance of this therapeutic mechanism, we investigated the synthesis of bifunctional molecules comprising Tri-GalNAc (an ASGPR binder) with a peptide inhibitor we previously reported. In addition to chemical synthesis, we report a novel method for the production of Tri-GalNAc-conjugated peptides, involving the use of enzymatically mediated ligation postsynthesis. We demonstrate that both the synthetic constructs and chemoenzymatic constructs have the intended structures and in vitro activities. While these molecules did not show cellular activities, the chemical and biochemical methods reported here could be broadly applied to the construction of LYTACs in general. One significant challenge that this work overcomes is the C-terminal attachment of Tri-GalNAc, which remains hitherto a difficult experimental task for not only peptides but also larger biologics in particular.