Enzyme-Assisted Synthesis and In Vitro Characterization of Bifunctional PCSK9 Inhibitors.

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key target for cholesterol reduction.
• PCSK9 inhibitors are clinically successful, but PCSK9 degradation offers an alternative therapeutic strategy.
• The asialoglycoprotein receptor (ASGPR) mediates lysosomal degradation of extracellular ligands, presenting a target for PCSK9 degradation.

Purpose of the Study:

• To synthesize bifunctional molecules linking Tri-GalNAc (an ASGPR binder) with a peptide inhibitor.
• To develop novel chemical and chemoenzymatic methods for producing Tri-GalNAc-conjugated peptides.
• To overcome challenges in C-terminal Tri-GalNAc attachment for peptides and biologics.

Main Methods:

• Chemical synthesis of Tri-GalNAc-peptide conjugates.
• Chemoenzymatic synthesis using enzymatically mediated ligation.
• Structural and in vitro activity validation of synthesized constructs.

Main Results:

• Successfully synthesized and characterized Tri-GalNAc-peptide conjugates using both chemical and chemoenzymatic approaches.
• Demonstrated in vitro activity of the synthesized constructs.
• Established methods for C-terminal Tri-GalNAc conjugation, a previously challenging task.

Conclusions:

• The developed chemical and biochemical methods are broadly applicable for constructing LYTACs (Lysosome-Targeting Chimeras).
• This work provides a foundation for novel therapeutic strategies targeting PCSK9 degradation.
• The methods overcome significant hurdles in conjugating ligands like Tri-GalNAc to peptides and biologics.