An amber obligate active site-directed ligand evolution technique for phage display

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Noncanonical amino acids (ncAAs) were first incorporated into phage libraries nearly two decades ago.
• The application of ncAAs in drug discovery has been limited due to library bias towards sense-containing phages.

Purpose of the Study:

• To develop a technique to enrich amber-containing phage clones, overcoming library bias.
• To utilize this technique for active site-directed ligand evolution of peptides containing ncAAs.
• To develop low nanomolar inhibitors of SIRT2 using phage-displayed peptide libraries.

Main Methods:

• A novel technique based on phage superinfection immunity was developed to enrich amber-containing clones.
• Phage-displayed peptide libraries containing genetically encoded butyryl lysine were constructed.
• These libraries were used to select ligands that bind SIRT2, followed by modification to create inhibitors.

Main Results:

• The developed technique successfully enriched amber-containing clones, avoiding library bias.
• Phage-displayed libraries with ncAAs were used to identify ligands for SIRT2.
• Modified ligands resulted in the development of low nanomolar inhibitors of SIRT2.

Conclusions:

• The superinfection immunity technique effectively overcomes limitations in incorporating ncAAs into phage libraries for drug discovery.
• This approach facilitates the evolution of targeted peptide ligands with ncAAs.
• The study demonstrates the successful development of potent SIRT2 inhibitors through ncAA-mediated phage display.