Genetically Encoding Propiolamide Warhead for the Construction of Phage Displayed Cyclic Peptide Library.

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Phage display is crucial for cyclic peptide ligand discovery.
• Genetically encoded electrophilic noncanonical amino acids (ncAAs) facilitate cyclic peptide library construction via crosslinking.
• The limited availability of electrophilic ncAAs restricts the application of this technology.

Purpose of the Study:

• To design and genetically encode novel propiolamide-bearing ncAAs for phage display.
• To demonstrate the utility of propiolamide warheads for creating stable cyclic peptide libraries.
• To discover cyclic peptide-based inhibitors targeting SARS-CoV-2 main protease.

Main Methods:

• Genetic encoding of propiolamide-bearing ncAAs into proteins.
• Demonstration of propiolamide conjugation to proximal Cysteine (Cys) via vinyl sulfide linkage.
• Construction and biopanning of large phage-displayed cyclic peptide libraries.

Main Results:

• Propiolamide ncAAs were successfully designed and genetically encoded.
• Stable vinyl sulfide connectivity was achieved between propiolamide and Cys.
• Large recombinant cyclic peptide libraries were constructed and displayed on phage.
• A cyclic peptide inhibitor targeting SARS-CoV-2 main protease was identified through biopanning.

Conclusions:

• Propiolamide-bearing ncAAs offer a versatile new tool for phage display and cyclic peptide library construction.
• This approach expands the utility of noncanonical amino acids in protein engineering and drug discovery.
• The identified inhibitor shows potential for further development against SARS-CoV-2.