MOrPH-PhD: A Phage Display System for the Functional Selection of Genetically Encoded Macrocyclic Peptides

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Macrocyclic peptides are valuable scaffolds for targeting biomolecules due to their high affinity and selectivity.
• Developing efficient methods for macrocycle diversification and functional selection is crucial for drug discovery.
• Genetically encoded cyclic peptides offer a powerful approach for library generation.

Purpose of the Study:

• To describe a novel phage display platform, MOrPH-PhD, for creating and functionally exploring genetically encoded cyclic peptide libraries.
• To provide detailed protocols for library production, screening, and isolation of functional macrocyclic peptides.
• To enable rapid identification of high-affinity binders for target proteins.

Main Methods:

• Integration of M13 bacteriophage display with a posttranslational peptide cyclization mechanism using a cysteine-reactive non-canonical amino acid.
• Creation of large combinatorial libraries of genetically encoded macrocyclic peptides displayed on phage particles (up to 10^10 members).
• Step-by-step protocols for library production, screening against immobilized protein targets, and isolation/characterization of binders.

Main Results:

• Successful development and implementation of the MOrPH-PhD platform.
• Demonstrated capability to generate and screen libraries of unprecedented size.
• Facilitated the isolation and characterization of functional macrocyclic peptides with high-affinity binding properties.

Conclusions:

• The MOrPH-PhD platform offers a robust and efficient system for the discovery of macrocyclic peptides.
• This method significantly advances the capabilities for functional selection of macrocycles in drug discovery.
• The described protocols provide a valuable resource for researchers in the field of peptide therapeutics.