In Vitro Selection of Macrocyclic l-α/d-α/β/γ-Hybrid Peptides Targeting IFN-γ/IFNGR1 Protein-Protein Interaction

Area of Science:

• Biochemistry
• Peptide Chemistry
• Drug Discovery

Background:

• Nonproteinogenic amino acids, including d-α-, β-, and γ-amino acids, are crucial for peptide bioactivity and stability.
• d-α-Amino acids (dαAA) enhance proteolytic stability, while cyclic β^2,3-amino acids (cβAA) and cyclic γ^2,4-amino acids (cγAA) improve binding affinity and prevent proteolysis, respectively.

Purpose of the Study:

• To develop a platform for synthesizing l-α/d-α/β/γ-hybrid peptides incorporating multiple nonproteinogenic amino acids.
• To apply these hybrid peptide libraries for in vitro selection against the interferon gamma receptor 1 (IFNGR1).

Main Methods:

• Engineered tRNA^Pro1E2 for ribosomal incorporation of multiple nonproteinogenic amino acids.
• Ribosomal synthesis of macrocyclic l-α/d-α/β/γ-hybrid peptide libraries.
• In vitro selection against interferon gamma receptor 1 (IFNGR1) and cellular assays.

Main Results:

• One hybrid peptide, IB1, demonstrated potent inhibition of IFN-γ/IFNGR1 protein-protein interaction (PPI) with an IC₅₀ of 12 nM, attributed to a cβAA.
• The presence of cγAAs and dαAAs contributed to enhanced serum stability of the peptides.
• Peptides effectively inhibited IFN-γ/IFNGR1 PPI at the cellular level, with the best IC₅₀ at 0.75 μM.

Conclusions:

• The developed platform enables the synthesis of complex hybrid peptides with enhanced stability and potency.
• These findings expand the chemical space for peptide-based drug discovery, particularly for targeting challenging protein-protein interactions.
• The study highlights the potential of l-α/d-α/β/γ-hybrid peptides in developing therapeutics for diseases involving the IFN-γ pathway.