RaPID Selection of α-Hydrazino Acid-Containing Macrocyclic Peptides.

Area of Science:

• Medicinal Chemistry
• Biotechnology
• Drug Discovery

Background:

• Incorporating alpha-hydrazino acid (α-Hza) into peptides enhances secondary structure stability, conformational rigidity, and proteolytic resistance.
• These properties are advantageous for developing therapeutic peptides with improved target binding affinity.
• However, de novo discovery of α-Hza-containing peptides against specific protein targets has not been successful.

Purpose of the Study:

• To construct diverse mRNA-encoded α-Hza-containing macrocyclic peptide libraries.
• To apply these libraries to the Random nonstandard Peptides Integrated Discovery (RaPID) selection system.
• To identify potent peptide binders against Janus kinase 2 (JAK2) and human factor XIIa (FXIIa).

Main Methods:

• Construction of diverse mRNA-encoded α-Hza-containing macrocyclic peptide libraries.
• Application of the RaPID selection system for affinity-based enrichment of ligands.
• Biochemical assays to evaluate binding affinity, inhibitory activity, target specificity, proteolytic stability, and membrane permeability.
• Mutational studies to elucidate the role of α-Hza residues.

Main Results:

• Identification of potent peptide binders with low-to-sub-nanomolar dissociation constants against JAK2 and FXIIa.
• Demonstration of potent inhibitory activity, target specificity, proteolytic stability, and membrane permeability in the identified binders.
• Mutational analyses confirmed the critical role of α-Hza residues in the functional potency of the macrocycles.

Conclusions:

• Establishment of a platform for de novo discovery of bioactive α-Hza-containing macrocyclic peptides.
• Expansion of the accessible chemical space for the RaPID system.
• Demonstration of the therapeutic potential of α-Hza-containing peptides for targeting enzymes like JAK2 and FXIIa.