Mixed Stereochemistry Macrocycle Acts as a Helix-Stabilizing Peptide N-Cap

Area of Science:

• Medicinal Chemistry
• Structural Biology
• Drug Discovery

Background:

• Protein-protein interactions involving alpha-helical peptides are key targets in drug discovery.
• Large interfaces of alpha-helices present challenges for developing inhibitors.
• Macrocyclic peptides show potential for stabilizing helical structures and inhibiting these interactions.

Purpose of the Study:

• To investigate N-terminal to side-chain thioether-cyclized peptides as inhibitors of the alpha-helix binding protein Mcl-1.
• To screen a trillion-scale library for potent Mcl-1 inhibitors.
• To characterize the structure-activity relationship of novel helical lariat (Heliat) inhibitors.

Main Methods:

• Screening of a trillion-scale library of modified peptides.
• Affinity determination using nanomolar (nM) assays.
• Structural analysis of macrocyclization effects on helical stability and peptide conformation.
• Synthesis of modified peptides incorporating a minimal macrocyclic motif.

Main Results:

• Identified 'Heliats' (helical lariats) that bind Mcl-1 with tens of nM affinity.
• Heliats effectively inhibit the interaction between Mcl-1 and its natural peptide ligands.
• Macrocyclization significantly enhances alpha-helical structure stability, affinity, and inhibitory potency.
• A minimal macrocyclic motif with D-stereochemistry at the N-terminal position demonstrated superior helix stabilization.

Conclusions:

• N-terminal to side-chain thioether-cyclized peptides (Heliats) are potent inhibitors of Mcl-1.
• Macrocyclization is a viable strategy to stabilize helical peptides and improve drug-like properties.
• The developed mixed-stereochemistry macrocyclic N-cap is synthetically accessible and enables rapid generation of helix-focused peptide libraries for *de novo* inhibitor discovery.