A simple method for developing lysine targeted covalent protein reagents.

Area of Science:

• Chemical Biology
• Biochemistry
• Drug Discovery

Background:

• Small molecules struggle to target shallow protein surfaces.
• Peptide-based covalent probes offer potential but require versatile modification strategies.
• Targeting broad residue ranges on proteins is challenging.

Purpose of the Study:

• To develop a versatile method for converting peptides and proteins into covalent binders.
• To enable targeting of shallow protein surfaces and a broad range of residues.
• To create potent and stable protein-based covalent binders.

Main Methods:

• Installation of thio-methacrylate ester-electrophiles on unprotected peptides and proteins via cysteine side chains.
• Selective reaction of these electrophiles with cysteine and lysine side chains on target proteins.
• Application of methacrylate-modified peptides and proteins to irreversibly label target proteins.

Main Results:

• Methacrylate phosphopeptides irreversibly labeled 14-3-3σ via lysine or cysteine residues.
• Methacrylate peptides targeting lysine showed pan-isoform binding of 14-3-3 proteins.
• A methacrylate-modified immunity protein irreversibly bound its target DNAse, increasing complex stability.

Conclusions:

• The thio-methacrylate ester approach provides a simple and versatile method for creating peptide and protein-based covalent binders.
• This strategy allows for efficient and selective covalent modification of target proteins.
• The developed covalent binders exhibit potent activity and enhanced stability, with potential applications in drug discovery.