Preventing Protein Self‐Association Through Strategic Covalent Modification

Protein self-interaction leading to aggregation is a major challenge facing protein pharmaceuticals. It leads to a range of problems, including increases in immunogenicity and loss of activity. In this work, we describe an approach for blocking or antagonizing the quaternary interactions that drive self-association. We applied the approach to glucagon, a therapeutic peptide known for its propensity to form fibrils due to self-interaction. We synthesized a regio-pure common feedstock that allowed easy modification with potential blocking peptides that represented a range of chemical types (anionic, cationic, polar, and nonpolar). From these synthesized materials, we identified two modified glucagons that showed significant stabilization against fibril formation compared with unmodified glucagon. This was confirmed by three complementary biophysical techniques. Both successful modifications introduced excess net charge to glucagon, consistent with overall electrostatic repulsion being at the root of the observed fibrillation resistance. This approach can potentially be applied to other therapeutic proteins that suffer from the problems associated with self-association.