Kinetics and Thermodynamics of Fibril Elongation and Dissociation of Dual Glucagon-like Peptide and Glucagon Receptor Agonists

Abstract

Obesity and type 2 diabetes require durable peptide therapeutics, yet oxyntomodulin (Oxm) and its analogue Aib2-Oxm exhibit short serum half-lives due to rapid enzymatic degradation. Fibrillar self-assembly markedly enhances their stability, motivating a systematic understanding of the thermodynamics and kinetics governing fibril formation and dissociation. Here, using quartz crystal microbalance with dissipation (QCM-D), we provide the first comprehensive quantitative analysis of these processes for Oxm and Aib2-Oxm. Both peptides form fibrils under the experimental conditions studied, with equilibrium free-peptide concentrations indicating that fibril incorporation is thermodynamically favored when soluble peptide concentration exceeds the equilibrium solubility. Oxm fibrils exhibit greater apparent thermodynamic stability than Aib2-Oxm fibrils, consistent with lower equilibrium free-peptide concentration, greater apparent lateral fibril association, and secondary-structure trends. Oxm peptides also exhibit faster elongation on preformed fibril seeds, whereas Aib2-Oxm fibril elongation is associated with a larger apparent activation barrier for conformational incorporation into the fibrillar state. Oxm peptides also nucleate and elongate more rapidly, whereas Aib2-Oxm fibrillation is slowed by a larger activation energy for peptide unfolding. Consequently, Aib2-Oxm fibrils dissociate faster than Oxm fibrils, predicting greater in-serum bioactivity due to enhanced peptide release. To enable controlled therapeutic deployment, we examined Aib2-Oxm fibrillation and dissociation across temperature, peptide concentration, and seed size. Aib2-Oxm assembly follows classical Arrhenius behavior, with fibrillation rates doubling between 23 and 37 °C. Peptide release likewise increases with temperature and is substantially slower at subcutaneous conditions (32 °C) than at core physiological temperatures (37–42 °C), supporting prolonged bioactive release following subcutaneous administration. This work provides the first integrated thermodynamic–kinetic framework for Oxm and Aib2-Oxm fibrils and establishes principles for engineering long-acting, protease-resistant peptide depots for obesity and type 2 diabetes therapy.