Mechanistic insights into the inhibition of insulin amyloidogenesis and associated toxicity by the p. M14L variant of exenatide

Insulin fibrillation and the subsequent formation of toxic amyloid aggregates are significant challenges in therapeutic development and are associated with cellular proteotoxicity. In this research, we explored how the p.M14L exenatide variant affects the formation of insulin amyloid fibrils. Applying a combination of spectroscopic and microscopic techniques, we demonstrated that this exenatide variant effectively shifts the aggregation pathway from mature fibrils toward off‐pathway amorphous aggregates. Atomic force microscopy (AFM) and dynamic light scattering (DLS) analyses revealed that in the presence of the exenatide variant, insulin is stabilized in off‐pathway amorphous aggregate structures with reduced hydrodynamic diameters. Molecular simulation studies further elucidated the molecular mechanisms underlying the interaction between insulin and the exenatide variant. Moreover, a cellular assay in SH‐SY5Y cells indicated that the aggregates formed in the presence of the exenatide variant significantly reduced cytotoxicity and apoptosis compared to mature fibrils. Therefore, the exenatide variant, through chaperone‐like behavior toward insulin, may hold potential as a candidate for improving its formulation stability and advancing the development of novel therapeutic approaches for amyloid‐related disorders.