Characterization of Peptide–Preservative Interaction by AlphaFold, Molecular Dynamics Simulation, and NMR Spectroscopy

The arrival of novel, more effective antiobesity medications has resulted in a surge in their use in the treatment of metabolic disorders including type 2 diabetes and weight-related comorbidities. Rapid growth in product demand has been witnessed in the United States and around the world. Ensuring that patients can access such life-saving medicines is vital to public health. One notable advancement in this field is the development of multiple-dose formulations containing antimicrobial preservatives. By enabling multiple doses from a single device or container, these products reduce the overall cost per dose and ease the supply chain bottleneck that causes product shortage, making medications more affordable and accessible. Interactions between a peptide and the preservative may induce structural perturbations and, as a result, impact the stability of the product. In the current study, we introduce a computational framework to investigate the interactions between a model therapeutic incretin peptide and antimicrobial preservatives, namely, benzyl alcohol and phenol. By integrating MD simulations with NMR data, we aim to elucidate how preservatives influence the stability of the peptide in the formulations. Our findings reveal that phenol displayed a significantly higher interaction frequency with the peptide compared to benzyl alcohol, particularly at the Trp cage and hydrophobic regions along the <i>N</i>- and <i>C</i>-termini. These interactions disrupt key stabilizing hydrogen bonds and increase the level of hydrophobic surface exposure, collectively heightening the stability risk of the peptide. Furthermore, phenol exhibited higher contact frequencies for residues such as Asp15, Ile17, Leu26, and Ile27, potentially explaining the differences in chemical stability observed with phenol-rich formulations. More importantly, the insights gained from simulations were independently corroborated by experimental results. This framework offers a strategy for developing robust multiple-dose preserved peptide formulations with the desired product stability throughout the shelf life.