Abstract
<jats:p> As of 2026, more than 150 peptides are in clinical development as therapeutic candidates and molecular biology tools, and more than 120 therapeutic and diagnostic peptides have already been approved, representing a growing area of medicine. Stapled peptides are chemically constrained to retain their secondary structure to provide enhancements in binding affinity, membrane permeability and proteolytic resistance, and have emerged as promising solutions for the disruption of protein-protein interactions implicated in a variety of diseases. Molecular dynamics simulations have proven to be a powerful tool for exploring peptide secondary structure. However, the lack of readily available accurate force field parameters for a broad range of staple chemistries, as well as the computing cost of predicting the secondary structure of stapled peptides, have limited the accessibility of stapled peptide MD simulations. To facilitate greater adoption, we have developed the Stapline pipeline for the parameterisation of popular stapled peptide moieties and implemented an efficient MD simulation protocol to obtain stapled peptide secondary structure preferences. The protocol’s applicability is demonstrated on a series of hDM2-binding stapled peptides, successfully reproducing experimental <jats:italic toggle="yes">α</jats:italic> -helicity trends. </jats:p>