Deconvolution of Permeability-Activity Relationships for Verticilide-Inspired Cyclic (Depsi)Peptides and Progression of the Structure-Activity Relationship Campaign for ent- V erticilide
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2025-07-01
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Abstract
Through a cross-disciplinary collaboration, it was discovered that ent-verticilide is a selective inhibitor of RyR2-mediated calcium release, including a preliminary study of efficacy in vivo. As a 24-membered cyclic depsipeptide with a molecular weight of 853 Da, ent-verticilide falls outside of the category of a traditional small molecule drug. While “Beyond Rule of 5” compounds with in vivo activity are growing in number, an understanding of their pharmacokinetics (PK) has lagged, thereby requiring new chemical tools and creative tactics to advance the field. By methodical structural changes to ent-verticilide, we have created an SAR-based feedback loop between permeability, activity, structure, and conformation. A strength of this approach is the combination of rigorous tools to study passive membrane permeability, and cardiomyocyte-based functional studies using both permeabilized and non-permeabilized cells to achieve an overall hypothesis-driven approach to discover how analogues of ent-verticilide travel through cellular membranes and ultimately target RyR2. Another strength is our positioning to prepare diverse analogues that include ring-chain variants likely to exhibit contrasting permeability. With increased understanding of the mechanism of action, we hypothesize that we can design analogues of ent-verticilide with improved potency and selectivity, thereby providing a potential therapeutic against fatal ventricular arrhythmias. Key findings of this work include that a single ester-to-N-H- amide point modification can enhance cell permeability significantly but several backbone modifications exhibit low activity and permeability; N-permethylation of ent-verticilide is critical for activity and permeability but in less critical with the ent-B1 oligomer; backbone cyclization enhances permeability; nat-verticilide is significantly less active and permeable than ent-
verticilide; and several side chain or backbone modifications results in loss of activity.
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permeability, macrocyclic peptides and depsipeptides, anti-arrhythmics, ent-verticilide, structure-activity relationships