Biased Equilibrium Drives Cyclosporine Membrane Permeability: The Goldilocks Energy Barriers

Conformational flexibility allows macrocyclic peptides like cyclosporine A (CycA) to cross membranes, yet drug design leveraging this property has largely failed. A key challenge is linking specific conformers to function, as different conformers govern permeability versus target binding. We reveal a mechanism that enhances CycA and alisporivir (ALI) permeability: <i>trans</i>-to-<i>cis</i> isomerization at MeVal11-MeBmt1 creates conformers that remain "soluble" in both membrane-like and aqueous environments. A biased equilibrium favors this conformer in protic environments, while a lipophilic conformer with <i>cis</i> MeLeu9-MeLeu10 dominates in aprotic conditions. This mechanism explains why CycH, Valspodar (VALSPO), and <i>O</i>-acetyl CycA (OAc-CycA) fail to cross membranes─they adopt similar states but lack this biased equilibrium. Our findings provide a new strategy for designing membrane-permeable <i>N</i>-methylated macrocycles and underscore the role of high-energy conformers as transition states between membrane permeability and target engagement─offering critical insights for drug development.