Synthesis, Conformation, and Permeability of Hydroxyl-containing Cyclosporin O Derivatives

Abstract

Cyclic peptides have been emerged as a promising scaffold for targeting intracellular protein-protein interaction (PPIs) which have been considered undruggable. However, membrane permeability is an obstacle for cyclic peptides to reach the intracellular targets. Physico-chemical properties and conformational characteristics, such as lipophilicity (i.e., AlogP) and chameleonic behavior to maximize solubility both in aqueous and lipid media, are known to be responsible for adequate membrane permeability of macrocyclic peptides. Here, we designed cyclic peptide library using cyclosporin O (CsO) scaffold and examined its structure-permeability relationship (SPR). Polar residue, which is often detrimental to cell permeability, was introduced, and the permeability of the CsO derivatives was compared with that of CsO. Parallel artificial membrane permeability assay (PAMPA) and nuclear magnetic resonance (NMR) spectroscopy were implemented to evaluate permeability and conformation of the peptides, respectively. Most cyclic peptides containing hydroxyl side chains had similar structures compared to CsA and CsO, but 1 and 2 had conformational heterogeneity due to lack of intramolecular hydrogen bonding. This observation indicates that the location of polar residue introduction is a significant element and significantly affect backbone structure. Decreased AlogP value due to hydroxyl side chain introduction was expected to result in low permeability, but 5 and 7 yielded similar or 4-fold higher permeability compared to CsO. This result suggests that the optimal lipophilicity of CsO scaffold for membrane permeability is same with CsA scaffold. Our results provide insights for designing membrane permeable macrocyclic peptides, which potentially target intracellular PPIs.