Hydration Changes Implicated in the Remarkable Temperature-Dependent Membrane Permeation of Cyclosporin A

Abstract

Cyclosporin A is a cyclic peptide believed to exist as multiple conformers in aqueous solution. Two major conformations, distinguished by a single cis-trans isomerization and the presence of four either intramolecular or intermolecular hydrogen bonds, have been confirmed depending on whether CsA is characterized in organic solvents or bound in aqueous complex with cyclophilin. The relationship between CsA conformation and its ability to penetrate biological membranes is currently unknown. Using Caco-2 cell monolayers, we documented a remarkable increase (more than 2 orders of magnitude) in the membrane permeation of the peptide as temperature was increased from 5 to 37 degrees C. The solubility of CsA was 72 microM at 5 degrees C, but decreased by more than an order of magnitude at 37 degrees C. Moreover, CsA partitioned into non-hydrogen bond donating solvents linearly as a function of increasing temperature, suggestive of a significant conformational change. However, while NMR spectra of CsA confirmed the previously predicted presence of multiple conformers in aqueous solution, the equilibrium between the two major species was not affected by changes in temperature. These NMR data indicated that the observed temperature-dependent changes in the membrane permeability of CsA do not originate from changes in the peptide backbone conformation. Sedimentation equilibrium analysis revealed that CsA behaves in a highly nonideal manner over the temperature range tested. We interpret this behavior as a change in the hydration state with a smaller (or weaker) hydration shell surrounding the peptide at higher temperatures. Such a change would result in lower peptide desolvation energy, thereby promoting partitioning into cellular membranes. We contend that changes in membrane penetration result from alterations in the hydration state of CsA and are not related to the interconversion of the defined conformations.

Molecular Switch Controlling the Binding of Anionic Bile Acid Conjugates to Human Apical Sodium-Dependent Bile Acid Transporter

“…In spite of cyclosporine A being a relatively large cyclic peptide, this drug shows high permeability across membrane and is well absorbed from the gut after oral administration. Drug conformation, including intramolecular hydrogen bonding, has been considered as a determinant in cyclosporine A's high permeability16.…”

YcaO-Dependent Posttranslational Amide Activation: Biosynthesis, Structure, and Function

“…Cyclosporine adopts a conformation where internal hydrogen bonds result in a conformation displaying a hydrophobic surface while masking polar regions to facilitate membrane transit. 575, 576 In the specific case of cyanobactins, macrocyclization also removes the zwitterionic N- and C-terminal charges. Heterocyclization further increases the hydrophobicity of the natural product.…”

Molecular aspects of cyclophilins mediating therapeutic actions of their ligands

“…It could be secreted together with certain extracellular proteins and it should be able to sequester some quantity of the in vivoadministered CsA. It was also shown that a modified form of CsA anchored to the surface of T cells could exert its immunomodulatory effects [100], as negatively charged CsA derivatives rest in the extracellular space [101] whereas CsA binds well to the membranes in a temperature-dependent fashion [102]. If CsA binds to the extracellular pool of hCyPB, then such a complex has two crucial attributes for entering into the cell's interior, namely the large exposed hydrophobic patch of CsA should have some affinity to the hydrophobic environment of the membranes while the positively charged patches of CyPB should induce a substantial driving force for entry into T cells.…”