Oral - Cyclosporines: A Deeper Look into Heterogeneity and Permeability

Macrocyclic peptides present an ideal therapeutic modality to target intracellular protein-protein interactions traditionally undruggable by small molecules. Many existing macrocyclic peptide drugs are natural product N-methylated macrocyclic peptides (N-MeMPs), exemplified by the cyclosporine family. Yet, the lack of comprehension regarding the chemical factors governing membrane permeability has significantly restricted the deployment of cyclic peptide medicinal agents. Over the past two decades, out of 18 cyclic peptides approved for clinical use, only 2 target intracellular proteins. To revolutionize the understanding of macrocyclic peptide structures and functions, we have determined a series of conformers required for robust passive membrane diffusion and those relevant to other functions, such as binding to protein targets or intermediates, in the presence of solvent additives. Nuclear magnetic resonance (NMR) and ion-mobility spectrometry-mass spectrometry (IMS-MS) are employed to characterize conformational heterogeneity and identify cis-amides relevant for good membrane permeability. In addition, ion mobility selected cyclic IMS-MS are conducted to evaluate the energy barriers between conformations. We study cyclosporine derivatives, such as cyclosporine H, cyclosporine C, cyclosporine A acetate, and alisporvirir, to draw conclusions about the properties that drive passive membrane permeation. Sifting through the conformational heterogeneity, we are discovering the importance of a complex conformational ensemble. It is demonstrated that conformational interconversion from the aqueous cis MeVal11–MeBmt1 state (A1) to the closed conformation featuring cis MeLeu9–MeLeu10 (C1) plays an essential role in facilitating membrane permeation. Additionally, we highlight that the transition from A1 to the all-trans open conformation (O1) is specifically triggered by the presence of CaCl2.