Design, synthesis, structural analysis and biochemical studies of stapled AIF (370-394) analogues as ligand of CypA

The neuronal apoptotic process requires the nuclear translocation of Apoptosis Inducing Factor (AIF) in complex with Cyclophilin A (CypA) with consequent chromatin condensation and DNA degradation events. Targeting CypA by delivering an AIF-blocking peptide (AIF(370–394)) provides a significant neuro protection, demonstrating the biological relevance of the AIF/CypA complex. To date pharmaceutical com pounds targeting this complex are missing. Methods: We designed and synthesized a set of mono and bicyclic AIF(370–394) analogs containing both dis ulfide and 1,2,3-triazole bridges, in the attempt to both stabilize the peptide conformation and improve its binding affinity to CypA. Peptide structures in solution and in complex with CypA have been studied by circular dichroism (CD), Nuclear Magnetic Resonance (NMR) and molecular modeling. The ability of stapled peptides to interact with CypA was evaluated by using Epic Corning label free technique and Isothermal Titration Calorimetry experiments. Results: We identified a stapled peptide analogue of AIF(370–394) with a ten-fold improved affinity for CypA. Molecular modeling studies reveal that the new peptide acquires β-turn/β-fold structures and shares with the parent molecule the same binding region on CypA. Conclusions: Data obtained provide invaluable assistance in designing new ligand of CypA for therapeutic ap proaches in neurodegenerative diseases. General significance: Due to the crucial role of AIF/CypA complex formation in neurodegeneration, identification of selective inhibitors is of high importance for targeted therapies. We describe new bicyclic peptide inhibitors with improved affinity for CypA, investigating the kinetic, thermodynamic and structural effects of conforma tional constraints on the protein−ligand interaction, and their utility for drug design.