Increased Conformational Flexibility of a Macrocycle–Receptor Complex Contributes to Reduced Dissociation Rates

Constraining a peptide in its bioactive conformation by macrocyclization represents a powerful strategy to design modulators of challenging biomolecular targets. This holds particularly true for the development of inhibitors of protein‐protein interactions which often involve interfaces lacking defined binding pockets. Such flat surfaces are demanding targets for traditional small molecules rendering macrocyclic peptides promising scaffolds for novel therapeutics. However, the contribution of peptide dynamics to binding kinetics is barely understood which impedes the design process. Herein, we report unexpected trends in the binding kinetics of two closely related macrocyclic peptides that bind their receptor protein with high affinity. Isothermal titration calorimetry, (19)F NMR experiments and molecular dynamics simulations reveal that increased conformational flexibility of the macrocycle–receptor complex reduces dissociation rates and contributes to complex stability. This observation has impact on macrocycle design strategies that have so far mainly focused on the stabilization of bioactive ligand conformations.