Conformational Plasticity and Binding Affinity Enhancement Controlled by Linker Derivatization in Macrocycles

Macrocycles are abundantly used by nature to enable cell‐permeable bioactive molecules. Synthetic non‐peptidic macrocycles are also increasingly considered as modalities for difficult‐to‐bind proteins but guidelines for macrocyclization are only beginning to emerge. Macrocycles are thought to constrain the available conformations but also to allow for residual flexibility, the latter being poorly understood. Here we show that even medium‐sized macrocycles display an unexpected high conformational plasticity, even when bound to their protein target. Minor modification of the linker region of macrocycles can shift the conformational ensemble to distinct conformational subclasses, each constituting distinct three‐dimensional scaffolds for further optimization. This led to several new ligands with improved affinity and beneficial physicochemical parameters for the FK506‐binding protein 51, a promising target for depression, obesity and chronic pain. Importantly, none of the beneficial modifications could have been identified by classical medicinal chemistry as they only work in the macrocyclic context. Our results show that macrocyclization can do more than keeping loose ends together but rather provide a platform for multiple series of macrocycles with distinct binding modes.