Conformationally Restricted Macrocycles as Improved FKBP51 Inhibitors Enabled by Systematic Linker Derivatization

Macrocycles are increasingly considered as promising modalities to target challenging intracellular proteins. However, strategies for transitioning from active linear starting points to improved macrocycles are still underdeveloped. Here we explored the derivatization of linkers as an approach for macrocycle optimization. Using the FK506‐binding protein 51 (FKBP51) as a model system we prepared >140 macrocycles with systematically derivatized linkers. Two backbones were identified as promising frameworks for subsequent optimization. Surprisingly, co‐crystal structure analyses revealed that these chemical templates represent an ensemble of three‐dimensional (3D) conformations that can give rise to several distinct 3D‐scaffolds. This resulted in a set of macrocycles with consistently improved affinity, plasma stability, and aqueous solubility compared to the linear precursors or the non‐functionalized macrocycles. Our results highlight linkers as an opportunity for macrocyclic drug development, show how linker derivatization can improve the performance of macrocycles, and emphasizes the need to track macrocyclic scaffold evolution at a three‐dimensional level.