Design and Synthesis of macrocyclic FKBP51 ligands

The FK506-binding protein 51 (FKBP51) emerged as a key player in several diseases like stress-related disorders, chronic pain, and obesity. Linear analogs of FK506 called SAFit1 and SAFit2, the most advanced FKBP51 inhibitors so far, were shown to be highly selective for FKBP51 over its closest homolog FKBP52, allowing the proof-of-concept studies in animal models. However, the SAFit ligands show several liabilities in their drug-like parameters and further modifications are needed for future drug developments. Due to the shallow binding site of FKBP51, the creation of low molecular mass inhibitors based on the SAFit scaffold remained unfruitful. Here, we pursued a different strategy and chose a structure-based approach to implement the SAFit scaffold into a macrocyclic framework. Macrocycles are a compound class known to be able to cope with difficult drug targets and key drug properties like solubility, metabolic stability, and oral bioavailability often benefit from the cyclization of linear ligands. Hence, we designed a synthesis strategy to vary linkage, linker length and functionalization and synthesized the first FKBP51-selective, macrocyclic ligand library. All 33 macrocycles retained high FKBP51 affinity and selectivity over FKBP52 and the incorporation of polar functionalities further enhanced affinity. Six high-resolution crystal structures of macrocyclic inhibitors in complex with FKBP51 confirmed the desired selectivity-enabling binding mode. Our results show that macrocyclization is a viable strategy to target the shallow FKBP51 binding site selectively. Furthermore, we present the synthesis of multi-gram amounts of SAFit2 as well as an inactive SAFit2 diastereomer and deuterated SAFit analogs. These compounds will contribute to high-quality research on FKBP51 in cellular and animal models and help to decipher its biochemical and biological role.