Enhancement by displacement. A single methyl group displaces a water molecule from the binding site of FKBPs, resulting in the most potent binders known, outperforming the natural products FK506 and rapamycin in biochemical and cellular assays.

The FK506-binding protein 51 (FKBP51) emerged as a key player in several diseases like stress-related disorders, chronic pain, and obesity. Linear analogues of FK506 called SAFit were shown to be highly selective for FKBP51 over its closest homologue FKBP52, allowing the proof-of-concept studies in animal models. Here, we designed and synthesized the first macrocyclic FKBP51-selective ligands to stabilize the active conformation. All macrocycles retained full 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.

Subtype selectivity represents ac hallenge in many drug discovery campaigns.Atypical example is the FK506 binding protein 51 (FKBP51), whichh as emerged as an attractive drug target. The most advanced FKBP51 ligands of the SAFit class are highly selective vs.F KBP52 but poorly discriminate against the homologs and off-targets FKBP12 and FKBP12.6. During am acrocyclization pilot study,w eo bserved that many of these macrocyclic analogs have unanticipated and unprecedented preference for FKBP51 over FKBP12 and FKBP12.6. Structural studies revealed that these macrocycles bind with anew binding mode featuring atransient conformation, which is disfavored for the small FKBPs.Using aconformation-sensitive assayweshow that this binding mode occurs in solution and is characteristic for this new class of compounds.T he discovered macrocycles are non-immunosuppressive,e ngage FKBP51 in cells,a nd blockt he cellular effect of FKBP51 on IKKa.O ur findings provide an ew chemical scaffold for improved FKBP51 ligands and the structural basis for enhanced selectivity.

Subtyp-Selektivitäti st oft eine Herausforderung bei der Wirkstoffentwicklung. Ein Beispiel ist das FK506-bindende Protein 51 (FKBP51) als Wirkstoffziel. Die am weitesten fortgeschrittenen FKBP51-Liganden der SAFit-Klasse sind hochselektiv gegenüber FKBP52, differenzieren aber kaum gegenüber den eng verwandten Proteinen FKBP12 und FKBP12.6. Eine Makrozyklisierungsstudie ergab,d ass viele dieser makrozyklischen Analoga eine unerwartete,n euartige Präferenz fürF KBP51 gegenüber FKBP12 und FKBP12.6 haben. Strukturellen Studien zufolge weisen diese Makrozyklen einen neuen Bindungsmodus auf,m it einer transienten Protein-Konformation, die fürd ie kleinen FKBPs ungünstig ist. Mithilfe eines konformationssensitiven Assaysz eigen wir, dass dieser Bindungsmodus in Lçsung auftritt und charakteristisch fürdiese neue Verbindungsklasse ist. Die Makrozyklen sind nichti mmunsuppressiv,b inden FKBP51 in Zellen und blockieren die zelluläre Wirkung von FKBP51 auf IKKa.Die Ergebnisse liefern ein neues chemisches Gerüst fürverbesserte FKBP51-Liganden und die strukturelle Grundlage füre ine erhçhte Selektivität.

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