The presented work covers two projects: the stabilization of the F67-out conformation for the FK506 binding protein 51 (FKBP51) and the generation of a ligand-based affinity matrix.
FKBP51 is a prominent example of a transient binding pocket that is key to achieve selectivity in ligand development. In FKBP51, the transient binding pocket is characterized by a rearrangement of residue F67 outward from the binding pocket, also called the F67-out conformation. The aim was to generate protein constructs with a stabilized F67-out
conformation to identify hits in screenings targeting this specific protein conformation. By rational protein engineering, I succeeded to generate FKBP51 variants with a stabilized F67-out conformation. Techniques used to covalently fix the conformation in this out conformation include crosslinking approaches such as photocrosslinking, click chemistry,
or cysteine-directed crosslinking, as well as chemical protein synthesis. The obtained variants showed improvement in binding affinity to a conformationally selective ligand tracer in the fluorescence polarization (FP) assay. Overall, the FKBP51FK1 F67C/K60C variant with a disulfide bridge emerged as the most suitable variant for fragment screening.
The reasons for this are the facile production of the protein variant in high quantity and purity, the improvement in binding affinity for conformationally selective ligands, and the possibility of crystallizing the protein in its apo form in a F67-out-like conformation.
Initial fragment-based screening by thermoshift assay followed by FP assay resulted in promising hits with an amine-substituted quinoline and isoquinoline-core structure. This is the first step on a new path to identifying new lead structures. In addition, a ligand-based affinity chromatography matrix was generated. This should allow FKBP51 to be enriched from complex biological mixtures as well as identify new
interaction partners and/or off-targets. In this work, four ligands were successfully immobilized and tested, with loading and elution conditions optimized. Two systems were found to be suitable for the enrichment of FKBP51 from complex biological mixtures. Either SAFit1 is directly immobilized and elution takes place under harsh conditions, or a
SAFit1 analog with low affinity (here called SAFit-LA) is immobilized so that elution can be performed competitively with SAFit1
