title: Targeted protein degradation of FK506-binding proteins
Creators: name: Geiger, Thomas Maximilian
Publisher: name: UNSPECIFIED
Year: 2024

description: The FK506-binding proteins FKBP12, FKBP51 and FKBP52 are peptidyl-prolyl isomerases and best known for their ability to enable the natural molecular glues FK506 and Rapamycin. Among them, the large FKBP51 and FKBP52 are co-chaperones in the Hsp90-machinery and key regulators of glucocorticoid receptor signaling, with FKBP51 inhibiting and FKBP52 potentiating GR activity. 

To support the development of FKBP-directed drugs, I first established a panel of assays to address key biochemical and molecular biological aspects of FKBPs. (i) I developed a HTRF-based binding assay for FKBP ligands. This assay allows precise affinity determination for a new generation of ultra-high affinity FKBP ligands. (ii) Next, a NanoBRET assay for FKBP ligand profiling in living cells was developed, optimized and semi-automated. With this assay, FKBP ligands can be rapidly profiled for intracellular target-engagement. (iii) To assess the key cellular functions of FKBP51 and FKBP52 in glucocorticoid receptor signaling, I established GR activity reporter gene assays. Additionally, I developed (iv) eGFP fusion-based FKBP12- and FKBP51-level reporter assays as well as (v) a HTRF-based FKBP52 level reporter assay to assess the degradation of the respective FKBPs.

Ligand affinity is a key parameter for ligand efficacy. The HTRF assay demonstrated that FKBP ligands biochemically bind to FKBPs with picomolar affinities. Yet, intracellular target occupation is critical for ligands to act in a cellular environment. The NanoBRET binding assays unambiguously demonstrated for the first time that FKBP ligands occupy human FKBPs in living cells. However, a general off-set between biochemical affinity and intracellular target engagement became evident. Additionally, the results showed the superiority in cellular potency of macrocycles compared to linear SAFit-type ligands with similar affinity. The NanoBRET assay bridges the gap between biochemical binding and cellular effects and will thereby further guide ligand optimization towards ligands with cellular potency.
In a cellular context, FKBP51 is a key regulator and inhibitor of glucocorticoid signaling, which constitutes the functional link to stress-related disorders. Using the glucocorticoid signaling reporter gene assays, I was able to demonstrate that the synthetic ligands SAFit2 and 18(S Me) did not reactivate FKBP51-suppressed GR signaling and were functionally silent, albeit they occupied the FK506-binding site as evidenced by the NanoBRET assay. This demonstrates that FKBP51 regulates the GR through scaffolding functions and that the binding pocket is dispensable for GR regulation. However, the immunosuppressive natural product FK506, which protrudes far further from the binding-site reactivated GR signaling. This shows that FK506, but not smaller ligands, can abrogate regulatory FKBP51:GR contacts. 

The reporter gene data show that the scaffolding functions of FKBP51 cannot be addressed by synthetic FKBP ligands. But ligands for non-functional binding sites can be used to generate PROTACs that degrade the protein and therefore abolish all protein functions. Since the development of PROTACs for FKBP51 turned out to be much more challenging than expected, a large number of PROTAC candidates needed to be profiled. Towards this aim, I established the eGFP-fusion based FKBP12- and FKBP51-level reporter assays and the HTRF-based FKBP52-level reporter assay to test 220 FKBP-directed PROTAC candidates for degradation of the respective FKBPs. Surprisingly, the PROTAC candidates had a strong degradation bias for FKBP12 over the close homologs FKBP51 and FKBP52. Among the candidates, a plethora of PROTACs was active for FKBP12, six for FKBP51 and none for FKBP52. Subsequent, FKBP12-FKBP51FK1 swap mutant analysis showed that the presence of the FK2 and TPR domain negatively influences the degradability. However, linker-based optimization of a first generation PROTAC with limited selectivity overcame the negative degradation bias for FKBP51 and lead to potent FKBP51 PROTAC SelDeg51 with improved cellular activity and selectivity. Ultimately, SelDeg51 is the best-in-class compound and efficiently depletes FKBP51. I established SelDeg51 as a useful functional tool through thorough cellular characterization and by confirming its mode of action. In subsequent reporter gene assays, I could show that FKBP51 depletion, but not inhibition, potently reactivated GR signaling. This demonstrated a fundamentally different pharmacology with enhanced efficacy compared to the functionally silent synthetic ligands. 

Nature repeatedly resorted to FKBPs as adapter proteins to enable molecular glues. Taken together with the high degradability of FKBP12 this indicated FKBP12 as ideal model system to discover molecular glue degraders. In favorable cases, ligands can be molecular glues that possess additional gain of function properties. In this work, I used the eGFP-fusion based FKBP12 reporter assays to discover FKBP12_eGFP degrading molecular glues through cellular testing of &gt;900 in-house FKBP ligands. The structure-activity relationship of the initial screening hits and inactive analogs enabled the rational optimization of the most promising hit into PPu670 with doubled cellular potency. Subsequent cellular analysis revealed a FK506-binding site- and proteasome- but not neddylation-dependent mode of action. Finally, a CRISPR-based genome-wide knockout screen identified UBR3 as the relevant engaged E3 ligase. This work demonstrates that target-focused libraries can contain molecular glue degraders and that the E3 ligase UBR3 is glue-able and can be (re)directed to neo-substrates.
Overall, the assays I established will be instrumental to guide future FKBP ligand development. SelDeg51 can be used to target all protein functions and answer the question if cellular FKBP51 levels matter in certain contexts. Finally, my work demonstrates that cellular testing of target-focused libraries can be a fruitful approach to discover molecular glue degraders.