Emilia Hietala, CCD Vault, UK 

PROteolysis TArgeting Chimeras (PROTACs) are chimeric molecules to induce the multistep degradation process of a protein of interest through the ubiquitin proteasomal system (UPS). Despite PROTACs are from rising interest in the drug-development field and entering the clinic, the rate limiting steps to determine their efficiency are largely unknown. Additionally, the major analysis of PROTAC-induced degradation is marked by endpoint assays like Western blotting or proteomics, which throughput and dynamic range limit the analysis of PROTAC SAR series. To tackle this, we developed an assay pipeline based on NanoLuciferase and fluorescently labelled HaloTag which allows measurement of ternary complex and degradation kinetics to test the influence of ternary complex formation and stability on degradation efficiency in live cells. Using this pipeline, we analyzed a set of chemically divers VHL recruiting WDR5 degraders to find a correlation between the steps in the degradation cascade where we were able to reveal a key role of ternary complex formation and stability over the binary PROTAC-WDR5 and PROTAC-E3 complexes. The developed pipeline outlines a strategy for rational PROTAC optimization in live cells for the early PROTAC induced degradation pathway.