Identification of KLHDC2 as an efficient proximity-induced degrader of K-RAS, STK33, ß-catenin, and FoxP3.

Targeted protein degradation (TPD), induced by enforcing target proximity to an E3 ubiquitin ligase using small molecules has become an important drug discovery approach for targeting previously undruggable disease-causing proteins. However, out of over 600 E3 ligases encoded by the human genome, just over 10 E3 ligases are currently utilized for TPD. Here, using the affinity-directed protein missile (AdPROM) system, in which an anti-GFP nanobody was linked to an E3 ligase, we screened over 30 E3 ligases for their ability to degrade 4 target proteins, K-RAS, STK33, ß-catenin, and FoxP3, which were endogenously GFP-tagged. Several new E3 ligases, including CUL2 diGly receptor KLHDC2, emerged as effective degraders, suggesting that these E3 ligases can be taken forward for the development of small-molecule degraders, such as proteolysis targeting chimeras (PROTACs). As a proof of concept, we demonstrate that a KLHDC2-recruiting peptide-based PROTAC connected to chloroalkane is capable of degrading HALO-GFP protein in cells.

Harnessing nanobodies for target protein degradation through the Affinity-directed PROtein Missile (AdPROM) system.

Targeted protein degradation (TPD) is a useful approach in dissecting protein function and therapeutics. Technologies such as RNA interference or gene knockout that are routinely used rely on protein turnover. However, RNA interference takes a long time to deplete target proteins and is not suitable for long-lived proteins, while a genetic knockout is irreversible, takes a long time to achieve and is not suitable for essential genes. TPD has the potential to overcome the limitations of RNA interference and gene editing approaches. We have established the Affinity directed PROtein Missile (AdPROM) system, which harnesses nanobodies or binders of target proteins to redirect E3 ubiquitin ligase activity to the target protein to induce TPD through the ubiquitin proteasome system. Here we provide a step-by-step protocol for using the AdPROM system for targeted proteolysis of endogenously GFP-tagged K-RAS through an anti-GFP nanobody. This protocol can be amended to target a wide range of different proteins of interest (POIs) either by replacing the anti-GFP nanobody with a nanobody recognising the POI or by endogenously tagging the POI with GFP through CRISPR/Cas9 genome editing.