Inducible Degradation of Target Proteins Through a Tractable Affinity-Directed PROtein Missile (AdPROM) System

https://papers.ssrn.com/sol3/papers.cfm?abstract_id=3552150 Previously we described an Affinity-directed PROtein Missile (AdPROM) system that utilises specific polypeptide binders of intracellular proteins of interest (POIs) conjugated to an E3 ubiquitin ligase moiety to enable targeted proteolysis of the POI. However, a chemically tuneable AdPROM system is more desirable. Here, we combine Halo-tag/VHL-recruiting proteolysis-targeting chimeras (HaloPROTACs) with AdPROM for ligand-inducible (L-AdPROM) degradation of POIs. When we express an L-AdPROM construct consisting of an anti-GFP nanobody conjugated to a Halo-tag, we achieve robust degradation of GFP-tagged POIs, including GFP-ULK1, FAM83D-GFP and SGK3-GFP, knocked in with a GFP-tag using CRISPR/Cas9, only upon treatment of cells with the HaloPROTAC. Substituting the anti-GFP nanobody for a monobody that binds H- and K-RAS, we achieve robust degradation of unmodified endogenous RAS proteins only in the presence of the HaloPROTAC. Through substitution of the polypeptide binder, the highly versatile L-AdPROM system is useful for the inducible degradation of potentially any intracellular POI.