A Systematic Direct-to-Biology Approach Identified Potent Cereblon HaloPROTACs

HaloTags have emerged as versatile tools for protein labeling, investigating target biology, and facilitating targeting protein degradation using synthetic ligands called HaloPROTACs. These chemical tools are heterobifunctional molecules consisting of a chloroalkane derivative linked to E3 ubiquitin ligase-recruiting moieties to artificially induce proximity between the Ligase and target protein of interest fused to the HaloTag construct. Through this induced proximity, HaloPROTACs can facilitate temporal- and dose-dependent degradation of target proteins, enabling efficient cellular protein knockdown without the need for target-specific ligands. In this study, we developed Cereblon (CRBN)-recruiting HaloPROTACs through the use of plate-based high-throughput synthesis and direct-to-biology screening. We evaluated over 100 structurally diverse CRBN HaloPROTACs in an FAK-tagged cell line, resulting in the identification of highly potent tools. Notably, HaloPROTACs incorporating dihydrouracil CRBN Binders exhibited superior potency and selectivity compared with their IMiD-based counterparts. These newly developed CRBN-based HaloPROTACs represent a valuable addition to the existing HaloPROTAC toolkit, offering enhanced capabilities for advancing research on targeted protein degradation.