A method to identify small molecule/protein pairs susceptible to protein ubiquitination by the CRBN E3 ligase

Although using DNA-encoded libraries (DELs) to find small molecule binders of target proteins is well-established, identifying molecules with functions beyond binding remains challenging in pooled screens. Here, we develop an approach for multiplexing functional screens that simultaneously evaluates encoded small molecules and encoded collections of protein targets in functional selections. We focus on ubiquitin (Ub) transfer with the cereblon-bound CRL4 E3 ligase because of its proven versatility in drug discovery. The functional selections recover small molecule/G-hairpin loop pairs based on their ability to promote Ub-transfer onto the G-hairpin loop. As Ub-transfer is the first step in tagging proteins for proteasomal destruction, finding small molecules capable of selectively reprogramming it is a significant challenge in contemporary drug development. Our work lays the foundation for functional DEL selections that match small molecule Ub-transfer catalysts with their optimal protein substrates.