A novel combinatorial high-throughput assay of small molecules and their protein targets

Understanding the molecular determinants of substrate specificity is essential for designing molecular glue degraders that engage new targets or avoid undesired off-targets. While prior studies have characterized degron motifs and structure-activity relationships of the thalidomide scaffold, decoding selective degradation requires profiling the effects of co-varying both sides of the interaction, and these efforts have been limited because we lack a scalable method to probe chemical and protein variation combinatorially. To address this need, we leveraged single-cell RNA sequencing and designed a molecular recording circuit to enable a platform for high-throughput screens of a chemical library and protein library combinatorially in live cells. Using this system, we quantified the degradation of 117 SALL4 point mutants across 138 glutaramide-containing compounds, generating 16,146 measurements that revealed key residue-functional group interactions. Together, these results inform rational design of molecular glue degraders with improved specificity, nominate promising leads for degraders of new neosubstrates, and establish a generalizable framework for mapping chemical-genetic interaction landscapes more broadly.