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

Simon P Shen, Brian B. Liau

Keywords: high-throughput screen, chemical genetics, targeted protein degradation

Abstract: 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.

Submission Number: 114