Selective ubiquitination of drug-like small molecules by the ubiquitin ligase HUWE1

Abstract The ubiquitin system regulates eukaryotic physiology by modifying myriad substrate proteins. Substrate specificity and the assembly of ubiquitin signals are determined by ubiquitin ligases, some of which also modify non-protein biomolecules. Here we expand this substrate realm, revealing that the human ligase HUWE1 can target drug-like small molecules. We demonstrate that compounds previously reported as HUWE1 inhibitors present substrates of their target ligase. Compound ubiquitination is driven by the canonical catalytic cascade, linking ubiquitin to the compound’s primary amino group. In vitro, the modification is selectively catalyzed by HUWE1, allowing the compounds to compete with protein substrates. We establish cellular detection methods, confirming HUWE1 promotes — but does not exclusively drive — compound ubiquitination in cells. Converting the existing compounds into specific HUWE1 substrates or inhibitors thus requires enhanced specificity. More broadly, our findings open avenues for harnessing the ubiquitin system to transform exogenous small molecules into novel chemical modalities within cells.