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Ubiquitin is a small, highly conserved protein that acts as a posttranslational modification in eukaryotes. Ubiquitination of proteins frequently serves as a degradation signal, marking them for disposal by the proteasome. Here we report a novel small molecule from a diversity-oriented synthesis library, BRD1732, that is directly ubiquitinated in cells, resulting in dramatic accumulation of inactive ubiquitin monomers and polyubiquitin chains, which causes broad inhibition of the ubiquitin-proteasome system. Ubiquitination of BRD1732 and its associated cytotoxicity are stereospecific and dependent on two homologous E3 ubiquitin ligases, RNF19A and RNF19B, and their shared E2 conjugating enzyme, UBE2L3. Our finding opens the possibility for indirect ubiquitination of a target through a ubiquitinated bifunctional small molecule and more broadly raises the potential for posttranslational modification in trans.