Xue, Y.; Zaczek, F.; Jansen, R.-P.

Different small-molecule drugs targeting the same protein can produce divergent clinical outcomes through poorly characterized interactome changes. Existing proximity labeling approaches for target identification suffer from background biotinylation independent of small-molecule recruitment, obscuring true drug targets and their binding partners. Here, we incorporate a destabilizing domain (DD) into the biotin targeting chimera (BioTAC) framework to create ddBioTAC, wherein the proximity labeling enzyme TurboID is selectively stabilized only upon binding of a bifunctional targeting molecule. Using the bromodomain-targeting molecule NICE-01 in HeLa cells, we demonstrate that, in the absence of the bifunctional targeting molecule the destabilized TurboID enzyme (TurboID-DD) exhibits reduced protein levels and biotinylation activity compared to the control TurboID-FKBP (FK506- binding protein), while recovering comparable activity upon NICE-01 treatment. This results in an eightfold improvement in specific enrichment of the known target bromodomain containing protein 4 (BRD4) and its interactors, including MED1 and EF1D. Proteome-wide mass spectrometry confirms that ddBioTAC more accurately discriminates drug targets and proximal interactors from non-specific background, advancing unbiased drug-induced interactome profiling.