A practical "preTACs-cytoblot" platform accelerates the streamlined development of PROTAC-based protein degraders.

With the growing importance of PROTAC-mediated protein degradation in drug discovery, robust synthetic methodologies and rapid screening assays are urgently needed. By harnessing the improved alkene hydroazidation reaction, we developed a novel strategy to introduce azido groups into the linker-E3 ligand conjugates and effectively created a range of prepacked terminal azide-labeled "preTACs" as PROTAC toolkit building blocks. Moreover, we demonstrated that preTACs are ready to conjugate to ligands targeting a protein of interest to generate libraries of chimeric degraders, which are subsequently screened for effective protein degradation directly from cultured cells with a cytoblot assay. Our study exemplifies that this practical "preTACs-cytoblot" platform allows efficient PROTAC assembly and rapid activity assessments. It may help industrial and academic investigators to accelerate their streamlined development of PROTAC-based protein degraders. [Display omitted] • A practical preTACs-Cytoblot platform is reported to streamline the PROTAC discovery. • Alkene hydroazidation is a useful method to synthesize the preTAC building blocks. • Improved methods to efficiently conjugate preTACs to the ligand of a target protein. • The Cytoblot assay was used to assess PROTAC activity directly from cells rapidly. • A potent CDK12 degrader V7 was identified with cell-selective antiproliferation activity. [ABSTRACT FROM AUTHOR]