An Intracellular Peptide Library Screening Platform Identifies Irreversible Covalent Transcription Factor Inhibitors

Abstract The development of an intracellular peptide library screening platform is described to identify covalent transcription factor (TF) antagonists. The Transcription Block Survival (TBS) assay and subsequent hit refinement previously produced potent but reversible antagonists of the oncogenic TF cJun. TBS moves beyond a target binding readout to ensure loss of TF function by blocking TF‐DNA binding. Here, the TBS methodology is significantly expanded to identify covalent and highly selective inhibitors. A 131,072‐member library is probed containing a Cys option at nine positions within a non‐reducing cell line. This identified a single Cys residue with the appropriate geometry for disulphide bond formation with cJun C269 in its DNA binding domain. The selection of a unique Cys in the antagonist indicates both target shutdown and concomitant disulphide formation in a single step, resulting in increased potency. Substituting Cys with an electrophile generates an irreversible yet highly selective covalent cJun inhibitor capable of penetrating human melanoma cells in culture and depleting oncogenic cJun levels to inhibit cell viability, with enhanced efficacy compared to a previous cJun‐targeting peptide. This enhanced covalent‐TBS screening pipeline provides a robust approach to profile target protein surfaces for ligandable cysteines, producing covalent and selective antagonists with appropriately positioned warheads.