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

We describe the development of an intracellular peptide library screening platform 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 we significantly expand the TBS methodology to identify covalent and highly selective inhibitors. We probed a 131,072-member library 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. Selection of a unique Cys in the antagonist indicated both target shutdown and concomitant disulphide formation in a single step, resulting in increased potency. Substituting Cys with an electrophile, generated 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.