Intracellular cyclization-coupled peptide library screening yields potent transcription factor antagonists

Transcription factors remain essential yet intractable targets for drug discovery owing to their flat, dynamic interfaces. We present an intracellular cyclization strategy that enables in-cell generation of conformationally constrained peptide libraries. Bis-alkylating reagents traverse bacterial membranes and selectively bridge cysteine pairs, permitting post-translational peptide stapling during in vivo screening. Integrated with the transcription block survival (TBS) assay, this intracellular-cyclization TBS (icTBS) platform simultaneously selects both peptide sequence and optimal constraint site, eliminating iterative synthesis. Libraries directed against
the oncogenic transcription factor CREB1 yielded three nanomolar-affinity antagonists, with cyclized variants selected by icTBS displaying enhanced functional activity in cellular assays. The lead peptide penetrated melanoma and colorectal cancer cells, suppressed CREB1-dependent transcription, reduced oncogenic protein
expression, and triggered apoptosis. icTBS thus provides a general, genetically encoded route to discover Q2 constrained peptide therapeutics that disrupt protein-DNA interfaces previously considered ‘‘undruggable.’’