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 constrained peptide therapeutics that disrupt protein-DNA interfaces previously considered "undruggable."
Brennan et al. (Sun,) studied this question.