Screening of genetically encoded peptide libraries has emerged as a powerful tool for the discovery of inhibitors of therapeutically relevant protein targets. By linking genotype to phenotype, these approaches screen millions of peptides simultaneously, with numerous library-derived hits currently advancing through therapeutic development pipelines. However, most current screening technologies identify peptides that simply bind a target protein rather than disrupt its biological activity. To address this challenge, we previously developed transcription block survival (TBS), a bacterial peptide selection methodology that directly links cellular growth to the inhibition of transcription factor (TF)–DNA interactions. In a TBS assay, a target TF engages inserted DNA-binding sites in the promoter of an essential gene to block growth, which is rescued only if a peptide expressed from a library disrupts this interaction. Unlike peptide display technologies that enrich for binders, TBS selects functional antagonists inside living cells, enabling the discovery of peptides that disrupt TF function. However, the original implementation of TBS had an important limitation: peptides were expressed recombinantly and therefore screened in their linear forms, which are typically less effective and stable. To enhance peptide efficacy, promising hits were chemically cyclized after screening, which required costly and iterative chemical syntheses.
Using icTBS, we identified cyclic peptide antagonists for the oncogenic TF CREB1, a challenging target with considerable therapeutic potential. The most potent peptides bound CREB1 with nanomolar affinity, suppressed CREB1-dependent transcription and induced cancer cell death. These findings demonstrate that cyclic peptide inhibitors discovered in bacteria can translate into functional modulators of mammalian cell biology. This strategy enables chemically constrained peptides to be discovered through functional selection, opening new opportunities to discover inhibitors of TFs and other challenging DNA-binding proteins.
