Abstract

mRNA display is a powerful technology to screen libraries of >1012 cyclic peptides against a protein target, enabling the rapid discovery of high affinity ligands. These cyclic peptides are particularly well suited to challenging protein targets that have been difficult to drug with small molecules. However, target choice can still be limited as screens are typically performed against purified proteins which often demands the use of isolated domains and precludes the use of aggregation‐prone targets. Here, we report a method to perform mRNA display selections in mammalian cell lysates without the need for prior target purification, vastly expanding the potential target scope of mRNA display. We have applied the methodology to identify low to sub‐nanomolar peptide binders for two targets: a NanoLuc subunit (LgBiT) and full‐length bromodomain‐containing protein 3 (BRD3). Our cyclic peptides for BRD3 were found to bind to the extraterminal (ET) domain of BRD3 and the closely related BRD proteins, BRD2 and BRD4. While many chemical probes exist for the bromodomains of BRD proteins, the ET domain is relatively underexplored, making these peptides valuable additions to the BRD toolbox.