A Versatile Strategy for Screening Custom‐Designed Warhead‐Armed Cyclic Peptide Inhibitors

Abstract Demand for peptide‐based pharmaceuticals has been steadily increasing, but only limited success has been achieved to date. To expedite peptide‐based drug discovery, we developed a general scheme for cell‐based screening of cyclic peptide inhibitors armed with a user‐designed warhead. We combined unnatural amino acid incorporation and split intein‐mediated peptide cyclization techniques and integrated a yeast‐based colorimetric screening assay to generate a new scheme that we call the custom‐designed warhead‐armed cyclic peptide screening platform (CWCPS). This strategy successfully discovered a potent inhibitor, CY5‐6Q, that targets human histone deacetylase 8 (HDAC8) with a K D value of 15 nM. This approach can be a versatile and general platform for discovering cyclic peptide inhibitors.