biostudies-literatureHaslbock SJapan Agency for Medical Research and DevelopmentMinistry of Education, Culture, Sports, Science and TechnologyJapan Society for the Promotion of Science368-378https://www.ebi.ac.uk/biostudies/studies/S-EPMC12814355biostudies-literatureUnknown148(1)De novo discovery of highly selective inhibitors of protein kinases (PKs) remains a significant challenge in drug discovery. Although ATP-site-directed bivalent inhibitors targeting a distinct binding site are an emerging alternative, their rational design is impeded by limited knowledge about allosteric binding sites unique to the target kinase and the complexity of the linker design. Here, we report a strategy that overcomes the above-mentioned issue using a macrocyclic peptide library containing an ATP-mimetic "warhead" amino acid, <i>p</i>-(pyrimidin-2-ylamino)phenylalanine (termed Z), performing the RaPID selection for the discovery of highly potent and selective inhibitors against the kinase GSK3β. The experiment led to the enrichment of macrocycles containing a sequence motif "ZRZ",Journal of the American Chemical SocietyDiscovery of Supra-Bivalent GSK3β Inhibitory Peptides Containing an ATP-Mimetic Amino Acid.PMC12814355JP20H05618JP25ama121001Sengoku TSuga HVinogradov AAHaslbock SOkada CFujimura NAikawa HfalseDiscovery of Supra-Bivalent GSK3β Inhibitory Peptides Containing an ATP-Mimetic Amino Acid.De novo discovery of highly selective inhibitors of protein kinases (PKs) remains a significant challenge in drug discovery. Although ATP-site-directed bivalent inhibitors targeting a distinct binding site are an emerging alternative, their rational design is impeded by limited knowledge about allosteric binding sites unique to the target kinase and the complexity of the linker design. Here, we report a strategy that overcomes the above-mentioned issue using a macrocyclic peptide library containing an ATP-mimetic "warhead" amino acid, <i>p</i>-(pyrimidin-2-ylamino)phenylalanine (termed Z), performing the RaPID selection for the discovery of highly potent and selective inhibitors against the kinase GSK3β. The experiment led to the enrichment of macrocycles containing a sequence motif "ZRZ",2026-01-01T00:00:00Z2026 Jan2026-06-06T15:38:48.346Z2026-06-02T03:09:02.107ZS-EPMC128143554148159810.1021/jacs.5c13788