Polo-like kinase 1(Plk1)is an evolutionarily conserved serine/threonine kinase in mammalian cells that participates in multiple steps in mitosis.Its N-terminal kinase domain(KD)controls cell signaling through phosphorylation.Inhibitors of Plk1 are potential anticancer drugs.Most known Plk1 KD inhibitors are ATP-competitive compounds,which may suffer from low selectivity.Until now,non-ATP-competitive inhibitors of Plk1 KD have not been reported.We discovered novel non-ATP-competitive Plk1 KD inhibitors by virtual screening and experimental studies.Potential binding sites in Plk1 KD were identified by using the protein binding site detection program Cavity.The identified site was subjected to molecular-docking-based virtual screening.The activities of top-ranking compounds were evaluated by in vitro enzyme assay with full-length Plk1 and direct binding assay with Plk1 KD.Several compounds showed inhibitory activity.The most potential compound with an IC50 value of 13.1±1.7 dμoeMs not compete with ATP or the peptide substrate.Our work provides new insight into the design of kinase inhibitors that target non-ATP binding sites.