Molecular modeling studies led to the identification of LFM-A13 (alpha-cyano-beta-hydroxy-beta-methyl-N-(2,5-dibromophenyl)propenamide) as a potent inhibitor of Polo-like kinase (Plk). LFM-A13 inhibited recombinant purified Plx1, the Xenopus homolog of Plk, in a concentration-dependent fashion, as measured by autophosphorylation and phosphorylation of a substrate Cdc25 peptide. LFM-A13 was a selective Plk inhibitor. While the human PLK3 kinase was also inhibited by LFM-A13 with an IC(50) value of 61 microM, none of the 7 other serine/threonine kinases, including CDK1, CDK2, CDK3, CHK1, IKK, MAPK1 or SAPK2a, none of the 10 tyrosine kinases, including ABL, BRK, BMX, c-KIT, FYN, IGF1R, PDGFR, JAK2, MET, or YES, or the lipid kinase PI3Kgamma were inhibited (IC(50) values >200-500 microM). The mode of Plk3 inhibition by LFM-A13 was competitive with respect to ATP with a K(i) value of 7.2 microM from Dixon plots. LFM-A13 blocked the cell division in a zebrafish (ZF) embryo model at the 16-cell stage of the embryonic development followed by total cell fusion and lysis. LFM-A13 prevented bipolar mitotic spindle assembly in human breast cancer cells and glioblastoma cells and when microinjected into living epithelial cells at the prometaphase stage of cell division, it caused a total mitotic arrest. Notably, LFM-A13-delayed tumor progression in the MMTV/neu transgenic mouse model of HER2 positive breast cancer at least as effectively as paclitaxel and gemcitabine. LFM-A13 showed a favorable toxicity profile in mice and rats. In particular there was no evidence of hematologic toxicity as documented by peripheral blood counts and bone marrow examinations. These results establish LFM-A13 as a small molecule inhibitor of Plk with in vitro and in vivo anti-proliferative activity against human breast cancer.