Anti-breast cancer activity of LFM-A13, a potent inhibitor of Polo-like kinase (PLK)

Molecular modeling studies led to the identification of LFM-A13 (α-cyano-β-hydroxy-β-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 6 μM, 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 PI3Kγ were inhibited (IC 50 values > 200–500 μM). The mode of Plk3 inhibition by LFM-A13 was competitive with respect to ATP with a K i value of 7.2 μM from Dixon plots. 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. 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. Molecular modeling studies led to the identification of LFM-A13 (α-cyano-β-hydroxy-β-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 μM, 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 PI3Kγ were inhibited (IC 50 values >200–500 μM). The mode of Plk3 inhibition by LFM-A13 was competitive with respect to ATP with a K i value of 7.2 μM 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. No