Dual G1 and G2/M phase inhibition by SC-alpha alpha delta 9, a combinatorially derived Cdc25 phosphatase inhibitor

Abstract

The Cdc25 dual specificity phosphatase family has a central role in controlling cell cycle progression and has been implicated in the etiology of cancer. One compound, 4-(benzyl-(2-[(2, 5-diphenyl-oxazole-4-carbonyl)-amino]-ethyl)-carbamoyl)-2-decanoylami no butyric acid (SC-alpha alpha delta 9), was previously identified as the most potent reported synthetic inhibitor of Cdc25 phosphatases in vitro. In the present study, we demonstrate that SC-alpha alpha delta 9 inhibited Cdc25-dependent cell cycle progression at both G1 and G2/M phase using tsFT210 cells, which express a temperature-sensitive Cdc2 mutant. SC-alpha alpha delta 9 blocked both G2/M transition and dephosphorylation of Cdc2 in a concentration-dependent manner. SC-alpha alpha delta 9 also enhanced tyrosine phosphorylation of both Cdk2 and Cdk4, and decreased Cdk4 kinase activity. Both of the kinases are potent regulators of G1 transition. Furthermore, closely related chemical analogs that lacked Cdc25 inhibitory activity failed to block cell cycle progression at both G1 and G2/M, and did not affect Cdc2 phosphorylation or Cdk4 kinase activity. SC-alpha alpha delta 9 did not alter p53, p21 or p16 levels. Our results support the hypothesis that the disruption in cell cycle transition caused by SC-alpha alpha delta 9 was due to intracellular Cdc25 inhibition. We propose that the SC-alpha alpha delta 9 pharmacophore could be useful in further clarifying the role of Cdc25 phosphatase-dependent pathways in checkpoint control, oncogenesis, and apoptosis.