Cdc25 inhibition and cell cycle arrest by a synthetic thioalkyl vitamin K analogue

A synthetic vitamin K analogue, 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoq uinone or compound 5 (Cpd 5), was found previously to be a potent inhibitor of tumor cell growth. We now demonstrate that Cpd 5 arrested cell cycle pr ogression at both G(1) and G(2)-M. Because of the potential arylating activ ity of Cpd 5, it might inhibit Cdc25 phosphatases, which contain a cysteine in the catalytic site. To test this hypothesis, we examined the inhibitory activity of Cpd 5 against several cell cycle-relevant protein tyrosine pho sphatases and found that Cpd 5 was a potent, selective, and partially compe titive inhibitor of Cdc25 phosphatases. Furthermore, Cpd 5 caused time-depe ndent, irreversible enzyme inhibition, consistent with arylation of the cat alytic cysteine in Cdc25. Treatment of cells with Cpd 5 blocked dephosphory lation of the Cdc25C substrate, Cdc2, and its kinase activity. Cpd 5 enhanc ed tyrosine phosphorylation of both potent regulators of G(1) transition, i .e., Cdk2 and Cdk4, and decreased the phosphorylation of Rb, an endogenous substrate for Cdk4 kinase. Furthermore, close chemical analogues that lacke d in vitro Cdc25 inhibitory activity failed to block cell cycle progression and Cdc2 kinase activity. Cpd 5 did not alter the levels of p53 or the end ogenous cyclin-dependent kinase inhibitors, p21 and p16. Our results suppor t the hypothesis that the disruption in cell cycle transition caused by Cpd 5 was attributable to intracellular Cdc25 inhibition. This novel thioalkyl K vitamin analogue could be useful for cell cycle control studies and may provide a valuable pharmacophore for the design of future therapeutics.