Discovery and biological evaluation of a new family of potent inhibitors of the dual specificity protein phosphatase Cdc25.

The Cdc25 dual specificity phosphatases have central roles in coordinating cellular signaling processes and cell proliferation, but potent and selective inhibitors are lacking. We experimentally examined the 1990 compound National Cancer Institute Diversity Set and then computationally selected from their 140 000 compound repository 30 quinolinediones of which 8 had in vitro mean inhibitory concentrations <1 microM. The most potent was 6-chloro-7-(2-morpholin-4-ylethylamino)quinoline-5,8-dione (NSC 663284), which was 20- and 450-fold more selective against Cdc25B(2) as compared with VHR or PTP1B phosphatases, respectively. NSC 663284 exhibited mixed competitive kinetics against Cdc25A, Cdc25B(2), and Cdc25C with K(i) values of 29, 95, and 89 nM, respectively. As compared with NSC 663284, the regioisomer 7-chloro-6-(2-morpholin-4-ylethylamino)quinoline-5,8-dione was 3-fold less active against Cdc25B(2) in vitro and less potent as a growth inhibitor of human breast cancer cells. Computational electrostatic potential mapping suggested the need for an electron-deficient 7-position for maximal inhibitor activity. Using a chemical complementation assay, we found that NSC 663284 blocked cellular Erk dephosphorylation caused by ectopic Cdc25A expression.

Spatial analysis of key signaling proteins by high-content solid-phase cytometry in Hep3B cells treated with an inhibitor of Cdc25 dual-specificity phosphatases.

Protein phosphorylation frequently results in the subcellular redistribution of key signaling molecules, and this spatial change is critical for their activity. Here we have probed the effects of a Cdc25 inhibitor, 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone, or Compound 5, on the spatial regulation and activation kinetics of tyrosine phosphorylation-dependent signaling events using two methods: (i) high-content, automated, fluorescence-based, solid-phase cytometry and (ii) a novel cellular assay for Cdc25A activity in intact cells. Immunofluorescence studies demonstrated that Compound 5 produced a concentration-dependent nuclear accumulation of phospho-Erk and phospho-p38, but not nuclear factor kappaB. Immunoblot analysis confirmed Erk phosphorylation and nuclear accumulation, and in vitro kinase assays showed that Compound 5-activated Erk was competent to phosphorylate its physiological substrate, the transcription factor Elk-1. Pretreatment of cells with the MEK inhibitor U-0126 prevented the induction by Compound 5 of phospho-Erk (but not phospho-p38) nuclear accumulation and protected cells from the antiproliferative effects of Compound 5. Overexpression of Cdc25A in whole cells caused dephosphorylation of Erk that was reversed by Compound 5. The data show that an inhibitor of Cdc25 increases Erk phosphorylation and nuclear accumulation and support the hypothesis that Cdc25A regulates Erk phosphorylation status.

Identification of new Cdc25 dual specificity phosphatase inhibitors in a targeted small molecule array.

Dual specificity protein phosphatases (DSPases) are key regulators of signal transduction, oncogenesis and the cell cycle. Few potent or specific inhibitors of DSPases, however, are readily available for these pharmacological targets. We have used a combinatorial/parallel synthetic approach to rigidify the variable core region and modify the side chains of 4-(benzyl-(2-[2,5-diphenyl-oxazole-4-carbonyl)-amino]-ethyl)-carbamoyl)- 2-decanoylamino butyric acid (or SC-alphaalphadelta9), which is the most active element in a previously described library of phosphatase inhibitors (Rice, R. L.; Rusnak, J. M.; Yokokawa, F.; Yokokawa, S.; Messner, D. J.; Boynton, A. L.; Wipf, P.; Lazo, J. S. Biochemistry 1997, 36, 15965). Several analogues were identified as effective inhibitors of the protein tyrosine phosphatase (PTPase) PTP1B and the DSPases VHR and Cdc25B2. Two compounds, FY3-alphaalpha09 and FY21-alphaalpha09, were partial competitive inhibitors of Cdc25B2 with Ki values of 7.6+/-0.5 and 1.6+/-0.2 microM, respectively. FY21-alphaalpha09 possessed only moderate activity against PTP1B. Consistent with its in vitro anti-phosphatase activity, FY21-alphaalpha09 inhibited growth in MDA-MB-231 and MCF-7 human breast cancer cell lines. FY21-alphaalpha09 also inhibited the G2/M transition in tsFT210 cells, consistent with Cdc25B inhibition. Several architectural requirements for DSPase inhibition were revealed through modification of the side chain moieties or variable core region of the pharmacophore, which resulted in decreased compound potency. The structure of FY21-alphaalpha09 provides a useful platform from which additional potent and more highly selective phosphatase inhibitors might be generated.

Small molecule inhibitors of dual specificity protein phosphatases.

One hallmark of neoplasia is the deregulation of cell cycle control mechanisms, which is secondary to altered protein phosphorylation. Dual specificity protein phosphatases uniquely dephosphorylate both phosphoserines/threonines and phosphotyrosines on the same protein substrate. As a class they regulate intracellular signaling through the mitogen activated and stress activated kinases and govern cellular movement through G1/S and G2/M cell cycle checkpoints by affecting the activity of cyclin-dependent kinases. In particular, the Cdc25 phosphatases, which dephosphorylate cyclin-dependent kinases, are overexpressed in many human tumors and this increased expression is associated with a poor prognosis. In addition to expression levels, the intracellular activity of Cdc25 phosphatases is determined by their subcellular distribution and physical proximity to substrates. Small molecules that either inhibit the catalytic activity or alter the subcellular distribution of these dual specificity protein phosphatases could provide effective tools to interrogate the role of phosphorylation pathways and may afford new approaches to the management of cancer.