Received for publication April 26, 2004.
Revised June 7, 2004.
Accepted for publication July 1, 2004.

Discovery and characterization of novel small molecule inhibitors of human Cdc25B dual specificity phosphatase

Abstract

Cdc25A and Cdc25B dual specificity phosphatases are key regulators of cell cycle transition and proliferation. They have oncogenic properties and are overexpressed in many human tumors. Since selective Cdc25 phosphatase inhibitors would be valuable biological tools and possible therapeutic agents, we have assayed a small molecule library for in vitro inhibition of Cdc25. We now report the identification of two new structurally distinct classes of Cdc25 inhibitors with cellular activity. The cyclopentaquinoline 5661118 (3a,4,5,9b-tetrahydro-1H-cyclopenta[c]quinoline-4,8-dicarboxylic acid) and the naphthofurandione 5169131 (3-benzoyl-naphtho[1,2-b]furan-4,5-dione) had in vitro IC₅₀ values of 2.5-11 µM against recombinant Cdc25 and were less potent inhibitors of other phosphatases. Unlike 5661118, 5169131 caused reversible inhibition of Cdc25B and displayed competitive inhibitor kinetics. No growth inhibitory activity was seen with 5661118, while 10-30 µM 5169131 caused G₁/S and G₂/M arrest. We also found that 5169131 inhibited human PC-3 prostate and MDA-MB-435 breast cancer cell proliferation. Concentration-dependent Tyr15 hyperphosphorylation was seen on cyclin dependent kinase 1 one hour after 5169131 treatment, consistent with Cdc25 inhibition. Cells resistant to DNA toposiomerase II inhibitors were as sensitive to 5169131 as parental cells indicating that this quinone compound does not inhibit topoisomerase II in vivo. Molecular modeling was used to predict a potential interaction site between the inhibitor and Cdc25B and to provide insights as to the molecular origins of the experimental observations. Based on its kinetic profile and cellular activity, we suggest 5169131 could be an excellent tool for further studies on the cellular roles of Cdc25.

Key words: Protein tyr Phosphatases, Protein Phosphatases (other), Structure-activity relationships and modeling, Protein targets, Mechanisms of cell killing/apoptosis, Oncogenes