Cdc25B dual-specificity phosphatase inhibitors identified in a high-throughput screen of the NIH compound library.
The University of Pittsburgh Molecular Library Screening Center (Pittsburgh, PA) conducted a screen with the National Institutes of Health compound library for inhibitors of in vitro cell division cycle 25 protein (Cdc25) B activity during the pilot phase of the Molecular Library Screening Center Network. Seventy-nine (0.12%) of the 65,239 compounds screened at 10 muM met the active criterion of > or =50% inhibition of Cdc25B activity, and 25 (31.6%) of these were confirmed as Cdc25B inhibitors with 50% inhibitory concentration (IC(50)) values <50 microM. Thirteen of the Cdc25B inhibitors were represented by singleton chemical structures, and 12 were divided among four clusters of related structures. Thirteen (52%) of the Cdc25B inhibitor hits were quinone-based structures. The Cdc25B inhibitors were further characterized in a series of in vitro secondary assays to confirm their activity, to determine their phosphatase selectivity against two other dual-specificity phosphatases, mitogen-activated protein kinase phosphatase (MKP)-1 and MKP-3, and to examine if the mechanism of Cdc25B inhibition involved oxidation and inactivation. Nine Cdc25B inhibitors did not appear to affect Cdc25B through a mechanism involving oxidation because they did not generate detectable amounts of H(2)O(2) in the presence of dithiothreitol, and their Cdc25B IC(50) values were not significantly affected by exchanging the dithiothreitol for beta-mercaptoethanol or reduced glutathione or by adding catalase to the assay. Six of the nonoxidative hits were selective for Cdc25B inhibition versus MKP-1 and MKP-3, but only the two bisfuran-containing hits, PubChem substance identifiers 4258795 and 4260465, significantly inhibited the growth of human MBA-MD-435 breast and PC-3 prostate cancer cell lines. To confirm the structure and biological activity of 4260465, the compound was resynthesized along with two analogs. Neither of the substitutions to the two analogs was tolerated, and only the resynthesized hit 26683752 inhibited Cdc25B activity in vitro (IC(50) = 13.83 +/- 1.0 microM) and significantly inhibited the growth of the MBA-MD-435 breast and PC-3 prostate cancer cell lines (IC(50) = 20.16 +/- 2.0 microM and 24.87 +/- 2.25 microM, respectively). The two bis-furan-containing hits identified in the screen represent novel nonoxidative Cdc25B inhibitor chemotypes that block tumor cell proliferation. The availability of non-redox active Cdc25B inhibitors should provide valuable tools to explore the inhibition of the Cdc25 phosphatases as potential mono- or combination therapies for cancer.
Johnston
Paul A
PA
University of Pittsburgh Drug Discovery Institute, Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, 3501 Fifth Avenue, Pittsburgh, PA 15260, USA. paj18@pitt.edu
Foster
Caleb A
CA
Tierno
Marni Brisson
MB
Shun
Tong Ying
TY
Shinde
Sunita N
SN
Paquette
William D
WD
Brummond
Kay M
KM
Wipf
Peter
P
Lazo
John S
JS
D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D045744:Cell Line, Tumor; D004353:Drug Evaluation, Preclinical; D054638:Dual Specificity Phosphatase 1 / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000302:isolation & purification; D054642:Dual Specificity Phosphatase 6 / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000302:isolation & purification; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D005260:Female; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D007202:Indicators and Reagents; D008297:Male; D054639:Mitogen-Activated Protein Kinase Phosphatases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000302:isolation & purification; D009316:National Institutes of Health (U.S.); D010084:Oxidation-Reduction; D054852:Small Molecule Libraries; D014481:United States; D020687:cdc25 Phosphatases / Q000037:antagonists & inhibitors*
D000970:Antineoplastic Agents; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D054852:Small Molecule Libraries; D006861:Hydrogen Peroxide; D054639:Mitogen-Activated Protein Kinase Phosphatases; C517019:DUSP1 protein, human; C517035:DUSP6 protein, human; D054638:Dual Specificity Phosphatase 1; D054642:Dual Specificity Phosphatase 6; D020687:cdc25 Phosphatases