Cell-active dual specificity phosphatase inhibitors identified by high-content screening.
Phosphorylation of extracellular signal-regulated kinase (Erk) is tightly controlled by dual specificity phosphatases (DSPases), but few inhibitors of Erk dephosphorylation have been identified. Using a high-content, fluorescence-based cellular assay and the National Cancer Institute's 1990 agent Diversity Set, we identified ten compounds (0.5%) that significantly increased phospho-Erk cytonuclear differences in intact cells. Three of the ten positive compounds inhibited the mitogen-activated protein kinase phosphatase-3 (MKP-3/PYST-1) in vitro without affecting VHR or PTP1B phosphatases. The most potent inhibitor of MKP-3 had an IC(50) of <10 microM and inhibited MKP-3 in a novel, fluorescence-based multiparameter chemical complementation assay. These results suggest that the phospho-Erk nuclear accumulation assay may be a useful tool to discover DSPase inhibitors with biological activity.
Vogt
Andreas
A
Department of Pharmacology, University of Pittsburgh, Pittsburgh, PA 15261, USA.
Cooley
Kathleen A
KA
Brisson
Marni
M
Tarpley
Michael G
MG
Wipf
Peter
P
Lazo
John S
JS
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D001572:Benzofurans / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D054642:Dual Specificity Phosphatase 6; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000032:analysis*; D005455:Fluorescent Antibody Technique / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D051379:Mice; D041681:NIH 3T3 Cells; D010766:Phosphorylation; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013379:Substrate Specificity; D020687:cdc25 Phosphatases / Q000037:antagonists & inhibitors* / Q000378:metabolism
D000970:Antineoplastic Agents; D001572:Benzofurans; D004791:Enzyme Inhibitors; D007093:Imidazoles; C483753:NSC 357756; C096335:B59 protein, human; C517035:DUSP6 protein, human; D054642:Dual Specificity Phosphatase 6; C517036:Dusp6 protein, mouse; D017027:Protein Tyrosine Phosphatases; D020687:cdc25 Phosphatases