The benzo[c]phenanthridine alkaloid, sanguinarine, is a selective, cell-active inhibitor of mitogen-activated protein kinase phosphatase-1.
Mitogen-activated protein kinase phosphatase-1 (MKP-1) is a dual specificity phosphatase that is overexpressed in many human tumors and can protect cells from apoptosis caused by DNA-damaging agents or cellular stress. Small molecule inhibitors of MKP-1 have not been reported, in part because of the lack of structural guidance for inhibitor design and definitive assays for MKP-1 inhibition in intact cells. Herein we have exploited a high content chemical complementation assay to analyze a diverse collection of pure natural products for cellular MKP-1 inhibition. Using two-dimensional Kolmogorov-Smirnov statistics, we identified sanguinarine, a plant alkaloid with known antibiotic and antitumor activity but no primary cellular target, as a potent and selective inhibitor of MKP-1. Sanguinarine inhibited cellular MKP-1 with an IC50 of 10 microM and showed selectivity for MKP-1 over MKP-3. Sanguinarine also inhibited MKP-1 and the MKP-1 like phosphatase, MKP-L, in vitro with IC50 values of 17.3 and 12.5 microM, respectively, and showed 5-10-fold selectivity for MKP-3 and MKP-1 over VH-1-related phosphatase, Cdc25B2, or protein-tyrosine phosphatase 1B. In a human tumor cell line with high MKP-1 levels, sanguinarine caused enhanced ERK and JNK/SAPK phosphorylation. A close congener of sanguinarine, chelerythrine, also inhibited MKP-1 in vitro and in whole cells, and activated ERK and JNK/SAPK. In contrast, sanguinarine analogs lacking the benzophenanthridine scaffold did not inhibit MKP-1 in vitro or in cells nor did they cause ERK or JNK/SAPK phosphorylation. These data illustrate the utility of a chemical complementation assay linked with multiparameter high content cellular screening.
Vogt
Andreas
A
Department of Pharmacology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA. avogt@pitt.edu
Tamewitz
Aletheia
A
Skoko
John
J
Sikorski
Rachel P
RP
Giuliano
Kenneth A
KA
Lazo
John S
JS
D000470:Alkaloids / Q000737:chemistry* / Q000494:pharmacology*; D053119:Benzophenanthridines; D020134:Catalytic Domain; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D045744:Cell Line, Tumor; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D054638:Dual Specificity Phosphatase 1; D054642:Dual Specificity Phosphatase 6; D015180:Electrophoresis, Gel, Two-Dimensional; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D015723:Gene Library; D006367:HeLa Cells; D006801:Humans; D017874:Immediate-Early Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D020128:Inhibitory Concentration 50; D007546:Isoquinolines; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D048670:MAP Kinase Kinase 4; D008856:Microscopy, Fluorescence; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D008956:Models, Chemical; D015233:Models, Statistical; D009369:Neoplasms / Q000188:drug therapy*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D010766:Phosphorylation; D010936:Plant Extracts / Q000494:pharmacology; D054645:Protein Phosphatase 1; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D020687:cdc25 Phosphatases / Q000378:metabolism
D000470:Alkaloids; D053119:Benzophenanthridines; D018797:Cell Cycle Proteins; D004791:Enzyme Inhibitors; D017874:Immediate-Early Proteins; D007546:Isoquinolines; D010936:Plant Extracts; C005705:sanguinarine; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D020929:Mitogen-Activated Protein Kinase Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; C517008:CDC25B protein, human; C517019:DUSP1 protein, human; C517035:DUSP6 protein, human; D054638:Dual Specificity Phosphatase 1; D054642:Dual Specificity Phosphatase 6; C516607:PTPN1 protein, human; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases; D020687:cdc25 Phosphatases