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J. Biol. Chem., Vol. 280, Issue 19, 19078-19086, May 13, 2005

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The Benzo[c]phenanthridine Alkaloid, Sanguinarine, Is a Selective, Cell-active Inhibitor of Mitogen-activated Protein Kinase Phosphatase-1^*

Andreas Vogt, Aletheia Tamewitz, John Skoko, Rachel P. Sikorski, Kenneth A. Giuliano, and John S. Lazo¶

From the Department of Pharmacology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

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 µM 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 µM, 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.

Received for publication, February 8, 2005

^* This work was supported by National Institutes of Health Grants CA78039 and CA52995 and the Fiske Drug Discovery Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Current address: Cellumen, Inc., 100 Technology Dr., Pittsburgh, PA 15219.

To whom correspondence may be addressed: Dept. of Pharmacology, The Hillman Cancer Center G.27a, University of Pittsburgh, Pittsburgh, PA 15213. Tel.: 412-623-1216; Fax: 412-623-1212; E-mail: avogt{at}pitt.edu. ¶ To whom correspondence may be addressed: Dept. of Pharmacology, Biomedical Science Tower E-1340, University of Pittsburgh, Pittsburgh, PA 15261. Tel.: 412-648-9319; Fax: 412-648-2229; E-mail: lazo{at}pitt.edu.

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