Abstract
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.
Highlights
The availability of a cell-active selective Mitogen-activated protein kinase phosphatase-1 (MKP-1) inhibitor would be a valuable tool for dissecting the complex regulatory processes involved in the attenuation of ERK, JNK, and p38 activation and for defining the contributions of MKP-1 and its cellular targets to the maintenance of the transformed phenotype
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
This may in part be due to difficulties in producing large amounts of recombinant enzyme and the need of MKPs to interact with their physiologphosphatase; ERK, extracellular signal regulated kinase; FITC, fluorescein isothiocyanate; gates for MKP (GFP), green fluorescent protein; GST, glutathione S-transferase; HCS, high content screening; JNK/SAPK, c-Jun terminal kinase/stress-activated protein kinase; KS, Kolmogorov-Smirnov; MAPK, mitogen-activated protein kinase; PAO, phenylarsine oxide; pERK, phospho-ERK; TPA, 12-O-tetradecanoylphorbol 13-acetate; VHR, VH-1 related phosphatase
Summary
The availability of a cell-active selective MKP-1 inhibitor would be a valuable tool for dissecting the complex regulatory processes involved in the attenuation of ERK, JNK, and p38 activation and for defining the contributions of MKP-1 and its cellular targets to the maintenance of the transformed phenotype. We have combined a definitive cellular assay for MKP-1 activity with HCS methodology and an unbiased statistical analysis of the HCS data set, and executed a small molecule screen for inhibitors of MKP-1 in intact mammalian cells. Compound credentialing was greatly facilitated by the employment of a cellular assay, which selected compounds with a defined biological activity These results reveal sanguinarine as the first reported selective inhibitor of MKP-1 and expose a primary cellular target for some of the biological activities of sanguinarine
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