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

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 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. 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. Mitogen-activated protein kinase phosphatase (MKP) 1The abbreviations used are: MKP, mitogen-activated protein kinase phosphatase; ERK, extracellular signal regulated kinase; FITC, fluorescein isothiocyanate; 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.1The abbreviations used are: MKP, mitogen-activated protein kinase phosphatase; ERK, extracellular signal regulated kinase; FITC, fluorescein isothiocyanate; 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.-1 is a dual specificity phosphatase that is overexpressed in many human tumors and protects cells from apoptosis by the anticancer agent cisplatin (1Sanchez-Perez I. Martinez-Gomariz M. Williams D. Keyse S.M. Perona R. Oncogene. 2000; 19: 5142-5152Crossref PubMed Scopus (125) Google Scholar), UV irradiation (2Franklin C.C. Srikanth S. Kraft A.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3014-3019Crossref PubMed Scopus (153) Google Scholar), and proteasome inhibitors (3Small G.W. Shi Y.Y. Edmund N.A. Somasundaram S. Moore D.T. Orlowski R.Z. Mol. Pharmacol. 2004; 66: 1478-1490Crossref PubMed Scopus (88) Google Scholar). Over the past five years, reports have become more numerous describing high levels of MKP-1 in human tumors. For example, high levels of MKP-1 have been found in prostate (4Magi-Galluzzi C. Mishra R. Fiorentino M. Montironi R. Yao H. Capodieci P. Wishnow K. Kaplan I. Stork P.J. Loda M. Lab. Investig. 1997; 76: 37-51PubMed Google Scholar), gastric (5Bang Y.J. Kwon J.H. Kang S.H. Kim J.W. Yang Y.C. Biochem. Biophys. Res. Commun. 1998; 250: 43-47Crossref PubMed Scopus (90) Google Scholar), breast (6Wang H. Cheng Z. Malbon C.C. Cancer Lett. 2003; 191: 229-237Crossref PubMed Scopus (156) Google Scholar), and pancreatic cancer (7Liao Q. Guo J. Kleeff J. Zimmermann A. Buchler M.W. Korc M. Friess H. Gastroenterology. 2003; 124: 1830-1845Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). In ovarian cancer samples, MKP-1 expression was correlated with decreased progression-free survival (8Denkert C. Schmitt W.D. Berger S. Reles A. Pest S. Siegert A. Lichtenegger W. Dietel M. Hauptmann S. Int. J. Cancer. 2002; 102: 507-513Crossref PubMed Scopus (93) Google Scholar). High levels of MKP-1 expression were also found in the early phases of prostate, colon, and bladder carcinogenesis (9Loda M. Capodieci P. Mishra R. Yao H. Corless C. Grigioni W. Wang Y. Magi-Galluzzi C. Stork P.J. Am. J. Pathol. 1996; 149: 1553-1564PubMed Google Scholar). Evidence that MKP-1 may actually support the transformed phenotype comes from a recent study by Liao et al. (7Liao Q. Guo J. Kleeff J. Zimmermann A. Buchler M.W. Korc M. Friess H. Gastroenterology. 2003; 124: 1830-1845Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) who showed that PANC-1 human pancreatic cancer cells stably transfected with a full-length MKP-1 antisense construct had longer doubling times, decreased ability to form colonies in soft agar, and were unable to form tumors in nude mice. The precise mechanism by which loss of MKP-1 expression affects tumorigenicity, however, remains elusive. Several reports have implicated JNK/SAPK and p38 as the primary mediators of MKP-1 mediated cytoprotection (1Sanchez-Perez I. Martinez-Gomariz M. Williams D. Keyse S.M. Perona R. Oncogene. 2000; 19: 5142-5152Crossref PubMed Scopus (125) Google Scholar, 2Franklin C.C. Srikanth S. Kraft A.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3014-3019Crossref PubMed Scopus (153) Google Scholar). On the other hand, the data presented by Liao et al. (7Liao Q. Guo J. Kleeff J. Zimmermann A. Buchler M.W. Korc M. Friess H. Gastroenterology. 2003; 124: 1830-1845Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) argue that the primary mechanism by which MKP-1 supports the transformed phenotype is mediated by ERK, but not JNK, as suppression of MKP-1 expression by antisense did not affect basal levels of phospho-JNK or phospho-p38, but instead increased basal ERK phosphorylation and prolonged ERK phosphorylation after epidermal growth factor stimulation in PANC-1 cells. 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. In light of recent findings, inhibitors of MKP-1 might also find applications as novel target-based antineoplastic therapies, either alone or in combination with clinically used antineoplastic agents (1Sanchez-Perez I. Martinez-Gomariz M. Williams D. Keyse S.M. Perona R. Oncogene. 2000; 19: 5142-5152Crossref PubMed Scopus (125) Google Scholar, 2Franklin C.C. Srikanth S. Kraft A.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3014-3019Crossref PubMed Scopus (153) Google Scholar, 3Small G.W. Shi Y.Y. Edmund N.A. Somasundaram S. Moore D.T. Orlowski R.Z. Mol. Pharmacol. 2004; 66: 1478-1490Crossref PubMed Scopus (88) Google Scholar). The search for MKP-1 inhibitors has been challenging for several reasons. In contrast to MKP-3, whose crystal structure has been reported and found to possess high structural similarity to the related VHR in its catalytic domain (10Stewart A.E. Dowd S. Keyse S.M. McDonald N.Q. Nat. Struct. Biol. 1999; 6: 174-181Crossref PubMed Scopus (144) Google Scholar), no structural information is available for MKP-1. This may in part be due to difficulties in producing large amounts of recombinant enzyme and the need of MKPs to interact with their physiological substrates to become fully active (11Camps M. Nichols A. Gillieron C. Antonsson B. Muda M. Chabert C. Boschert U. Arkinstall S. Science. 1998; 280: 1262-1265Crossref PubMed Scopus (430) Google Scholar). The conformational changes associated with phosphatase-substrate interactions may render assays based on dephosphorylation of small molecule substrates or short peptides inaccurate predictors of effective phosphatase inhibitors in a physiological context. Cellular assays provide a potentially powerful alternative to in vitro screening for phosphatase inhibitors. The advent and maturation of cellular assay technologies such as high content screening (HCS) (12Giuliano K.A. DeBiasio R.L. Dunlay T. Gough A. Volosky J.M. Zock J. Pavlakis G.N. Taylor D.L. J. Biomol. Screen. 1997; 2: 249-259Crossref Scopus (186) Google Scholar) permit the construction of rapid and quantitative discovery assays with definitive phenotypic endpoints. In this report, 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. By analyzing a relatively small number of diverse natural products, we identified a plant alkaloid, sanguinarine, as a cell-active inhibitor of MKP-1. Compound credentialing was greatly facilitated by the employment of a cellular assay, which selected compounds with a defined biological activity. These results not only reveal sanguinarine as the first reported selective inhibitor of MKP-1 but also expose a primary cellular target for some of the biological activities of sanguinarine. Compounds—The MicroSource Natural Products Library (Discovery Systems, Inc., Gaylordsville, CT) is a 720-compound collection of pure natural products and their derivatives. The collection includes simple and complex oxygen heterocycles, alkaloids, sesquiterpenes, diterpenes, pentacyclic triterpenes, sterols, and many other diverse representatives. Compounds were supplied as 10 mm stocks of Me2SO, stored at –20 °C and thawed immediately before analysis. Aliquots were dissolved to a final concentration of 50 μm in complete growth medium prior to cell treatment. Lipofectamine 2000 was from Invitrogen. All other chemicals, including the sanguinarine analogs, were purchased from Sigma-Aldrich. Antibodies and Plasmids—Mouse monoclonal anti-phospho-ERK (E10), rabbit polyclonal phospho-ERK, phospho-JNK, and pan-JNK/SAPK antibodies were from Cell Signaling Technology (Beverly, MA). Rabbit polyclonal ERK antibody and mouse monoclonal anti-c-myc (9E10) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary antibodies were AlexaFluor 488-conjugated goat antimouse, AlexaFluor 546-, 555-, and 647-conjugated anti-rabbit IgG (Molecular Probes, Eugene, OR), and horseradish peroxidase-conjugated goat-anti rabbit IgG and goat-anti mouse IgG (Jackson ImmunoResearch, West Grove, PA). cDNA encoding myc-tagged human MKP-3 (PYST-1) in a pSG5 mammalian expression vector (37Groom L.A. Sneddon A.A. Alessi D.R. Dowd S. Keyse S.M. EMBO J. 1996; 15: 3621-3632Crossref PubMed Scopus (365) Google Scholar, 38Dowd S. Sneddon A.A. Keyse S.M. J. Cell Sci. 1998; 111: 3389-3399Crossref PubMed Google Scholar) and CL100 in a pET15b bacterial expression vector were gifts from Dr. Stephen Keyse (CRUK, Dundee, UK). A plasmid encoding myc-tagged mouse MKP-1 in a pCEP4 mammalian expression vector was a gift from Dr. Nicholas Tonks (Cold Spring Harbor Laboratories). To improve transfection efficiency, a BamH1 the was from the pCEP4 vector and expression for human and were gifts from Dr. of and Dr. of The expression vector was from The of cDNA were by Cell and cells were from and were in medium and in a of at High for MKP-1 cells were in the of and to were transfected with or and Lipofectamine 2000 in medium as after were and medium was cells were in for with or of and and with a of anti-phospho-ERK and anti-c-myc antibodies as A. K.A. M. P. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). and were with and AlexaFluor All were on a 2000 Inc., CA). For and selectivity cells were transfected with MKP-3, or GFP, for In some was AlexaFluor or AlexaFluor were by analysis for and in an defined by the on the PA). were in an at of or cells were for phospho-ERK, or For the of cells was from for and cells. of HCS of the Kolmogorov-Smirnov to Kolmogorov-Smirnov was to by of the Google and by A. R. Google The used was a of the of the in The of Scholar). the two-dimensional data physiological from a multiparameter HCS assay after were with the two-dimensional data from of cells. data was defined by the and values from the cell data The two-dimensional was found by to find the the of cells in and the of cells in were with the statistical Inc., of HCS and cell were as A for cells was defined as the from a and the of cells in the MKP-1 cell whose the was Using this the of cells was in cells, and high to in cells In MKP-3, and as as the catalytic domain of were as from VHR and phosphatase were as from a vector as R.L. J.M. S. P. 1997; PubMed Scopus Google Scholar). and were from human MKP-1 was from a pET15b vector and on activity assays were a assay with fluorescein as R.L. J.M. S. P. 1997; PubMed Scopus Google Scholar, K.A. B. A. P. J. PubMed Scopus Google Scholar). were at at its for of and by cells were in the of a to and for with phenylarsine or were and with a of anti-phospho-ERK mouse and antibodies by with AlexaFluor 488-conjugated goat IgG and AlexaFluor 647-conjugated goat anti-rabbit were with and AlexaFluor and AlexaFluor were on the in an defined by the as To for values were for in the of primary antibodies and from the in the of primary cells were in to compounds for by and Cell were on and to were with phospho-JNK, and antibody were peroxidase-conjugated antibodies (Jackson ImmunoResearch, West Grove, and an enhanced to For of protein expression levels, were on a a High of a Natural Products Library Using a for MKP-1 in a definitive assay for MKP-1 inhibition in intact mammalian cells as a high content cellular The which we have A. A. J. K. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar) and for high content screening A. K.A. M. P. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), is based on the of ERK phosphorylation in MKP-1 and cell after transfection with mammalian expression to that used to inhibitors of MKP-3 A. K.A. M. P. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) cells were in and transfected with a mammalian expression plasmid encoding MKP-1. after cells were with to a and and with a of anti-c-myc and anti-phospho-ERK and cells were with AlexaFluor and AlexaFluor Using this showed that cells had levels of with cells for a of the identified in the natural products from the MKP-1 high content screen were for and and cells were for and from with PAO, or sanguinarine from the cells were identified and and were to MKP-1 levels in cells. and sanguinarine in of a available natural products for compounds that levels in the MKP-1 cells to that in cells. with cells, with cells transfected with vector or GFP, with cells transfected with MKP-1 and with and with cells transfected with MKP-1 and with All were for with alone or in combination with compounds in at a concentration of 50 The protein-tyrosine phosphatase PAO, was as a A of cells in were on an for and and and cell by a for based on the AlexaFluor of This an HCS data for cell that was used to transfection and with report, we found transfection of to for MKP-1 and to for GFP, which was as of we used a two-dimensional analysis with the data have been as a to the of cell J. PubMed Scopus Google Scholar, 2000; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, C. PubMed Scopus Google Scholar), and to be a valuable tool for the of HCS data K.A. Taylor D.L. J. Biomol. Screen. 2004; PubMed Scopus Google Scholar). The two-dimensional over data in a in search of a two-dimensional data of the Google Scholar, A. R. Google Scholar). The values can a and To fully the information in multiparameter high content we an that combined the with analysis of PubMed Scopus Google Scholar) of AlexaFluor (MKP-1) and AlexaFluor in a of cells in on a data from transfected but not were for and used to a two-dimensional data The two-dimensional data of was with the data and a was for A based on two-dimensional values was to the of screening Using this we found that the two-dimensional for was This that the biological assay was with would be based on the by J. PubMed Scopus Google Scholar), a of on the and in By the two-dimensional for cells with the PAO, was By agents that showed a in two-dimensional values over the of data we identified compounds of as compounds from the screen were for of levels in cells. in which MKP-1 cells were identified on the of and in the In the of small molecule cells did not to with increased ERK phosphorylation. the phosphatase PAO, and the active in ERK phosphorylation in the MKP-1 cells, the results from the two-dimensional analysis. of MKP-1 transfected cells with sanguinarine in a of cells in the cell the data were to the of cells in the concentration of sanguinarine for a was 10 μm Sanguinarine MKP-1 but MKP-3 in sanguinarine was selective for MKP-1 over the related MKP-3, cells were transfected with MKP-1 or MKP-3 and the chemical complementation assay as in the A and and A and showed that MKP-1 and MKP-3 ERK phosphorylation in the cells. of sanguinarine ERK phosphorylation in MKP-1 but not in MKP-3 A and that sanguinarine inhibited but not MKP-3 in intact cells. cell two-dimensional that MKP-1 and MKP-3 caused a of cells in MKP-1 and MKP-3 and with sanguinarine increased the of cells in the MKP-1 but did not affect cells In contrast, the protein-tyrosine phosphatase PAO, and cells and of the of cells a that sanguinarine increased the of cells in but not in cell that the inhibited MKP-1 but not MKP-3 in intact cells. the concentration sanguinarine to which to its on levels in cells. In and the ability of sanguinarine to inhibit MKP-1 and related in vitro and found to be a potent and selective inhibitor of MKP-1 with MKP-3, VHR, and protein-tyrosine phosphatase exploited the availability of sanguinarine analogs from and five compounds a of in the benzophenanthridine scaffold chelerythrine, which is to sanguinarine, was also found to be a inhibitor of MKP-1 of the sanguinarine such as and of the scaffold or the structure to the related MKP-1 activity that a was for MKP-1 inhibition. for cellular inhibition of we found only sanguinarine and were active in the chemical complementation assay with their in vitro MKP-1 of dual specificity by IC50 from IC50 from protein in a vitro inhibition of MKP-1 and by sanguinarine IC50 from IC50 from IC50 from in a of MKP-1 by sanguinarine analogs in intact cells. The of sanguinarine analogs in was for inhibition of MKP-1 in cells the chemical complementation the were as a as the of cells in MKP-1 and from a that has been with the compounds only compounds with in vitro MKP-1 sanguinarine and chelerythrine, inhibited MKP-1 in intact Compounds with MKP-1 ERK and in Cancer MKP-1 ERK, JNK, and p38 Y. R. K. J. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). the ability of MKP-1 inhibitors on the phosphorylation of ERK and JNK/SAPK as a phenotypic with MKP-1 inhibition. PANC-1 human pancreatic cancer cells because they were to high levels of MKP-1 (7Liao Q. Guo J. Kleeff J. Zimmermann A. Buchler M.W. Korc M. Friess H. Gastroenterology. 2003; 124: 1830-1845Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). that sanguinarine increased and phospho-JNK levels in PANC-1 cells, as by and analysis. In contrast, sanguinarine analogs of MKP-1 activity to either or phospho-JNK in PANC-1 cells with MKP-1 activity ERK and phosphorylation in human pancreatic cancer cells. PANC-1 cells were with sanguinarine or analogs for at the and for and levels by as in the to the from at HCS a potentially powerful phenotypic to small with cellular In of an HCS A. K.A. M. P. Biol. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), we found that of the natural compounds caused a in of with the A of however, that only sanguinarine, a phenotype in which cells had levels of with cells a large number of were identified with the By two-dimensional values as the primary we were to a more and the natural phosphatase The two-dimensional the number of in the chemical complementation assay by with used the ability to the active Sanguinarine is a alkaloid from the plant the of which have been used in The plant is in the 2002; Scholar) as for the of of sanguinarine and antitumor activity in Pharmacol. 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