A Cell-permeable Stat3 SH2 Domain Mimetic Inhibits Stat3 Activation and Induces Antitumor Cell Effects in Vitro

Given the role of constitutively active Signal Transducer and Activator of Transcription (Stat) 3 in human tumors, Stat3 inhibitors would be useful as novel therapeutics and as tools for probing Stat3-mediated tumor processes. We herein report that a 28-mer peptide, SPI, derived from the Stat3 SH2 domain, replicates Stat3 biochemical properties. Studies show SPI and Stat3 (or Stat3 SH2 domain) bind with similar affinities to known Stat3-binding phosphotyrosine (pY) peptide motifs, including those of the epidermal growth factor receptor (EGFR) and the high-affinity, IL-6R/gp130-derived pY-peptide, GpYLPQTV-NH2. Consequently, SPI functions as a potent and selective inhibitor of Stat3 SH2 domain:pTyr interactions and disrupts the binding of Stat3 to the IL-6R/gp130 peptide, GpYLPQTV-NH2. Fluorescence imaging and immunofluorescence staining/laser-scanning confocal microscopy show SPI is cell membrane-permeable, associates with the cytoplasmic tail of EGFR in NIH3T3/hEGFR, and is present in the cytoplasm, but strongly localized at the plasma membrane and in the nucleus in malignant cells harboring persistently active Stat3. Moreover, SPI specifically blocks constitutive Stat3 phosphorylation, DNA binding activity, and transcriptional function in malignant cells, with little or no effect on the induction of Stat1, Stat5, and Erk1/2MAPK pathways, or on general pTyr profile at the concentrations that inhibit Stat3 activity. Significantly, treatment with SPI of human breast, pancreatic, prostate, and non-small cell lung cancer cells harboring constitutively active Stat3 induced extensive morphology changes, associated with viability loss and apoptosis. Our study identifies SPI as a novel molecular probe for interrogating Stat3 signaling and that functions as a selective inhibitor of Stat3 activation with antitumor cell effects. Given the role of constitutively active Signal Transducer and Activator of Transcription (Stat) 3 in human tumors, Stat3 inhibitors would be useful as novel therapeutics and as tools for probing Stat3-mediated tumor processes. We herein report that a 28-mer peptide, SPI, derived from the Stat3 SH2 domain, replicates Stat3 biochemical properties. Studies show SPI and Stat3 (or Stat3 SH2 domain) bind with similar affinities to known Stat3-binding phosphotyrosine (pY) peptide motifs, including those of the epidermal growth factor receptor (EGFR) and the high-affinity, IL-6R/gp130-derived pY-peptide, GpYLPQTV-NH2. Consequently, SPI functions as a potent and selective inhibitor of Stat3 SH2 domain:pTyr interactions and disrupts the binding of Stat3 to the IL-6R/gp130 peptide, GpYLPQTV-NH2. Fluorescence imaging and immunofluorescence staining/laser-scanning confocal microscopy show SPI is cell membrane-permeable, associates with the cytoplasmic tail of EGFR in NIH3T3/hEGFR, and is present in the cytoplasm, but strongly localized at the plasma membrane and in the nucleus in malignant cells harboring persistently active Stat3. Moreover, SPI specifically blocks constitutive Stat3 phosphorylation, DNA binding activity, and transcriptional function in malignant cells, with little or no effect on the induction of Stat1, Stat5, and Erk1/2MAPK pathways, or on general pTyr profile at the concentrations that inhibit Stat3 activity. Significantly, treatment with SPI of human breast, pancreatic, prostate, and non-small cell lung cancer cells harboring constitutively active Stat3 induced extensive morphology changes, associated with viability loss and apoptosis. Our study identifies SPI as a novel molecular probe for interrogating Stat3 signaling and that functions as a selective inhibitor of Stat3 activation with antitumor cell effects. IntroductionThe binding of cytokines or growth factors to cognate receptors initiates a cascade of molecular events that culminate in the activation of the Signal Transducer and Activator of Transcription (STAT) family of proteins (1Bromberg J. Breast Cancer Res. 2000; 2: 86-90Crossref PubMed Scopus (98) Google Scholar, 2Darnell Jr., J.E. Nat. Rev. Cancer. 2002; 2: 740-749Crossref PubMed Scopus (941) Google Scholar). Among these is the recruitment of STATs, via the SH2 domain, to the receptor phosphotyrosine (pTyr) 2The abbreviations used are: pTyr or pYphosphorylated tyrosineSH2Src homology 2EMSAelectrophoretic mobility shift assayEGFRepidermal growth factor receptorErkMAPKextracellular signal-regulated kinase-mitogen-activated protein kinaseGOLDgenetic optimization for ligand dockingSPRsurface plasmon resonanceFPfluorescence polarization. peptide motifs, which brings them into close proximity for phosphorylation on a key tyrosyl residue by growth factor receptor tyrosine kinases, Janus kinases (Jaks), and the Src family kinases. Consequently, dimerization between two STAT monomers is promoted through a reciprocal pTyr-SH2 domain interaction, and the active STAT dimers in the nucleus bind to specific DNA-response elements in the promoters of target genes and regulate gene expression. In response to growth factors and cytokines, normal STAT signaling promotes cell growth and differentiation, development, inflammation, and immune responses.The STAT proteins are modular in structure and contain N-terminal domain, coiled-coil domain, DNA-binding domain, SH2 domain, and a transcriptional activation domain, with each domain engaging in important molecular events for promoting STAT functions. In particular, the SH2 domain mediates crucial interactions with specific pTyr peptide motifs, including promoting the association with receptors and holding up two activated STAT monomers together in a reciprocal SH2 domain:pTyr interactions in STAT:STAT dimerization. Among the STAT family members, Stat3 and Stat5 have been strongly implicated in malignant transformation and tumorigenesis (3Yu H. Jove R. Nat. Rev. Cancer. 2004; 4: 97-105Crossref PubMed Scopus (1919) Google Scholar, 4Yue P. Turkson J. Expert Opin. Investig. Drugs. 2009; 18: 45-56Crossref PubMed Scopus (337) Google Scholar, 5Turkson J. Expert Opin. Ther. Targets. 2004; 8: 409-422Crossref PubMed Scopus (255) Google Scholar, 6Turkson J. Jove R. Oncogene. 2000; 19: 6613-6626Crossref PubMed Scopus (551) Google Scholar, 7Darnell J.E. Nat. Med. 2005; 11: 595-596Crossref PubMed Scopus (249) Google Scholar) and have become valid targets for anticancer drug design. In general, given the role of the SH2 domain as an important motif in signal transduction, in relation to engaging in interactions with pTyr peptide modules (8Yaffe M.B. Nat. Rev. Mol. Cell Biol. 2002; 3: 177-186Crossref PubMed Scopus (283) Google Scholar), there are considerable efforts to design probes that disrupt these interactions for potential application as drugs (9Sawyer T.K. Biopolymers. 1998; 47: 243-261Crossref PubMed Scopus (138) Google Scholar, 10Burke Jr., T.R. Luo J. Yao Z.J. Gao Y. Zhao H. Milne G.W. Guo R. Voigt J.H. King C.R. Yang D. Bioorg. Med. Chem. Lett. 1999; 9: 347-352Crossref PubMed Scopus (41) Google Scholar, 11Burke Jr., T.R. Smyth M.S. Otaka A. Nomizu M. Roller P.P. Wolf G. Case R. Shoelson S.E. Biochemistry. 1994; 33: 6490-6494Crossref PubMed Scopus (197) Google Scholar, 12Dharmawardana P.G. Peruzzi B. Giubellino A. Burke Jr., T.R. Bottaro D.P. Anticancer Drugs. 2006; 17: 13-20Crossref PubMed Scopus (61) Google Scholar). For Stat3, pTyr peptide mimetics have been shown to suppress its functions. Thus, the Stat3 SH2 domain:pTyr peptide interaction has become an attractive target in many drug design strategies intended to identify small molecule inhibitors as new therapeutics for cancers in which aberrant Stat3 activity is implicated (4Yue P. Turkson J. Expert Opin. Investig. Drugs. 2009; 18: 45-56Crossref PubMed Scopus (337) Google Scholar, 5Turkson J. Expert Opin. Ther. Targets. 2004; 8: 409-422Crossref PubMed Scopus (255) Google Scholar, 13Turkson J. Kim J.S. Zhang S. Yuan J. Huang M. Glenn M. Haura E. Sebti S. Hamilton A.D. Jove R. Mol. Cancer Ther. 2004; 3: 261-269PubMed Google Scholar, 14Turkson J. Ryan D. Kim J.S. Zhang Y. Chen Z. Haura E. Laudano A. Sebti S. Hamilton A.D. Jove R. J. Biol. Chem. 2001; 276: 45443-45455Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar, 15Song H. Wang R. Wang S. Lin J. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 4700-4705Crossref PubMed Scopus (441) Google Scholar, 16Siddiquee K. Zhang S. Guida W.C. Blaskovich M.A. Greedy B. Lawrence H.R. Yip M.L. Jove R. McLaughlin M.M. Lawrence N.J. Sebti S.M. Turkson J. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 7391-7396Crossref PubMed Scopus (609) Google Scholar, 17Siddiquee K.A. Gunning P.T. Glenn M. Katt W.P. Zhang S. Schrock C. Schroeck C. Sebti S.M. Jove R. Hamilton A.D. Turkson J. ACS Chem. Biol. 2007; 2: 787-798Crossref PubMed Scopus (155) Google Scholar, 18Coleman 4th, D.R. Ren Z. Mandal P.K. Cameron A.G. Dyer G.A. Muranjan S. Campbell M. Chen X. McMurray J.S. J. Med. Chem. 2005; 48: 6661-6670Crossref PubMed Scopus (115) Google Scholar, 19Ren Z. Cabell L.A. Schaefer T.S. McMurray J.S. Bioorg Med. Chem. Lett. 2003; 13: 633-636Crossref PubMed Scopus (118) Google Scholar, 20Schust J. Sperl B. Hollis A. Mayer T.U. Berg T. Chem. Biol. 2006; 13: 1235-1242Abstract Full Text Full Text PDF PubMed Scopus (766) Google Scholar, 21Gunning P.T. Glenn M.P. Siddiquee K.A. Katt W.P. Masson E. Sebti S.M. Turkson J. Hamilton A.D. Chembiochem. 2008; 9: 2800-2803Crossref PubMed Scopus (42) Google Scholar, 22Fletcher S. Turkson J. Gunning P.T. Chem. Med. Chem. 2008; 3: 1159-1168Crossref Scopus (82) Google Scholar).Whereas the focus of the existing Stat3 drug discovery efforts have been on disrupting the Stat3 SH2 domain:pTyr peptide interactions for a good reason, the approaches have largely been directed at SH2 domain antagonists, which are pTyr peptide mimics that compete for the binding to the Stat3 SH2 domain (4Yue P. Turkson J. Expert Opin. Investig. Drugs. 2009; 18: 45-56Crossref PubMed Scopus (337) Google Scholar, 5Turkson J. Expert Opin. Ther. Targets. 2004; 8: 409-422Crossref PubMed Scopus (255) Google Scholar, 22Fletcher S. Turkson J. Gunning P.T. Chem. Med. Chem. 2008; 3: 1159-1168Crossref Scopus (82) Google Scholar). One of the major limitations of this approach has been finding a membrane-permeable, optimum pTyr substitute that retains the high binding affinities of the native pTyr peptide motifs, against which these antagonists will be competing for the binding to the Stat3 SH2 domain. To eliminate this issue, we have taken the converse approach of identifying a suitable Stat3 SH2 domain-mimic. Key structural information from the computational modeling of the native pTyr peptide, PpYLKTK bound to the Stat3 SH2 domain, per the crystal structure of Stat3β (23Becker S. Groner B. Müller C.W. Nature. 1998; 394: 145-151Crossref PubMed Scopus (656) Google Scholar) facilitated the design of a 28-mer peptide, SPI from the Stat3 SH2 domain. Studies presented herein show that SPI retains the binding characteristics of the SH2 domain. In vitro biochemical and biophysical studies indicate SPI, like Stat3 binds to cognate pTyr-peptide motifs with a similar affinity. Accordingly, SPI blocks the binding of Stat3 (or Stat3 SH2 domain) to cognate pTyr peptide motifs, and hence functions as a selective inhibitor of constitutive Stat3 activation in human breast, prostate, pancreatic, and non-small cell lung cancer cells, with antitumor cell effects.DISCUSSIONProtein-protein interactions are a common molecular event important in signal transduction and many other physiological processes. In the case of Stat3, the recruitment via the SH2 domain to cognate receptor pTyr peptide motifs is a key initial step for phosphorylation. Data herein show that the Stat3 SH2 domain-derived 28-mer peptide, SPI alone is sufficient to reproduce some aspects of the biochemical properties of Stat3 (or the Stat3 SH2 domain), thereby serving as a critical motif that engages in the inter-molecular interactions with the key residues of the cognate pTyr peptides to which Stat3 binds. Using biophysical analysis, such as SPR, we demonstrate similarities in the binding characteristics of Stat3 (or Stat3 SH2 domain) and SPI to known cognate pTyr peptides, including the native IL-6R/gp-130 derived peptide, GpYLPQTV-NH2, the Stat3 peptide, pY705Stat3, and the EGFR motif, pY1068EGFR, which Stat3 and SPI interact relatively stronger, and to the EGFR motif, pY1086EGFR and the Stat1 peptide, pY701Stat1, which they bind to with rather low affinities. The similarity in the binding characteristics of SPI and Stat3 is further evident by the SPR analysis that further suggests rather unfavorable interactions with the native Stat5 phosphopeptide, pY694Stat5, with affinities that are in milli-molar concentrations (KD of 1–7 mm). Together, present studies indicate SPI, like Stat3, shows preferential binding to different cognate pTyr peptide motifs, specifically showing stronger binding to the Stat3 phosphopeptide, compared with weaker binding to the Stat1 phosphopeptide. Given the observed differences in the affinities, we surmise that the type and number of binding partners to which Stat3 (or Stat3 SH2 domain) or SPI would interact with would be strongly influenced by their intracellular concentrations. Fluorescence polarization analysis based on the binding to the gp130-derived peptide (as 5-carboxyfluorescein-GpYLPQTV-NH2), (40Chen J. Bai D. Z. C. Zhang J. H. Wang S. ACS Med. Chem. Lett. PubMed Scopus Google Scholar), further the similarities in the binding characteristics between SPI and to the SH2 domain and interact with pTyr peptide motifs the that SPI compete against Stat3. is by the polarization studies that SPI strongly against Stat3 for the binding to pTyr peptide is presented that SPI at concentrations in the of intracellular Stat3 phosphorylation, DNA-binding and transcriptional The of intracellular Stat3 activation be in by the to disrupt as has been observed for other Stat3 dimerization inhibitors J. Kim J.S. Zhang S. Yuan J. Huang M. Glenn M. Haura E. Sebti S. Hamilton A.D. Jove R. Mol. Cancer Ther. 2004; 3: 261-269PubMed Google Scholar, 14Turkson J. Ryan D. Kim J.S. Zhang Y. Chen Z. Haura E. Laudano A. Sebti S. Hamilton A.D. Jove R. J. Biol. Chem. 2001; 276: 45443-45455Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar, 16Siddiquee K. Zhang S. Guida W.C. Blaskovich M.A. Greedy B. Lawrence H.R. Yip M.L. Jove R. McLaughlin M.M. Lawrence N.J. Sebti S.M. Turkson J. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 7391-7396Crossref PubMed Scopus (609) Google Scholar, 17Siddiquee K.A. Gunning P.T. Glenn M. Katt W.P. Zhang S. Schrock C. Schroeck C. Sebti S.M. Jove R. Hamilton A.D. Turkson J. ACS Chem. Biol. 2007; 2: 787-798Crossref PubMed Scopus (155) Google Scholar), and on the of the potential that by with receptor pTyr motifs, SPI Stat3-binding to and hence phosphorylation. Thus, the treatment of cells with the protein the of activated Stat3 constitutive to the of by protein the of cells to SPI of activated Stat3. Moreover, the that SPI preferential of Stat3 activation to Stat1, the physiological of a the two STAT family are activated by suggests SPI have for Stat3 Stat1, but that activated Stat3 protein to a activated with of Stat3 via binding to cognate pTyr peptide motifs, is in converse to the of Stat3 by many of the existing which are peptide mimetics and bind to the Stat3 SH2 (4Yue P. Turkson J. Expert Opin. Investig. Drugs. 2009; 18: 45-56Crossref PubMed Scopus (337) Google Scholar, 5Turkson J. Expert Opin. Ther. Targets. 2004; 8: 409-422Crossref PubMed Scopus (255) Google Scholar, 13Turkson J. Kim J.S. Zhang S. Yuan J. Huang M. Glenn M. Haura E. Sebti S. Hamilton A.D. Jove R. Mol. Cancer Ther. 2004; 3: 261-269PubMed Google Scholar, 14Turkson J. Ryan D. Kim J.S. Zhang Y. Chen Z. Haura E. Laudano A. Sebti S. Hamilton A.D. Jove R. J. Biol. Chem. 2001; 276: 45443-45455Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar, 15Song H. Wang R. Wang S. Lin J. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 4700-4705Crossref PubMed Scopus (441) Google Scholar, 19Ren Z. Cabell L.A. Schaefer T.S. McMurray J.S. Bioorg Med. Chem. Lett. 2003; 13: 633-636Crossref PubMed Scopus (118) Google Scholar, 22Fletcher S. Turkson J. Gunning P.T. Chem. Med. Chem. 2008; 3: 1159-1168Crossref Scopus (82) Google Scholar, J. Bai D. Z. C. Zhang J. H. Wang S. ACS Med. Chem. Lett. PubMed Scopus Google Scholar, D. K. T. C. B. Lin J. P.K. Bioorg Med. Chem. Lett. 2008; 18: PubMed Scopus Google Scholar), or to the approaches that have been for the of other SH2 such as the P.G. Peruzzi B. Giubellino A. Burke Jr., T.R. Bottaro D.P. Anticancer Drugs. 2006; 17: 13-20Crossref PubMed Scopus (61) Google Scholar), the biochemical of the would be the which is to disrupt domain there are many protein with an SH2 that are in promoting signal transduction and other biochemical (8Yaffe M.B. Nat. Rev. Mol. Cell Biol. 2002; 3: 177-186Crossref PubMed Scopus (283) Google Scholar, T.K. Biopolymers. 1998; 47: 243-261Crossref PubMed Scopus (138) Google Scholar), present of for SPI which is in the of effect on Stat5 activation and transcriptional activity, the or on Stat1 activation at concentrations that inhibit Stat3 activity. Accordingly, antitumor cell of SPI are observed at concentrations that inhibit Stat3 activity and are with the of aberrant Stat3 activation (4Yue P. Turkson J. Expert Opin. Investig. Drugs. 2009; 18: 45-56Crossref PubMed Scopus (337) Google Scholar, 5Turkson J. Expert Opin. Ther. Targets. 2004; 8: 409-422Crossref PubMed Scopus (255) Google Scholar, 13Turkson J. Kim J.S. Zhang S. Yuan J. Huang M. Glenn M. Haura E. Sebti S. Hamilton A.D. Jove R. Mol. Cancer Ther. 2004; 3: 261-269PubMed Google Scholar, 14Turkson J. Ryan D. Kim J.S. Zhang Y. Chen Z. Haura E. Laudano A. Sebti S. Hamilton A.D. Jove R. J. Biol. Chem. 2001; 276: 45443-45455Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar, 15Song H. Wang R. Wang S. Lin J. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 4700-4705Crossref PubMed Scopus (441) Google Scholar, J. Bai D. Z. C. Zhang J. H. Wang S. ACS Med. Chem. Lett. PubMed Scopus Google Scholar, D. K. T. C. B. Lin J. P.K. Bioorg Med. Chem. Lett. 2008; 18: PubMed Scopus Google Scholar). human breast, pancreatic, prostate, and non-small cell lung cancer cells harboring aberrant Stat3 activity are to We an of SPI in malignant cells, which we will a stronger of of activated Stat3 and Stat3 transcriptional activity. in the EGFR in which Stat3 is SPI is localized to the cell The for the and the and there are specific factors that the in malignant cells to be is a Stat3 SH2 that functions as an inhibitor of Stat3 The approach to the of Stat3 activation by SPI the known of the pTyr for the existing SH2 The in vitro activity of SPI a suitable for a for further of in effects. SPI as a molecular probe for interrogating aberrant Stat3 functions in tumor and for in vitro peptide binding IntroductionThe binding of cytokines or growth factors to cognate receptors initiates a cascade of molecular events that culminate in the activation of the Signal Transducer and Activator of Transcription (STAT) family of proteins (1Bromberg J. Breast Cancer Res. 2000; 2: 86-90Crossref PubMed Scopus (98) Google Scholar, 2Darnell Jr., J.E. Nat. Rev. Cancer. 2002; 2: 740-749Crossref PubMed Scopus (941) Google Scholar). Among these is the recruitment of STATs, via the SH2 domain, to the receptor phosphotyrosine (pTyr) 2The abbreviations used are: pTyr or pYphosphorylated tyrosineSH2Src homology 2EMSAelectrophoretic mobility shift assayEGFRepidermal growth factor receptorErkMAPKextracellular signal-regulated kinase-mitogen-activated protein kinaseGOLDgenetic optimization for ligand dockingSPRsurface plasmon resonanceFPfluorescence polarization. peptide motifs, which brings them into close proximity for phosphorylation on a key tyrosyl residue by growth factor receptor tyrosine kinases, Janus kinases (Jaks), and the Src family kinases. Consequently, dimerization between two STAT monomers is promoted through a reciprocal pTyr-SH2 domain interaction, and the active STAT dimers in the nucleus bind to specific DNA-response elements in the promoters of target genes and regulate gene expression. In response to growth factors and cytokines, normal STAT signaling promotes cell growth and differentiation, development, inflammation, and immune responses.The STAT proteins are modular in structure and contain N-terminal domain, coiled-coil domain, DNA-binding domain, SH2 domain, and a transcriptional activation domain, with each domain engaging in important molecular events for promoting STAT functions. In particular, the SH2 domain mediates crucial interactions with specific pTyr peptide motifs, including promoting the association with receptors and holding up two activated STAT monomers together in a reciprocal SH2 domain:pTyr interactions in STAT:STAT dimerization. Among the STAT family members, Stat3 and Stat5 have been strongly implicated in malignant transformation and tumorigenesis (3Yu H. Jove R. Nat. Rev. Cancer. 2004; 4: 97-105Crossref PubMed Scopus (1919) Google Scholar, 4Yue P. Turkson J. Expert Opin. Investig. Drugs. 2009; 18: 45-56Crossref PubMed Scopus (337) Google Scholar, 5Turkson J. Expert Opin. Ther. Targets. 2004; 8: 409-422Crossref PubMed Scopus (255) Google Scholar, 6Turkson J. Jove R. Oncogene. 2000; 19: 6613-6626Crossref PubMed Scopus (551) Google Scholar, 7Darnell J.E. Nat. Med. 2005; 11: 595-596Crossref PubMed Scopus (249) Google Scholar) and have become valid targets for anticancer drug design. In general, given the role of the SH2 domain as an important motif in signal transduction, in relation to engaging in interactions with pTyr peptide modules (8Yaffe M.B. Nat. Rev. Mol. Cell Biol. 2002; 3: 177-186Crossref PubMed Scopus (283) Google Scholar), there are considerable efforts to design probes that disrupt these interactions for potential application as drugs (9Sawyer T.K. Biopolymers. 1998; 47: 243-261Crossref PubMed Scopus (138) Google Scholar, 10Burke Jr., T.R. Luo J. Yao Z.J. Gao Y. Zhao H. Milne G.W. Guo R. Voigt J.H. King C.R. Yang D. Bioorg. Med. Chem. Lett. 1999; 9: 347-352Crossref PubMed Scopus (41) Google Scholar, 11Burke Jr., T.R. Smyth M.S. Otaka A. Nomizu M. Roller P.P. Wolf G. Case R. Shoelson S.E. Biochemistry. 1994; 33: 6490-6494Crossref PubMed Scopus (197) Google Scholar, 12Dharmawardana P.G. Peruzzi B. Giubellino A. Burke Jr., T.R. Bottaro D.P. Anticancer Drugs. 2006; 17: 13-20Crossref PubMed Scopus (61) Google Scholar). For Stat3, pTyr peptide mimetics have been shown to suppress its functions. Thus, the Stat3 SH2 domain:pTyr peptide interaction has become an attractive target in many drug design strategies intended to identify small molecule inhibitors as new therapeutics for cancers in which aberrant Stat3 activity is implicated (4Yue P. Turkson J. Expert Opin. Investig. Drugs. 2009; 18: 45-56Crossref PubMed Scopus (337) Google Scholar, 5Turkson J. Expert Opin. Ther. Targets. 2004; 8: 409-422Crossref PubMed Scopus (255) Google Scholar, 13Turkson J. Kim J.S. Zhang S. Yuan J. Huang M. Glenn M. Haura E. Sebti S. Hamilton A.D. Jove R. Mol. Cancer Ther. 2004; 3: 261-269PubMed Google Scholar, 14Turkson J. Ryan D. Kim J.S. Zhang Y. Chen Z. Haura E. Laudano A. Sebti S. Hamilton A.D. Jove R. J. Biol. Chem. 2001; 276: 45443-45455Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar, 15Song H. Wang R. Wang S. Lin J. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 4700-4705Crossref PubMed Scopus (441) Google Scholar, 16Siddiquee K. Zhang S. Guida W.C. Blaskovich M.A. Greedy B. Lawrence H.R. Yip M.L. Jove R. McLaughlin M.M. Lawrence N.J. Sebti S.M. Turkson J. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 7391-7396Crossref PubMed Scopus (609) Google Scholar, 17Siddiquee K.A. Gunning P.T. Glenn M. Katt W.P. Zhang S. Schrock C. Schroeck C. Sebti S.M. Jove R. Hamilton A.D. Turkson J. ACS Chem. Biol. 2007; 2: 787-798Crossref PubMed Scopus (155) Google Scholar, 18Coleman 4th, D.R. Ren Z. Mandal P.K. Cameron A.G. Dyer G.A. Muranjan S. Campbell M. Chen X. McMurray J.S. J. Med. Chem. 2005; 48: 6661-6670Crossref PubMed Scopus (115) Google Scholar, 19Ren Z. Cabell L.A. Schaefer T.S. McMurray J.S. Bioorg Med. Chem. Lett. 2003; 13: 633-636Crossref PubMed Scopus (118) Google Scholar, 20Schust J. Sperl B. Hollis A. Mayer T.U. Berg T. Chem. Biol. 2006; 13: 1235-1242Abstract Full Text Full Text PDF PubMed Scopus (766) Google Scholar, 21Gunning P.T. Glenn M.P. Siddiquee K.A. Katt W.P. Masson E. Sebti S.M. Turkson J. Hamilton A.D. Chembiochem. 2008; 9: 2800-2803Crossref PubMed Scopus (42) Google Scholar, 22Fletcher S. Turkson J. Gunning P.T. Chem. Med. Chem. 2008; 3: 1159-1168Crossref Scopus (82) Google Scholar).Whereas the focus of the existing Stat3 drug discovery efforts have been on disrupting the Stat3 SH2 domain:pTyr peptide interactions for a good reason, the approaches have largely been directed at SH2 domain antagonists, which are pTyr peptide mimics that compete for the binding to the Stat3 SH2 domain (4Yue P. Turkson J. Expert Opin. Investig. Drugs. 2009; 18: 45-56Crossref PubMed Scopus (337) Google Scholar, 5Turkson J. Expert Opin. Ther. Targets. 2004; 8: 409-422Crossref PubMed Scopus (255) Google Scholar, 22Fletcher S. Turkson J. Gunning P.T. Chem. Med. Chem. 2008; 3: 1159-1168Crossref Scopus (82) Google Scholar). One of the major limitations of this approach has been finding a membrane-permeable, optimum pTyr substitute that retains the high binding affinities of the native pTyr peptide motifs, against which these antagonists will be competing for the binding to the Stat3 SH2 domain. To eliminate this issue, we have taken the converse approach of identifying a suitable Stat3 SH2 domain-mimic. Key structural information from the computational modeling of the native pTyr peptide, PpYLKTK bound to the Stat3 SH2 domain, per the crystal structure of Stat3β (23Becker S. Groner B. Müller C.W. Nature. 1998; 394: 145-151Crossref PubMed Scopus (656) Google Scholar) facilitated the design of a 28-mer peptide, SPI from the Stat3 SH2 domain. Studies presented herein show that SPI retains the binding characteristics of the SH2 domain. In vitro biochemical and biophysical studies indicate SPI, like Stat3 binds to cognate pTyr-peptide motifs with a similar affinity. Accordingly, SPI blocks the binding of Stat3 (or Stat3 SH2 domain) to cognate pTyr peptide motifs, and hence functions as a selective inhibitor of constitutive Stat3 activation in human breast, prostate, pancreatic, and non-small cell lung cancer cells, with antitumor cell effects.