Investigation of the Binding Determinants of Phosphopeptides Targeted to the Src Homology 2 Domain of the Signal Transducer and Activator of Transcription 3. Development of a High-Affinity Peptide Inhibitor

Abstract

Signal transducer and activator of transcription 3 (Stat3) is a cytosolic transcription factor that relates signals from the cell membrane directly to the nucleus where it, in complex with other proteins, initiates the transcription of antiapoptotic and cell cycling genes, e.g., Bcl-x(L) and cyclin D1. In normal cells Stat3 transduces signals from cytokines such as IL-6 and growth factors such as the epidermal growth factor. Stat3 is constitutively activated in a number of human tumors. Antisense and dominant negative gene delivery result in apoptosis and reduced cell growth, thus this protein is an attractive target for anticancer drug design. As part of our research on the design of Src homology 2 (SH2) directed peptidomimetic inhibitors of Stat3, in this paper we describe structure-activity relationship studies that provide information on the nature of peptide-protein interactions of a high-affinity phosphopeptide inhibitor of Stat3 dimerization and DNA binding, Ac-Tyr(PO3H2)-Leu-Pro-Gln-Thr-Val-NH2, peptide 1. There is a hydrophobic surface on the SH2 domain that can accommodate lipophilic groups on the N-terminus. Of the amino acids tested, leucine provided the highest affinity at pY+1 and its main chain NH is involved with a hydrogen bond with Stat3, presumably Ser636. cis-3,4-Methanoproline is optimal as a backbone constraint at pY+2. The side chain amide protons of Gln are required for high-affinity interactions. The C-terminal dipeptide, Thr-Val, can be replaced with groups ranging in size from methyl to benzyl. We synthesized a phosphopeptide incorporating groups that provided increases in affinity at each position. Thus, hydrocinnamoyl-Tyr(PO3H2)-Leu-cis-3,4-methanoPro-Gln-NHBn, 50, was the highest affinity peptide, exhibiting an IC50 of 125 nM versus 290 nM for peptide 1 in a fluorescence polarization assay.

Potent Targeting of the STAT3 Protein in Brain Cancer Stem Cells: A Promising Route for Treating Glioblastoma

“…While phosphopeptides have been reported to bind to STAT3 with nanomolar potency, these agents have not shown in vivo efficacy. 22 Comparative SPR analysis against STAT5, an analogous member of the STAT family proteins, showed modest selectivity (K D = 464 ± 22 nM). Since the BTSCs evaluated do not harbor hyperactivated STAT5 (Supporting Information), we cannot attribute the observed activity to anti-STAT5 activity.…”

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

“…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 (4, 5,(13)(14)(15)(16)(17)(18)(19)(20)(21)(22).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 (4, 5,22). 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.…”

“…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 (4, 5,(13)(14)(15)(16)(17)(18)(19)(20)(21)(22).…”

Toll-Like Receptors as Novel Therapeutic Targets for the Treatment of Pancreatic Cancer

“…Like NF-B, STAT3 also activates anti-apoptotic genes such as Bcl-xL, Bcl-2, and c-IAP2 (Yu & Jove, 2004;Chang et al, 2006;Rebouissou et al, 2009), cell cycle and proliferation genes such as Cyclin D1 and cMyc (Levy & Darnell, 2002;Naugler & Karin, 2008;Bollrath et al, 2009), and members of the AP-1 family such as c-Jun and c-Fos (Hirano et al, 2000;Yang et al, 2005b;Yang et al, 2007), whereas others such as Mcl-1 and Survivin are STAT-3-dependent (Yu & Jove, 2004). Thus, STAT3 is an attractive target for anti-cancer therapy and numerous strategies have been employed to inhibit constitutive STAT3 signaling in cancer cells (Meydan et al, 1996;Turkson et al, 2001;Coleman et al, 2005;Song et al, 2005;Duan et al, 2006;Schust et al, 2006;Iwamaru et al, 2007;Siddiquee et al, 2007;Goel et al, 2008;Hatcher et al, 2008;Lin et al, 2010a;Lin et al, 2010b). …”