Signal transducers and activators of transcription (STATs) comprise a family of cytoplasmic signaling proteins that participates in normal cellular responses to cytokines and growth factors. Frequently, however, constitutive activation of certain STAT family members, particularly Stat3, has accompanied a wide variety of human malignancies. To identify small molecule inhibitors of Stat3, we investigated the ability of the Stat3 SH2 domain-binding peptide, PY*LKTK (where Y* represents phosphotyrosine), to disrupt Stat3 activity in vitro. The presence of PY*LKTK, but not PYLKTK or PFLKTK, in nuclear extracts results in significant reduction in the levels of DNA binding activities of Stat3, to a lesser extent of Stat1, and with no effect on that of Stat5. Analyses of alanine scanning mutagenesis and deletion derivatives of PY*LKTK reveal that the Leu residue at the Y؉1 position and a substituent at the Y؊1 position (but not necessarily Pro) are essential for the disruption of active Stat3, thereby mapping the minimum active sequence to the tripeptide, XY*L. Studies involving bead-coupled PY*LKTK peptide demonstrate that this phosphopeptide directly complexes with Stat3 monomers in vitro, suggesting that PY*LKTK disrupts Stat3:Stat3 dimers. As evidence for the functional importance of peptide-directed inhibition of Stat3, PY*LKTK-mts (mts, membrane translocating sequence) selectively inhibits constitutive and ligand-induced Stat3 activation in vivo. Furthermore, PY*LKTK-mts suppresses transformation by the Src oncoprotein, which has been shown previously to require constitutive Stat3 activation. Altogether, we have identified a minimal peptide that inhibits Stat3 signaling and provides the conceptual basis for use of this peptide as a lead for novel peptidomimetic drug design. STATs1 were originally discovered as latent cytoplasmic transcription factors that mediate cellular responses to diverse cytokines and growth factors (1-6). Following ligand binding and activation of cytokine or growth factor receptors, STATs are recruited to the cytoplasmic portion of receptors where they are tyrosine-phosphorylated. Tyrosine kinases that mediate STAT phosphorylation and activation include growth factor receptors and cytoplasmic tyrosine kinases, particularly Janus kinase and Src kinase families. Once tyrosine-phosphorylated, two STAT monomers form dimers through reciprocal phosphotyrosine-SH2 interactions, translocate to the nucleus, and bind to STAT-specific DNA-response elements of target genes to induce gene expression. To date, there are seven STAT family members identified in mammals, designated Stat1, Stat2, Stat3, Stat4, Stat5a, Stat5b, and Stat6. STATs have diverse normal biological functions, which include roles in cell differentiation, proliferation, development, apoptosis, and inflammation (7-18). In addition to their roles in normal cellular processes, some STATs have been observed to participate in signaling events that lead to oncogenic transformation. Constitutively active Stat3 is observed in transformation by...

Serine, threonine-derived (4S)-oxazolidine-4-carboxylic acid, and cysteine-derived (4R)-thiazolidinecarboxylic acid, denoted pseudo-proline (Xaa[ΨR 1 ,R 2 pro]), serve as structure disrupting, solubilizing building blocks in peptide synthesis. Variation of the 2-C substituents within the heterocyclic system results in different physicochemical and conformational properties. NMR studies of a series of pseudo-proline (ΨPro)-containing peptides reveal a pronounced effect of the 2-C substituents upon the cis to trans ratio of the adjacent amide bond in solution. 2-C unsubstituted systems show a preference similar to that of the proline residue for the trans form, whereas 2,2-dimethylated derivatives adopt the cis amide conformation in high content. For 2-monosubstituted ΨPro, the cis−trans distribution depends on the 2-C chirality. For the 2-(S)-diastereoisomer, both forms are similarly populated in solution, whereas the 2-(R)-epimer adopts preferentially the trans form. The results are supported by conformational energy calculations and suggest that, by tailoring the degree of substitution, pseudo-prolines may serve as a temporary proline mimetic or as a hinge in peptide backbones.

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