Article Figures & Data
Figures
- Figure 1.
EMSA analyses of STAT DNA-binding activities showing effects of peptidomimetics. Nuclear extracts containing activated Stat1, Stat3, and Stat5 are treated with the indicated concentrations of peptidomimetics ISS 493, ISS 610, ISS 637, ISS 593, ISS 221, or ISS 610NP for 30 min at room temperature before incubation with radiolabeled oligonucleotide probes. Stat1 and Stat3 binding to hSIE probe (A) and Stat1 and Stat5 binding to MGFe probe (B). Positions of STAT:DNA complexes in gel are labeled. Control lanes, nuclear extracts from NIH3T3 cells stimulated with EGF but not treated with peptidomimetics.
- Figure 2.
Evidence for dissociation of STAT dimers by phosphopeptide or peptidomimetic. Cell lysates contain either only activated Stat1 (Stat1), Stat3 (Stat3), or both [pooled lysates (Stat1 + Stat3, 0–300)] and are treated (Stat1 + Stat3, 30–300) with the indicated concentrations of PY*LKTK (A) or ISS 610 (B) for 30 min at room temperature before incubation with radiolabeled hSIE oligonucleotide probe. Positions of STAT:DNA complexes in gel are labeled. Cell lysates were prepared from recombinant baculovirus-infected Sf-9 cells as described in Materials and Methods.
- Figure 3.
Evaluation of peptidomimetic effects on Stat3 activation and Stat3-mediated gene expression in intact cells, and on Src transformation. A and B, luciferase activities in extracts prepared from ISS 610- or ISS 610NP-treated v-Src-transformed mouse fibroblasts that stably express Stat3-dependent (NIH3T3/v-Src/pLucTKS3) and Stat3-independent (NIH3T3/v-Src/pRLSRE) luciferase reporters. Columns, means of three independent assays; bars, SD. EMSA analyses of Stat3 DNA-binding activities (using hSIE oligonucleotide probe) in nuclear extracts prepared from v-Src-transformed NIH3T3/v-Src (C and D), and human breast carcinoma MDA-MB-435, MDA-MB-468, MDA-MB-231, and non-small cell lung carcinoma A549 (E). F, effect of ISS 610 on soft-agar growth of v-Src-transformed fibroblasts (NIH3T3/v-Src) and their v-Ras-transformed counterparts (NIH3T3/v-Ras). Transformed cells were seeded in soft agar and treated every 2–3 days with or without ISS 610 until large colonies were evident. Columns, means of three independent assays; bars, SD.
- Figure 4.
Evaluation of peptidomimetic effects on cell proliferation. Growth curves for transformed and tumor cells. Normal and transformed fibroblasts (NIH3T3, NIH3T3/v-Src, or NIH3T3/v-Ras) as well as human breast carcinoma (MDA-MB-231, MDA-MB-435, or MDA-MB-453) cells were treated with or without compounds and counted by trypan blue exclusion on each of 4 days. Cells were untreated (dotted lines) or treated with 1 mm ISS 610 or PY*LKTK-MTS (solid lines). Points, means of four independent determinations; bars, SD.
- Figure 5.
Computer modeling of ISS 610 bound to the SH2 pocket. Comparison of the lowest GOLD (51) docked conformation of ISS 610 (green), and the COOH-terminal phosphotyrosine peptide, AAPY*LK (orange), of the associated Stat3β monomer determined from the crystal structure (49), in the SH2 domain of Stat3β (pale blue).
Tables
- Table 1.
Disruption of Stat3 DNA-binding activity by peptidomimetics
Compound R′ IC50(μM)a Prolylphosphotyrosylleucine 
182±15 Alanylphosphotyrosylleucine 217±55 ISS 248 ne ISS 265 ne ISS 375 ne ISS 610 42±23 ISS 637 55±35 ISS 219 232±16 ISS 221 75±36 ISS 593 48±32 ISS 223 225±15 ISS 249 ne ISS 493 38±16 ISS 352 410±15 ISS 353 650±22 ISS 355 ne ISS 360 420±35 ISS 363 643±43 -
Note: Nuclear extracts containing active Stat3 were preincubated for 30 min with or without peptidomimetics before incubation with radiolabeled hSIE probe and analysis by EMSA.
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↵a Values are the means and SDs of at least three independent assays. ne, no effect at 1 mm. Results are representative peptidomimetics from over 80 that have been evaluated. Structural formula of compounds: R′Y*L (where R′ is defined as shown).
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- Table 2.
Selective disruption of STAT family members by peptidomimetics
Mimetics IC50 Values (μm) against STAT Dimersa Stat3:Stat3 Stat1:Stat3 Stat1:Stat1 Stat5:Stat5 ISS 221 75 ± 36 455 ± 97 310 ± 74 50 ± 12 ISS 493 38 ± 16 230 ± 22 273 ± 22 300 ± 23 ISS 593 48 ± 32 87 ± 15 175 ± 65 10 ± 6 ISS 610 42 ± 23 125 ± 15 310 ± 145 285 ± 32 ISS 637 55 ± 35 195 ± 12 255 ± 55 695 ± 123 -
Note: Nuclear extracts containing active Stat1, Stat3, and Stat5 were preincubated with or without peptidomimetics for 30 min before incubation with radiolabeled hSIE probe and EMSA analysis.
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a Values are the means and SDs of at least three independent assays, ne, no effect at 1 mm.Results are representative peptidomimetics from over 80 that have been evaluated.
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- Table 3.
Flow cytometric analysis of cells treated with PY*LKTK-MTS and ISS 610
NIH3T3 NIH3T3/v-Src Control 2.1% 1.8% 1 mm PYLKTK-MTS 1.2% 0.4% 1 mm PY*LKTK-MTS 3.7% 27.6% 1 mm ISS 610 0.3% 21.4% -
Note: Cells were treated with compounds for 48 h, labeled with Apo-BrdUrd and analyzed by flow cytometry for percentage apoptotic cells. Control represents no treatment.
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Volume 3, Issue 3
