A novel small-molecule disrupts Stat3 SH2 domain–phosphotyrosine interactions and Stat3-dependent tumor processes

Abstract

The molecular modeling of the phosphotyrosine (pTyr)-SH2 domain interaction in the Stat3:Stat3 dimerization, combined with in silico structural analysis of the Stat3 dimerization disruptor, S3I-201, has furnished a diverse set of analogs. We present evidence from in vitro biochemical and biophysical studies that the structural analog, S3I-201.1066 directly interacts with Stat3 or the SH2 domain, with an affinity (KD) of 2.74 nM, and disrupts the binding of Stat3 to the cognate pTyr-peptide, GpYLPQTV-NH2, with an IC50 of 23 μM. Moreover, S3I-201.1066 selectively blocks the association of Stat3 with the epidermal growth factor receptor (EGFR), and inhibits Stat3 tyrosine phosphorylation and nuclear translocation in EGF-stimulated mouse fibroblasts. In cancer cells that harbor aberrant Stat3 activity, S3I-201.1066 inhibits constitutive Stat3 DNA-binding and transcriptional activities. By contrast, S3I-201.1066 has no effect on Src activation or the EGFR-mediated activation of the Erk1/2MAPK pathway. S3I-201.1066 selectively suppresses the viability, survival, and malignant transformation of the human breast and pancreatic cancer lines and the v-Src-transformed mouse fibroblasts harboring persistently-active Stat3. Treatment with S3I-201.1066 of malignant cells harboring aberrantly-active Stat3 down-regulated the expression of c-Myc, Bcl-xL, Survivin, the matrix metalloproteinase 9, and VEGF. The in vivo administration of S3I-201.1066 induced significant antitumor response in mouse models of human breast cancer, which correlates with the inhibition of constitutively-active Stat3 and the suppression of known Stat3-regulated genes. Our studies identify a novel small-molecule that binds with a high affinity to Stat3, blocks Stat3 activation and function, and thereby induces antitumor response in human breast tumor xenografts harboring persistently-active Stat3.

STAT3 upregulation in pituitary somatotroph adenomas induces growth hormone hypersecretion

“…The evidence for elevated somatotroph adenoma STAT3 and STAT3 regulation of GH suggests STAT3 as a potential cellular target to treat acromegaly. Small-molecule STAT3 inhibitors have been used in preclinical and clinical studies (17,24,25,(42)(43)(44)(45), and we tested S3I-201, a cell-permeable amidosalicylic acid compound that selectively inhibits STAT3 DNA-binding activity in vitro (IC 50 = 86 ± 33 μM) (24,25). Here, we show that, using a dose of 5 mg/kg, S3I-201 inhibited rat GH3 pituitary cell growth in vitro and attenuated growth of somatotroph tumor xenografts in Wistar Furth rats.…”

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

“…Cloning and Protein Expression-The molecular cloning, expression, and the purification of His-tagged Stat3 and Histagged Stat3 SH2 domain were carried out as we have previously reported (27). Clones were sequenced to verify the correct sequences and orientation.…”

“…Fluorescence Polarization Assay-Fluorescence Polarization (FP) Assay was conducted as previously reported (20,27) using the labeled phosphopeptide, 5-carboxyfluoresceinGpYLPQTV-NH 2 (where pY represents phospho-Tyr) as probe and Stat3 or SPI. For saturation curves, a fixed concentration of the fluorescently labeled peptide probe (10 nM) was incubated with increasing concentration of Stat3 (0 -0.8 M) or SPI (0 -400 M) for 30 min at room temperature in the buffer, 50 mM NaCl, 10 mM HEPES, 1 mM EDTA, 0.1% Nonidet P-40, and the fluorescent polarization measurements were determined using the POLARstar Omega (BMG LABTECH, Durham, NC), with the set gain adjustment at 35 mP.…”

“…Surface Plasmon Resonance Analysis-SensiQ and its analysis software Qdat (ICX Technologies, Oklahoma City, OK) were used to analyze the interaction between known Stat3-binding pTyr peptide motifs (analyte) and Stat3 or the Stat3 SH2 domain (target) and to determine the binding affinity, as previously reported (27). Purified Stat3 (50 g), Stat3 SH2 domain (30 g), or SPI (25 g) was immobilized on a Carboxyl Sensor Chip (for SPI) or a HisCap Sensor Chip (for Stat3 and the Stat3 SH2 domain) by injecting the peptide or protein onto the chip.…”

“…The interaction with SPI could block the binding of Stat3 to its cognate pTyr peptide motifs. Fluorescence polarization (FP) study has previously been used to demonstrate the binding of Stat3 (or Stat3 SH2 domain) to the high-affinity peptide, GpYLPQTV-NH 2 (19,20,27). This assay was therefore used to verify SPI binding to the high-affinity pTyr peptide motif and to further assess the potential that such a binding could disrupt the association of Stat3 with the same peptide probe.…”