In vitro activity: Stattic inhibits binding of a phosphotyrosine-containing peptide derived from the gp130 receptor to the STAT3 SH2 domain in a strongly temperature-dependent manner. Stattic only has a very weak effect on binding of a tyrosinephosphorylated peptide to the SH2-domain of the tyrosine kinase Lck. And it doesn’t inhibit dimerization of two other dimeric transcription factors (c-Myc/Max and Jun/Jun). It also inhibits fluorescein-labeled phosphopeptides to the SH2 domains of STAT1 and STAT5b. Stattic selectively inhibits DNA binding of STAT3 homodimers at a concentration of 10 μM. Shown to inhibit cellular phosphorylation of STAT3 at Tyr705 with little effect towards STAT1 phosphorylation at Tyr701 (in HepG2 cells) or the phosphorylation of JAK1, JAK2, and c-Src (in MDA-MB-231 and MDA-MB-235S cells). Stattic increases the apoptotic rate of STAT3-dependent breast cancer cell lines.

Kinase Assay: Screening is performed at approximately 30 °C. The specificity of screening hits is validated in analogous assays for binding of the test compounds to the SH2 domains of STAT1, STAT5, and Lck. The final concentration of buffer components used for all FP assays is 10 mM HEPES (pH 7.5), 1 mM EDTA, 0.1% Nonidet P-40, 50 mM NaCl, and 10% DMSO. The absence of dithiothreitol is essential for inhibitory activity. The sequences of the peptides are: STAT3, 5-carboxyfluorescein-GY(PO3H2)LPQTV-NH2; STAT1, 5-carboxyfluorescein-GY(PO3H2)DKPHVL; STAT5, 5-carboxyfluorescein-GY(PO3H2)LVLDKW; and Lck, 5-carboxyfluorescein-GY(PO3H2)EEIP. For specificity analysis at 30 °C, proteins are used at 150 nM (STAT1, STAT3, and STAT5). For specificity analysis at 37 °C, proteins are used at 370 nM (STAT3) or 100 nM (Lck). Proteins are incubated with test compounds in Eppendorf tubes at the indicated temperatures for 60 min prior addition of the respective 5-carboxyfluorescein labeled peptides (final concentration: 10 nM). Before measurement at room temperature, the mixtures are allowed to equilibrate for at least 30 min. Test compounds are used at the indicated concentrations diluted from a 20× stock in DMSO. Binding curves and inhibition curves are fitted with SigmaPlot. All competition curves are repeated three times in independent experiment.

Cell Assay: Stattic significantly inhibited STAT3 activation and expression, leading to decreased cell survival and proliferation as well as increased radiosensitivity. The Stattic treatment also reduced STAT3-mediated HIF-1 expression.

Cancer Res. 2011 May 1;71(9):3182-8; Chem Biol. 2006 Nov;13(11):1235-42.