Identification of a natural product-like STAT3 dimerization inhibitor by structure-based virtual screening

Abstract

STAT3 regulates a variety of genes involved with cell proliferation, differentiation, apoptosis, angiogenesis, metastasis, inflammation, and immunity. The purpose of this study was to apply molecular docking techniques to identify STAT3 inhibitors from a database of over 90000 natural product and natural product-like compounds. The virtual screening campaign furnished 14 hit compounds, from which compound 1 emerged as a top candidate. Compound 1 inhibited STAT3 DNA-binding activity in vitro and attenuated STAT3-directed transcription in cellulo with selectivity over STAT1 and with comparable potency to the well-known STAT3 inhibitor S3I-201. Furthermore, compound 1 inhibited STAT3 dimerization and decreased STAT3 phosphorylation in cells without affecting STAT1 dimerization and phosphorylation. Compound 1 also exhibited selective anti-proliferative activity against cancer cells over normal cells in vitro. Molecular docking analysis suggested that compound 1 might putatively function as an inhibitor of STAT3 dimerization by binding to the SH2 domain. This study also validates the use of in silico techniques to identify inhibitors of protein-protein interactions, which are typically considered difficult to target with small molecules.

Figure 1

Structures of compounds 1–14 identified in the high-throughput virtual screening chosen for biological validation

Figure 2

STAT3 DNA-binding inhibitory activity of compounds 1–14 as determined by ELISA. Microtitre plates coated with the STAT3 consensus sequence were incubated with nuclear extracts containing activated STAT3 and 10 μM of compounds. STAT3 binding was detected using anti-STAT3 primary antibody and horseradish peroxidase-conjugated secondary antibody. Results are representative of three independent experiments

Figure 3

Compound 1 inhibits the DNA-binding activity of STAT3 in a dose-dependent manner as measured by ELISA. Microtitre plates coated with the STAT3 consensus sequence were incubated with HepG2 nuclear extracts containing EGF-activated STAT3 and 1 or S3I-201 at the indicated concentrations. STAT3 binding was detected using anti-STAT3 primary antibody and horseradish peroxidase-conjugated secondary antibody. Results are representative of three independent experiments. Error bars represent the S.D. of triplicate results. Estimated IC₅₀ values: compound 1: 15 μM, S3I-201: 10 μM

Figure 4

Low-energy-binding conformations of compound 1 bound to the STAT3β homodimer generated by virtual ligand docking. Compound 1 is depicted as a ball-and-stick model showing carbon (yellow) and oxygen (red) atoms. Hydrogen bonds are depicted as dotted lines. The binding pocket of the STAT3β is represented as a translucent green surface

Figure 5

Effect of compound 1 on STAT3-driven transcription activity, STAT3 dimerization and STAT3 phosphorylation. (a) Compound 1 inhibits STAT3-driven transcription activity in EGF-stimulated HeLa cells as measured using a luciferase reporter assay. Estimated IC₅₀ values: compound 1: 30 μM, S3I-201: 30 μM. (b) Compound 1 inhibits the dimerization of STAT3 in HEK293T cells. (c) Quantification of STAT3 dimerization inhibition by densitometry analysis. (d) Compound 1 inhibits STAT3 Y705 phosphorylation but not total STAT3 content in HepG2 cells. (e) Quantification of STAT3 Y705 phosphorylation inhibition by densitometry analysis. (f) Densitometry analysis for the cell-based western blot shows no inhibition of compound 1 on total STAT3. Error bars represent the S.D. of triplicate results. *P<0.05, **P<0.001

Figure 6

Effect of compound 1 on IFN-α-induced STAT3 Y705 phosphorylation and IFN-α-induced STAT1 Y701 phosphorylation in HeLa cells. (a) Compound 1 inhibits IFN-α-induced STAT3 Y705 phosphorylation but not IFN-α-induced total STAT3. (b) Quantification of STAT3 phosphorylation inhibition by densitometry analysis. (c) Quantification of total STAT3 expression by densitometry analysis. (d) Compound 1 has no effect on IFN-α-induced STAT1 Y701 phosphorylation and IFN-α-induced total STAT1. (e) Quantification of STAT1 phosphorylation inhibition by densitometry analysis. (f) Quantification of total STAT1 expression by densitometry analysis. Error bars represent the S.D. of triplicate results. *P<0.05, **P<0.001

Figure 7

Cytotoxicity of compound 1 on cell viability as determined by the MTT assay. LO2, HepG2, RAW264.7, Caco2 were treated with the indicated concentrations of compound 1 for 72 h. The data are expressed as the percentage of living cells compared with the negative control. Results are representative of three independent experiments. Error bars represent the S.D. of triplicate results