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. 2019 Nov 22;9(1):17335.
doi: 10.1038/s41598-019-53856-1.

Pyrrothiogatain Acts as an Inhibitor of GATA Family Proteins and Inhibits Th2 Cell Differentiation in Vitro

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Free PMC article

Pyrrothiogatain Acts as an Inhibitor of GATA Family Proteins and Inhibits Th2 Cell Differentiation in Vitro

Shunsuke Nomura et al. Sci Rep. .
Free PMC article

Abstract

The transcription factor GATA3 is a master regulator that modulates T helper 2 (Th2) cell differentiation and induces expression of Th2 cytokines, such as IL-4, IL-5, and IL-13. Th2 cytokines are involved in the protective immune response against foreign pathogens, such as parasites. However, excessive production of Th2 cytokines results in type-2 allergic inflammation. Therefore, the application of a GATA3 inhibitor provides a new therapeutic strategy to regulate Th2 cytokine production. Here, we established a novel high-throughput screening system for an inhibitor of a DNA-binding protein, such as a transcription factor, and identified pyrrothiogatain as a novel inhibitor of GATA3 DNA-binding activity. Pyrrothiogatain inhibited the DNA-binding activity of GATA3 and other members of the GATA family. Pyrrothiogatain also inhibited the interaction between GATA3 and SOX4, suggesting that it interacts with the DNA-binding region of GATA3. Furthermore, pyrrothiogatain significantly suppressed Th2 cell differentiation, without impairing Th1 cell differentiation, and inhibited the expression and production of Th2 cytokines. Our results suggest that pyrrothiogatain regulates the differentiation and function of Th2 cells via inhibition of GATA3 DNA binding activity, which demonstrates the efficiency of our drug screening system for the development of novel small compounds that inhibit the DNA-binding activity of transcription factors.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Establishment of the high-throughput assay system to directly detect a DNA–protein interaction. (A) Immunoblot analysis of FLAG-tagged recombinant GATA3 (FLAG-GATA3) synthesized by the wheat cell-free system. The whole translational mixture (W) and the supernatant (S), obtained after centrifugation, were analysed using anti-FLAG M2 antibody. (B) A schematic diagram of the high-throughput biochemical DNA-binding assay system to detect the direct binding between GATA3 and its target DNA. When FLAG-tagged GATA3 binds the DNA labelled with biotin at the 5 prime-terminal, AlphaScreen beads generate luminescent signal. (C) The binding assay of GATA3 with its consensus DNA-binding motif. The binding assay between the crude translation mixture of FLAG-tagged GATA3 (1 µL) and biotinylated DNA (10 nM) was performed in the presence of various concentrations of NaCl (100 to 150 mM). An oligonucleotide with a mutated GATA-binding site was used as control for this assay. (D) The binding assay as described in (C) was performed in the presence of indicated concentrations of biotinylated DNA. A reaction mixture containing FLAG-tagged GATA3 and 150 mM NaCl prepared under the same conditions as those in (C) was mixed with 1 to 10 nM biotinylated DNA. (E) Competition assay with non-labelled GATA consensus DNA. The binding assay with the same conditions as those in (D) was mixed with biotinylated DNA (4 nM) and non-labelled GATA consensus DNA (0 to 250 nM) as a control. (F) Validation of the quality of the binding assay using AlphaScreen. The ‘Z’ factor was calculated from the binding reaction of GATA3 with the GATA consensus DNA (positive control, n = 20) or its GATA-binding site mutant (negative control, n = 20). In (C–E), all data are expressed as individual points of three independent experiments with error bars indicating standard deviation.
Figure 2
Figure 2
Identification of a GATA3 inhibitor using the wheat cell-free drug screening system. (A) A flow chart of the high-throughput screening to identify the 3-(2,5-dimethyl-1H-pyrrol-1-yl)thiophene-2-carboxylic acid that inhibits the DNA-binding activity of GATA3. The 3-(2,5-dimethyl-1H-pyrrol-1-yl)thiophene-2-carboxylic acid was named pyrrothiogatain (pyrrole and thiofuran containing GATA3 inhibitor). (B) The results of the first screen using 9,600 compounds. In the assay, each chemical compound was used at a final concentration of 10 µM. (C) The results of the second screening that show the inhibition rate of pyrrothiogatain. The inhibition assay of GATA3 DNA-binding activity was performed in the presence of various concentrations of pyrrothiogatain (0 to 10 µM). Biotinylated FLAG-peptide was used as a control to measure the interference of compounds in the AlphaScreen assay. In addition, a binding assay of RelA and its target DNA was used as control for GATA3. (D) Electrophoretic mobility shift assay (EMSA) of GATA3 with its consensus motif containing oligonucleotide in the presence of various concentrations of pyrrothiogatain (0 to 100 µM). GATA3 consensus oligonucleotide labelled with 32P was detected by autoradiography. (E) MTS assay to confirm the cytotoxicity of pyrrothiogatain. Jurkat cells were cultured with various concentrations of pyrrothiogatain (0 to 100 µM) for 3 days and subjected to MTS assay. (F) HEK293T cells were transfected with firefly luciferase reporter for the Il-5 promoter plus renilla luciferase reporter in the presence (+) or absence (−) of GATA3-expressing plasmid. Then, the cells were left unstimulated (Med.) or stimulated (Stim.) with the phorbol ester PMA (30 ng/mL) in the presence of pyrrothiogatain (0 to 30 µM). The luciferase activity is presented relative to renilla luciferase activity. In (B,D,E), all data are expressed as individual points of three independent experiments with error bars indicating standard deviation.
Figure 3
Figure 3
Effect of pyrrothiogatain on the DNA-binding activity of GATA family proteins. (A) Immunoblot analysis of FLAG-tagged recombinant GATA family proteins (GATA1-GATA6) synthesized by the wheat cell-free system. The whole translational mixture (W) and the supernatant (S) were analysed using an anti-FLAG M2 antibody. (B) The AlphaScreen assay to detect the binding between the GATA family and DNA containing the GATA consensus binding sequence using the same protocol as that in Fig. 1F. (C) The inhibition assays for the DNA-binding activity of GATA family proteins (GATA2-GATA5) were performed in the presence of various concentrations of pyrrothiogatain (0 to 200 µM). The binding activity is represented by the relative AlphaScreen signal. (D) The results of EMSA of the binding GATA family proteins (GATA2-GATA5) in the presence of pyrrothiogatain (0 to 100 µM). GATA binding to the DNA labelled with 32P was detected by autoradiography. In (B,C), all data are expressed as individual points of three independent experiments with error bars indicating standard deviation.
Figure 4
Figure 4
Pyrrothiogatain inhibits the GATA3–SOX4 interaction. (A) Immunoblot analysis of biotin fused recombinant SOX4 (Biotin-SOX4) synthesized by the wheat cell-free system. The whole translational mixture (W) and the supernatant (S) were analysed using an anti-biotin antibody. (B) A schematic diagram of the binding assay to detect the direct binding between GATA3 and SOX4. When FLAG-tagged GATA3 binds with biotin fused SOX4, AlphaScreen beads generate a luminescent signal. (C) The binding assay of GATA3 and SOX4. The luminescence signals were detected by AlphaScreen technology. FLAG-DHFR was used as a control. (D) The inhibition assay of the binding of GATA3 and SOX4 in the presence of various concentrations of pyrrothiogatain (0 to 100 µM). The binding activity was calculated from the AlphaScreen signal. (E) The binding of recombinant GATA3 with SOX4 in the presence of pyrrothiogatain (0 to 100 µM) was assessed by immunoprecipitation assay. AGIA-tagged GATA3 and FLAG-tagged SOX4 were synthesized by the wheat cell system and subjected to an immunoprecipitation assay using an anti-FLAG-affinity agarose gel. In (C,D), all data are expressed as individual points of three independent experiments with error bars indicating standard deviation.
Figure 5
Figure 5
Pyrrothiogatain inhibits Th2 cell differentiation and production of Th2 cytokines. (A) Intracellular staining of IL-5/IL-13, IL-4/IFN-γ, and IL-2/IFN-γ in naive CD4+ T cells cultured under Th2 conditions in the presence or absence of pyrrothiogatain (30 and 80 µM) for five days. (B) Cytokine production induced in the pyrrothiogatain treated Th2 cells shown in panel (A) was determined by ELISA. (C) Quantitative RT-PCR analysis of the pyrrothiogatain-treated Th2 cells shown in panel (A). (D) Immunoblot analysis of GATA3 in naive CD4+ T cells cultured under Th2 conditions in the presence or absence of pyrrothiogatain (80 µM) for 5 days. (E) Cytokine production from Th2 cells stimulated with immobilized anti-TCRβ mAb for 16 h in the presence or absence of pyrrothiogatain (0 to 100 µM). The amounts of IL-4, IL-5 and IL-13 in the culture supernatants were determined by ELSA. In (B), (C) and (E), all data are expressed as individual points of three independent experiments with error bars indicating standard deviation.
Figure 6
Figure 6
Pyrrothiogatain inhibits GATA3 DNA-binding to Th2 cytokine gene locus. (A) The GATA3 binding sites in Th2 cells are shown as a schematic diagram. Chromatin immunoprecipitation analysis of GATA3 in naive CD4+ T cells cultured under Th2 conditions in the presence or absence of pyrrothiogatain (80 µM) for 2 days. All data are expressed as mean values of three independent experiments with error bars indicating standard deviation.

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