A small-molecule inhibitor shows that pirin regulates migration of melanoma cells

Journal name:
Nature Chemical Biology
Volume:
6,
Pages:
667–673
Year published:
DOI:
doi:10.1038/nchembio.423
Received
Accepted
Published online

Abstract

The discovery of small molecules that bind to a specific target and disrupt the function of proteins is an important step in chemical biology, especially for poorly characterized proteins. Human pirin is a nuclear protein of unknown function that is widely expressed in punctate subnuclear structures in human tissues. Here, we report the discovery of a small molecule that binds to pirin. We determined how the small molecule bound to pirin by solving the cocrystal structure. Either knockdown of pirin or treatment with the small molecule inhibited melanoma cell migration. Thus, inhibition of pirin by the small molecule has led to a greater understanding of the function of pirin and represents a new method of studying pirin-mediated signaling pathways.

At a glance

Figures

  1. Identification of TPh A as a pirin binder.
    Figure 1: Identification of TPh A as a pirin binder.

    (a) The slide was treated with HEK293T cell lysates that overexpressed DsRed or DsRed-fused pirin. A fluorescent image is shown by merged display analysis of a glass slide. The positive signal of the area that spotted with TPh A is indicated by white arrows. TPh A has three phenyl groups (S1–S3) and a sulfilimine moiety. (b) TPhs A–D (10 mM) were immobilized onto a glass slide. The slide was incubated with HEK293T cell lysates that overexpressed DsRed-fused pirin. A fluorescent image is shown, and the total fluorescent intensity was corrected for background intensity. The error bars denote the s.d. of three replicates. The structures of TPhs B–D are also shown. (c) A solution of TPh A (10 mM) was spotted onto glass slides, and the slides were incubated with HEK293T cell lysates that overexpressed DsRed-fused pirin in the absence or presence of various concentrations of free TPh A. (d) The ITC profile of TPh A for purified His6-pirin. A solution of TPh A (20 μM), containing 50 mM NaH2PO4-HCl (pH 8.0), 300 mM NaCl and 6% (v/v) DMSO was titrated with a solution of His6-pirin (400 μM) in the same buffer.

  2. Crystal structure of the complex formed by pirin and TPh A.
    Figure 2: Crystal structure of the complex formed by pirin and TPh A.

    Pirin is colored green and cyan for the N- and C-terminal regions, respectively. The N- and C-terminal domains are cross-linked, with a single iron ion (brown) in the N-terminal domain. TPh A is shown as a stick model. (a) Overall view of crystal structure of pirin complexed with TPh A. (b) A molecule of TPh A and the surrounding residues of pirin. The phenyl groups in the S1 and S2 positions of TPh A are stabilized by stacking interactions with His56 and Phe53, respectively. The 2FoFc electron density map around TPh A is contoured at 1.0 σ. (c) The nitrogen atom of TPh A interacts with a water molecule surrounding an iron ion through a hydrogen-bonding network. The iron ion is coordinated by His56, His58, His101, Glu103 and two water molecules. (d) The phenyl group in the S3 position of TPh A inserts into a hydrophobic pocket composed of Phe45, Met73, Leu67, Ser65 and Met85.

  3. Inhibition of the interaction between pirin and Bcl3 by TPh A in vitro and in cells.
    Figure 3: Inhibition of the interaction between pirin and Bcl3 by TPh A in vitro and in cells.

    (a) His6-pirin, GST and GST-Bcl3 ARD were synthesized in E. coli and purified (left). His6-pirin was incubated with glutathione-Sepharose beads that were conjugated to GST or GST-Bcl3 ARD. Pull-down assay was performed in the presence or absence of TPh A (100 μM). GST-Bcl3 ARD–bound His6-pirin was detected by western blot with anti-pirin antibody (right). (b) HEK293T cells were transfected with vectors that encoded Bcl3-Myc and pirin-His6. After 43 h of transfection, the cells were treated with TPh A (20 μM) for the next 5 h and lysed, and pulldown was performed with Ni-NTA agarose beads. The expression levels of Bcl3-Myc, pirin-His6 and α-tubulin in the total cell lysates and the complexes were detected by western blot with the indicated antibodies. Uncropped images of blots are shown in Supplementary Figure 9.

  4. Inhibition of melanoma cell migration by TPh A.
    Figure 4: Inhibition of melanoma cell migration by TPh A.

    (a) Expression of pirin in human cell lines. Total cell lysates were prepared, and western blot was performed with the indicated antibodies. Uncropped images of blots are shown in Supplementary Figure 9. (b) An artificial wound was created by a pipette tip. The WM266-4 cells were treated with 1% (v/v) DMSO (control) or various concentrations of TPh A and B. After 18 h, photographs were taken using phase-contrast microscopy. (c) Percentage of wound repair after 18 h in the absence or presence of various concentrations of TPh A or B (open bars and closed bars indicate cell proliferation and scratch closure, respectively). The relative cell number was calculated after 48 h (open bars). *P < 0.05. (d) WM266-4 cells were untreated or treated with siRNAs targeting pirin (siRNA-pirin#1, #2 and #3) or luciferase (siRNA-luc). The expression of pirin was detected by western blot using the indicated antibodies. (e) After treatment with siRNA-luc or siRNA-pirin, the cells were harvested and used to seed new dishes. The relative cell number was calculated for the next 48 h (open bars), and the percentage of wound repair was measured after 18 h (closed bars). *P < 0.05. (f) WM266-4 cells were untreated or treated with siRNAs targeting Bcl3 (siRNA-Bcl3#1 and siRNA-Bcl3#2) or siRNA-luc. The expression of Bcl3 was measured by semiquantitative RT-PCR. (g) After treatment with siRNA-luc or siRNA-Bcl3, the cells were harvested and used to seed new dishes. The relative cell number was calculated for the next 48 h (open bars), and the percentage wound repair was measured after 18 h (closed bars). *P < 0.05.

  5. Pirin controls melanoma cell migration through the regulation of SNAI2 expression.
    Figure 5: Pirin controls melanoma cell migration through the regulation of SNAI2 expression.

    (ac) Regulation of SNAI2 expression by pirin and Bcl3. WM266-4 cells were treated with TPh A or B or DMSO (control) for 24 h (a); untreated or treated with siRNA-pirin#1 or siRNA-GFP (b); and untreated or treated with siRNA-Bcl3#1 or siRNA-GFP (c) and total RNA was extracted. The expression of several genes was determined by semiquantitative RT-PCR. (d) Suppression of melanoma cell migration by knockdown of SNAI2. WM266-4 cells were untreated or treated with siRNAs targeting SNAI2 (siRNA-SNAI2#1 and siRNA-SNAI2#2) or siRNA-luc, and total RNA was extracted. The expression of several genes was determined by semiquantitative RT-PCR. (e) After treatment with siRNA-luc or siRNA-SNAI2, the cells were harvested and used to seed new dishes. The relative cell number was calculated after 48 h (open bars), and the percentage of wound repair was measured after 18 h (closed bars). *P < 0.05.

  6. Regulation of SNAI2 promoter activity by pirin.
    Figure 6: Regulation of SNAI2 promoter activity by pirin.

    (a) WM266-4 cells were pretreated with 10 μM or 50 μM TPh A or B for 2 h. The cells were transfected with pGL3 or pGL3-SNAI2 and phRL-promoterless reporter vectors. After 12 h of transfection, the luminescence of the cell lysates was measured. The value of DMSO-treated SNAI2 promoter activity was defined as 100%. *P < 0.05. (b) WM266-4 cells were transfected with luciferase reporter vectors and pCI-neo (Neo) or pCI-pirin-His6 (pirin) expression vector. After 12 h of transfection, the luminescence of the cell lysates was measured. The value of pCI-neo-transfected SNAI2 promoter activity was defined as 100%. *P < 0.05.

Compounds

8 compounds View all compounds
  1. N-{[4-(Benzyloxy)phenyl](methyl)-λ4-sulfanylidene}-4-methylbenzenesulfonamide
    Compound 1 N-{[4-(Benzyloxy)phenyl](methyl)-λ4-sulfanylidene}-4-methylbenzenesulfonamide
  2. 4-{1-[(3-Chlorobenzoyl)imino]-1-methyl-λ4-sulfanyl}phenyl 4-chlorobenzoate
    Compound 2 4-{1-[(3-Chlorobenzoyl)imino]-1-methyl-λ4-sulfanyl}phenyl 4-chlorobenzoate
  3. 4-(1-{[(2-Chlorophenyl)sulfonyl]imino}-1-methyl-λ4-sulfanyl)phenyl 4-chlorobenzoate
    Compound 3 4-(1-{[(2-Chlorophenyl)sulfonyl]imino}-1-methyl-λ4-sulfanyl)phenyl 4-chlorobenzoate
  4. 4-(1-{[(4-Chlorophenyl)sulfonyl]imino}-1-methyl-λ4-sulfanyl)phenyl 4-chlorobenzoate
    Compound 4 4-(1-{[(4-Chlorophenyl)sulfonyl]imino}-1-methyl-λ4-sulfanyl)phenyl 4-chlorobenzoate
  5. 4-Chloro-N-[4-(methylthio)phenyl]benzamide
    Compound 5a 4-Chloro-N-[4-(methylthio)phenyl]benzamide
  6. N-{[4-(p-Chlorobenzamide)phenyl](methyl)-λ4-sulfanylidene}-4-methylbenzenesulfonamide
    Compound 5 N-{[4-(p-Chlorobenzamide)phenyl](methyl)-λ4-sulfanylidene}-4-methylbenzenesulfonamide
  7. 1-Chloro-4-{[4-(methylthio)phenoxy]methyl}benzene
    Compound 6a 1-Chloro-4-{[4-(methylthio)phenoxy]methyl}benzene
  8. N-{[4-(p-Chlorobenzyloxy)phenyl](methyl)-λ4-sulfanylidene}-4-methylbenzenesulfonamide
    Compound 6 N-{[4-(p-Chlorobenzyloxy)phenyl](methyl)-λ4-sulfanylidene}-4-methylbenzenesulfonamide

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Author information

Affiliations

  1. Chemical Library Validation Team, Chemical Biology Core Facility, Chemical Biology Department, RIKEN Advanced Science Institute, Wako, Saitama, Japan.

    • Isao Miyazaki,
    • Siro Simizu,
    • Hideo Okumura,
    • Satoshi Takagi &
    • Hiroyuki Osada

Contributions

I.M., S.S. and H. Osada designed the research; I.M., H. Okumura and S.T. performed the research; and I.M., S.S., H. Okumura and H. Osada wrote the manuscript.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

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    Supplementary Methods, Supplementary Figures 1–9 & Supplementary Tables 1–3

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