XPB, a subunit of TFIIH, is a target of the natural product triptolide

Journal name:
Nature Chemical Biology
Volume:
7,
Pages:
182–188
Year published:
DOI:
doi:10.1038/nchembio.522
Received
Accepted
Published online

Abstract

Triptolide (1) is a structurally unique diterpene triepoxide isolated from a traditional Chinese medicinal plant with anti-inflammatory, immunosuppressive, contraceptive and antitumor activities. Its molecular mechanism of action, however, has remained largely elusive to date. We report that triptolide covalently binds to human XPB (also known as ERCC3), a subunit of the transcription factor TFIIH, and inhibits its DNA-dependent ATPase activity, which leads to the inhibition of RNA polymerase II–mediated transcription and likely nucleotide excision repair. The identification of XPB as the target of triptolide accounts for the majority of the known biological activities of triptolide. These findings also suggest that triptolide can serve as a new molecular probe for studying transcription and, potentially, as a new type of anticancer agent through inhibition of the ATPase activity of XPB.

At a glance

Figures

  1. Triptolide is a new type of inhibitor of RNAPII-mediated transcription.
    Figure 1: Triptolide is a new type of inhibitor of RNAPII-mediated transcription.

    (a) Chemical structure of triptolide. (b) Inhibition of protein, RNA and DNA synthesis after 1-h treatment with triptolide and a 1-h pulse with [32S]-methionine, [3H]-uridine or [3H]-thymidine. Mean values ± s.e.m. (error bars) from three independent experiments are shown. IC50 value is listed next to corresponding curves ± s.e.m. (c) Western blot analysis of the degradation and dephosphorylation of RNA polymerase II largest subunit after treatment of HeLa cells with different transcription inhibitors. Upper band is phosphorylated RNAPII. (d,e) In vitro transcription assays using HeLa nuclear extracts show that triptolide selectively inhibits RNA synthesis driven by RNA polymerase II promoter. Relative transcription activity was quantified with a PhosphorImager. (f) Effects of triptolide and α-amanitin on transcription of a tailed template with purified calf RNA polymerase II. Abbreviations: TPL, triptolide; α-Aman, α-amanitin; ActD, actinomycin D.

  2. Triptolide inhibits TFIIH-dependent basal transcription and nucleotide excision repair.
    Figure 2: Triptolide inhibits TFIIH-dependent basal transcription and nucleotide excision repair.

    (a) In vitro transcription assays with purified or recombinant basal transcription factors on linear and supercoiled templates in the absence and presence of 10 μM triptolide. (b) Effect of triptolide (3.3 μM) on in vitro transcription from a bubbled template. (c) Inhibition of TFIIH- and TFIIE-dependent enhancement of abortive initiation by triptolide (3.3 μM). (d) Inhibition of nucleotide excision repair by triptolide. A representative sequencing gel is shown. See Supplementary Figure 7 for quantitation.

  3. Triptolide inhibits the DNA-dependent ATPase activity of TFIIH without affecting its DNA helicase activity.
    Figure 3: Triptolide inhibits the DNA-dependent ATPase activity of TFIIH without affecting its DNA helicase activity.

    (a) Effect of triptolide on the two helicase activities of TFIIH in a bidirectional helicase assay with the holo TFIIH complex. (b,c) Inhibition of DNA-dependent ATPase of the holo (B) and core (C) complex of TFIIH (1 nM) by triptolide. 100% activity corresponds to hydrolysis of 14.3% of ATP (1 μM starting concentration) for core TFIIH and 27.8% for holo TFIIH. Relative ATPase activity was quantified with a PhosphorImager.

  4. Triptolide binds covalently to XPB subunit of TFIIH and correlation between inhibition of TFIIH ATPase activity and inhibition of cell proliferation by triptolide analogs.
    Figure 4: Triptolide binds covalently to XPB subunit of TFIIH and correlation between inhibition of TFIIH ATPase activity and inhibition of cell proliferation by triptolide analogs.

    (a) [3H]-triptolide binds covalently and selectively to a 90-kDa protein in HeLa nuclear extract that can be immunoprecipitated by anti-XPB antibody. (b) The 90-kDa protein labeled by [3H]-triptolide comigrates with XPB on SDS-PAGE gels. (c) [3H]-triptolide binds covalently to recombinant his-XPB which can be competed away with excess unlabeled triptolide. (d) Triptolide inhibits DNA-dependent ATPase activity of recombinant his-XPB (100 nM). 100% activity corresponds to hydrolysis of 23% of ATP (1 μM starting concentration). Relative ATPase activity was quantified with a PhosphorImager. (e) Correlation between inhibition of TFIIH ATPase activity and inhibition of cell proliferation by analogs of triptolide. Data points were fitted to the linear equation y = 1.0691x + 1.55 with R2 = 0.95184. Correlation coefficient (r) is calculated using the CORREL function in Excel.

Compounds

11 compounds View all compounds
  1. Triptolide
    Compound 1 Triptolide
  2. 14-epi-Triptolide
    Compound 2 14-epi-Triptolide
  3. 5α-Hydroxytriptolide
    Compound 3 5α-Hydroxytriptolide
  4. Triptonide
    Compound 4 Triptonide
  5. 5α-Hydroxytriptonide
    Compound 5 5α-Hydroxytriptonide
  6. 14-(1H-Imidazole-1-carbothioyl)triptolide
    Compound 7 14-(1H-Imidazole-1-carbothioyl)triptolide
  7. 14-Deoxy-14α-fluorotriptolide
    Compound 8 14-Deoxy-14α-fluorotriptolide
  8. (14S)-Triptolide-14-spiro-1'-oxirane
    Compound 9 (14S)-Triptolide-14-spiro-1'-oxirane
  9. (14S)-5α-Hydroxytriptolide-14-spiro-1'-oxirane
    Compound 10 (14S)-5α-Hydroxytriptolide-14-spiro-1'-oxirane
  10. (14S)-12-Deoxy-12β-chloro-13α-hydroxytriptolide-14-spiro-1'-oxirane
    Compound 11 (14S)-12-Deoxy-12β-chloro-13α-hydroxytriptolide-14-spiro-1'-oxirane
  11. 7-Deoxy-8β-hydroxytriptolide
    Compound 12 7-Deoxy-8β-hydroxytriptolide

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Author information

Affiliations

  1. Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

    • Denis V Titov,
    • Qing-Li He,
    • Shridhar Bhat,
    • Woon-Kai Low,
    • Yongjun Dang &
    • Jun O Liu
  2. Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado, USA.

    • Benjamin Gilman,
    • Jennifer F Kugel &
    • James A Goodrich
  3. Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, USA.

    • Michael Smeaton &
    • Paul S Miller
  4. Charles A. Dana Research Institute for Scientists Emeriti, Drew University, Madison, New Jersey, USA.

    • Arnold L Demain
  5. Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

    • Jun O Liu
  6. Present: Department of Pharmaceutical Sciences, College of Pharmacy, St. John's University, Queens, New York, USA (W.-K.L.).

    • Woon-Kai Low

Contributions

D.V.T., J.A.G., P.S.M. and J.O.L. designed the experiments. D.V.T., B.G., Q.-L.H., S.B., W.-K.L. and M.S. performed the experiments. W.-K.L., A.L.D., P.S.M., J.F.K., Y.D. and J.A.G. contributed reagents. D.V.T. and J.O.L. wrote the manuscript.

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The authors declare no competing financial interests.

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    Supplementary Methods and Supplementary Figures 1–14.

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