xpb, a subunit of tfiih, is a target of the natural …...2011/01/30 · pressive activities....

182 NATURE CHEMICAL BIOLOGY | VOL 7 | MARCH 2011 | www.nature.com/naturechemicalbiology

ARTICLEPUBLISHED ONLINE: 30 JANUARY 2011 | DOI: 10.1038/NCHEMBIO.522

Natural products have played an important role in the discov-ery and development of drugs1. In recent years, they have also become important molecular probes for studying different

cellular processes by virtue of their ability to bind to specific protein targets and interfere with their cellular functions. For example, the identification of calcineurin as the target of the immunosuppressive drugs cyclosporine A and FK506 (ref. 2) and of TOR as the target of rapamycin3 opened the gateways to the subsequent studies of calcium-calcineurin and TOR signaling pathways, respectively. More recently, we and others identified the type 2 methionine aminopeptidase as the target for the potent angiogenesis inhibitors fumagillin and ovalicin4 and the eukaryotic translation initiation factor 4A as the target for the marine sponge–derived antitumor natural product pateamine A5. These studies have led to the exploitation of MetAP2 and eIF4A1 as new molecular targets for discovering and developing new antiangio-genic and anticancer drugs, respectively. Thus, natural products can serve as bridges between chemistry, biology and medicine, constitut-ing a major tool set for chemical biologists today. Elucidation of the mechanisms of action of natural products not only offers new insights into the cellular functions of their protein targets but also facilitates the ensuing use of natural products as leads in drug development.

Triptolide6 is a diterpene triepoxide purified from Tripterygium wilfordii Hook F, commonly known as lei gong teng or thunder god vine, a medicinal plant whose extracts have been used in tradi-tional Chinese medicine for treating a wide variety of diseases from inflammation to arthritis for centuries7. It is structurally distinct in that it contains three epoxide groups next to each other (Fig. 1a). It also possesses a unique profile of biological activities. Triptolide has been shown to have potent antiproliferative and immunosup-pressive activities. Preclinical studies have revealed that triptolide is effective against cancer, collagen-induced arthritis, skin allograft rejection and bone marrow transplantation in animal models8–10. Triptolide and derivatives have entered human clinical trials for cancer, among other diseases11.

Extensive scrutiny of its mechanism of action in the past few decades has yielded important insights. At the cellular level, triptolide shows strong antiproliferative activity, inhibiting the proliferation of all 60 US National Cancer Institute cancer cell lines with half-maximal inhibitory concentration (IC50) values in the low nanomolar range (average IC50 = 12 nM). It also induces apoptosis in a number of cancer cell lines. At the molecular level, triptolide was shown to interfere with a number of transcription factors including NF- B, p53, NF-AT and HSF-1 (refs. 12–14). An interesting common feature of the effects of triptolide on all those transcription factors is that it seems to block their trans-activation activity without affecting DNA binding. More recently, it was shown that triptolide inhibits de novo RNA synthesis, which was suggested to be because of indirect inhibition of tran-scription mediated by RNA polymerases I and II (refs. 15–18). Attempts to isolate the molecular target of triptolide have led to the identification of a calcium channel polycystin-2 and detec-tion of a 90-kDa nuclear protein as potential molecular targets of triptolide15,19. However, polycystin-2 cannot account for most of the aforementioned biological activities of triptolide, and the identity of the 90-kDa putative nuclear triptolide-binding pro-tein has remained unknown.

In this study, we took a ‘top-down’ approach to identifying the molecular target of triptolide. Taking advantage of the extensive prior knowledge of eukaryotic transcription initiation, we system-atically examined the effects of triptolide on different steps and players involved and eventually identified the molecular target of triptolide as the XPB subunit of the general transcription factor TFIIH. Inhibition of XPB by triptolide offers a unified molecular mechanism for the diverse biological activities of triptolide. Our preliminary evidence also revealed a previously unknown activity of triptolide: inhibition of nucleotide excision repair, which has important implications in the application of triptolide for the treatment of cancer.

1Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. 2Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado, USA. 3Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, USA. 4Charles A. Dana Research Institute for Scientists Emeriti, Drew University, Madison, New Jersey, USA. 5Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. 6Present: Department of Pharmaceutical Sciences, College of Pharmacy, St. John’s University, Queens, New York, USA (W.-K.L.). *e-mail: [email protected]

XPB, a subunit of TFIIH, is a target of the natural product triptolideDenis V Titov1, Benjamin Gilman2, Qing-Li He1, Shridhar Bhat1, Woon-Kai Low1,6, Yongjun Dang1, Michael Smeaton3, Arnold L Demain4, Paul S Miller3, Jennifer F Kugel2, James A Goodrich2 & Jun O Liu1,5*

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.

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NATURE CHEMICAL BIOLOGY | VOL 7 | MARCH 2011 | www.nature.com/naturechemicalbiology 183

ARTICLENATURE CHEMICAL BIOLOGY DOI: 10.1038/NCHEMBIO.522

RESULTSTriptolide inhibits RNAPII-dependent transcriptionTo determine whether triptolide has a specific effect on transcription, we examined its effects on global protein, RNA synthesis and DNA synthesis using incorporation of [35S]-methionine, [3H]- uridine and [3H]-thymidine as readouts, respectively. Treatment of HeLa cells with triptolide for 1 h led to a dose-dependent inhibition of [3H]-uridine incorporation with an IC50 of 109 nM, which is in agreement with previous results in A549 cells16 (Fig. 1b). In contrast, protein and DNA synthesis were not affected under these conditions (Fig. 1b). Inhibition of protein and DNA synthesis required longer incubation with triptolide. Notably, the IC50 value for the inhibition of HeLa cell proliferation by triptolide is significantly lower than 109 nM (IC50 = 5 nM, Supplementary Results and Supplementary Fig. 1). It turned out that the effects of triptolide on RNA synthesis and cell proliferation were time dependent, and the apparent difference in IC50 values was due to different drug treatment times for these assays. An increase in triptolide treatment time from 1 h to 3 h led to a 50% decrease in IC50 for [3H]-uridine incorporation (from 109 nM to 62 nM, Fig. 1b and Supplementary Figs. 2 and 3). Similarly, an increase in treatment from 1 h to 3 h to 24 h led to a progressive decrease in IC50 for [3H]-thymidine incorporation (1 h, no effect; 3 h, 41 nM; 24 h, 12 nM. see Supplementary Fig. 4). Thus, temporally, triptolide showed earlier inhibition of RNA synthesis. Nevertheless, the IC50 values for inhibition of RNA synthesis (62 nM) and DNA synthesis as a measure of cell proliferation (41 nM) are similar after 3-h treatment (Supplementary Figs. 3 and 4).

We next sought to compare the effects of triptolide with those of known inhibitors of RNA synthesis. In comparison with three other transcription inhibitors, -amanitin (RNAPII inhibitor)20, fla-vopiridol (a CDK9 inhibitor)21 and actinomycin D (a DNA-binding ligand)22, the dose-response curve for triptolide is similar to that of flavopiridol in that it levels off at the higher concentration range with the maximal inhibition staying at around 80% (90% for flavopiri-dol), suggesting that, unlike actinomycin D, it does not inhibit the synthesis of all RNA species in cells (Supplementary Figs. 2 and 3). As expected, methionine incorporation was not significantly inhib-ited by any of those compounds (Supplementary Figs. 5 and 6). Notably, -amanitin was unable to inhibit RNA synthesis until its concentration reached 50 M, which was unexpected, consider-ing that it has subnanomolar Kd for RNAPII and its lack of cellular activity is likely due to previously reported low cell permeability23. Next, we determined and compared the effects of triptolide on phosphorylation and degradation of the largest subunit of RNA polymerase II with those of other known transcription inhibitors, including 5,6-dichloro-1- -D-ribofuranosylbenzimidazole (DRB) (another CDK9 inhibitor)24, actinomycin D and -amanitin. Unlike

DRB, triptolide had no effect on the phosphorylation of RNAPII, suggesting that it is not an inhibitor of CDK9 (Fig. 1c). As previ-ously reported17, after prolonged treatment (4 h), triptolide induced degradation of the catalytic subunit of RNAPII, similar to the effects of DRB and -amanitin. Prolonged treatment with triptolide also led to faster disappearance of the transcribing-phosphorylated form of RNAPII than nontranscribing-unphosphorylated form, which suggested that it might inhibit transcription initiation or promote transcription termination. The disappearance of the phosphorylated RNAPII was previously attributed to Cdk9 inhibition, which is not the case, judging from the kinetics and magnitude of this effect com-pared to DRB25. In contrast, actinomycin D had no obvious effect on the stability of RNAPII and seemed to shift the equilibrium toward the phosphorylated form compared to control (Fig. 1c). Together, these results suggested that the effect of triptolide on RNAPII is distinct from those of Cdk9 inhibitors (DRB and flavopiridol) and actinomycin D but similar to that of -amanitin.

We next determined the effect of triptolide on transcription mediated by all three RNA polymerases in vitro. Triptolide inhib-ited RNAPII-mediated transcription in a dose-dependent manner with an IC50 of about 200 nM (Fig. 1d). Using the same in vitro transcription assay with templates under the control of RNAPI and RNAPIII specific promoters, we found that triptolide had no effect on RNAPI- or RNAPIII-dependent transcription at concentrations up to 10 M, suggesting that triptolide is a selective inhibitor of the RNA polymerase II transcription machinery (Fig. 1e). To deter-mine whether triptolide works by directly binding to and inhibit-ing RNAPII catalytic activity like -amanitin, we used purified calf RNAPII to direct the RNA synthesis from a DNA template with a poly(dC) overhang in the absence of general transcription factors26. As previously reported17, triptolide had no effect on the catalytic activity of purified RNAPII (Fig. 1f), ruling out the possibility that triptolide shares the same molecular target as -amanitin and sug-gesting that triptolide targets one of the general transcription factors associated with RNAPII.

Triptolide inhibits TFIIH-dependent transcription and repairTo identify which general transcription factor is targeted by trip-tolide, we began by determining the effect of triptolide in a recon-stituted in vitro transcription assay with purified or recombinant general transcription factors and RNAPII (ref. 27). Triptolide inhibited transcription from a linear template with the adenovi-rus major late promoter (−53 to +10) fused to a 380-basepair (bp) G-less cassette in the presence of TFIIB, TBP, TFIIH, TFIIE, TFIIF and RNAPII (ref. 28) (Fig. 2a, lane 2 versus 1 and lane 4 versus 3). Surprisingly, transcription from a negatively supercoiled template was refractory to inhibition by triptolide (Fig. 2a, lane 8 versus 7).

100

80

60

[3 H/35

S]nu

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atio

n, %

Triptolide (1)

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Uridine

109 8 nM

DMSODMSO DMSO

200 nM TPL

500 nM ActD50 M DRB

40 M -Aman

400 M -Aman10 M TPL

10 M TPL

1,000 bp

300 bp400 bp600 bp800 bp

1 M -Aman10 M-Aman

Thymidine

10–11

[TPL] (M)10–410–510–610–710–810–910–10

Methionine

Tubulin

RNAPll

RNAPlllRNAPl RNAPll

––––

– – – – – – – ––––––––

––– –

–– – – – –

–– –

–– – – – – – –

––––––

– –

––– –– – – – ––

–– – – – –

–––

–

––– –

–––––– – – – –++

++

+ ++

++

+

++

++ +

++

+

++

+

++

++

4 h DMSO 30 100TPL (nM)

1,000 3,000300

1.00 0.80 0.62Relative transcription activity

0.020.100.36

1 h

ba c d e f

O

OH

O

O

O

OH

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 -amanitin on transcription of a tailed template with purified calf RNA polymerase II. Abbreviations: TPL, triptolide; -Aman, -amanitin; ActD, actinomycin D.

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ARTICLE NATURE CHEMICAL BIOLOGY DOI: 10.1038/NCHEMBIO.522

It is known that transcription from supercoiled templates can bypass the need for TFIIH and TFIIE, suggesting that triptolide might inhibit the function of TFIIH or TFIIE27. In addition, it has been previously shown that a linear template containing a preexisting bubble at the transcription start site with mismatched sequence from −9 to +3 of the AdMLP can also undergo transcription initiation independent of TFIIH and TFIIE29, much as with a negatively supercoiled tem-plate. Indeed, transcription from this bubble- containing template is likewise insensitive to triptolide and to TFIIH and TFIIE (Fig. 2b). To further dissect the site of action of triptolide on transcription, we exploited the ability of transcription initiation complexes to synthesize multiple copies of 3mers in a process known as abortive initiation30. Abortive initiation can occur in the absence of TFIIH and TFIIE but is strongly stimulated by them. Although triptolide had no effect on basal abortive initiation in the absence of TFIIH and TFIIE, it inhibited the enhancement of abortive initiation by TFIIH and TFIIE (Fig. 2c). Together, these results narrowed down the target for triptolide to either TFIIH or TFIIE.

Although TFIIH and TFIIE work in concert to enable RNAPII-mediated transcription, TFIIH, unlike TFIIE, is also required for nucleotide excision repair (NER)31. We thus determined whether triptolide affected NER. We took advantage of the ability of HeLa whole-cell extract to support NER in vitro. An oligonucleotide containing one cisplatin-damaged site with a 32P label 5 to the site was incubated with HeLa cell extract, leading to accumula-tion of 22- to 25-nucleiotide–long excised fragments as a result of the NER activity (Fig. 2d and Supplementary Fig. 7). Triptolide inhibited the NER activity at concentrations similar to those required for in vitro transcription. Given the dependence of NER on TFIIH, this result suggested that TFIIH, but not TFIIE, is likely the target of triptolide.

Triptolide binds to and inhibits ATPase activity of XPBTFIIH contains a total of ten subunits, four of which have detect-able enzymatic activities. Cdk7 possesses RNAPII kinase activity, both XPB and XPD have DNA helicase and ATPase activities and p44 has ubiquitin ligase activity32. As triptolide had no effect on the phosphorylation of the catalytic subunit of RNAPII (Fig. 1c), Cdk7 was ruled out as a target. Ubiquitin ligase activity of p44 was ruled out because it was shown to not be important for either in vitro transcription or NER32. We then determined whether

triptolide affected the DNA helicase and ATPase activities of XPB and XPD. Using immunoaffinity-purified TFIIH holocomplex con-taining all ten subunits (Supplementary Fig. 8), we were able to observe both the 3 -5 DNA helicase activity associated with XPB and the 5 -3 helicase activity associated with XPD. Neither helicase activity of TFIIH was affected by triptolide (Fig. 3a). In contrast, triptolide inhibited the DNA-dependent ATPase activity of the TFIIH holocomplex at concentrations similar to those required for in vivo [3H]-uridine incorporation, in vitro transcription and in vitro NER assays (Fig. 3b and Supplementary Fig. 9). To further deter-mine whether triptolide inhibited the ATPase activity arising from XPB or XPD subunit of TFIIH, we purified the TFIIH core complex that is devoid of the XPD subunit and used it in the ATPase activity assay in the absence and presence of triptolide33 (Supplementary Fig. 8). Similar to that of the holocomplex, the DNA-dependent ATPase activity of the core complex of TFIIH is also inhibited by triptolide at similar concentrations (Fig. 3c and Supplementary Fig. 10), suggesting that XPB is the molecular target for triptolide. It is noteworthy that triptolide selectively inhibited the ATPase activity of both the core and holo-TFIIH complexes without affect-ing the DNA helicase activity.

It was previously reported that triptolide binds covalently to a 90-kDa protein, and coincidentally, this molecular weight is closest to that of XPB among all the subunits of TFIIH15. We thus deter-mined whether triptolide binds to XPB. HeLa cell nuclear extract was incubated with [3H]-triptolide and then immunoprecipitated with an anti-XPB antibody. The protein covalently labeled by [3H]-triptolide is quantitatively immunoprecipitated by the anti-XPB anti-body with no labeled protein remaining in the supernatant (Fig. 4a, lane 3 versus 1). The binding of [3H]-triptolide to the protein was sensitive to competition by excess unlabeled triptolide (Fig. 4a, lane 3 versus 4). Western blot analysis revealed that under those condi-tions XPB was nearly quantitatively pulled down from the cell lysate by the anti-XPB antibody (Supplementary Fig. 11), suggesting that XPB is the triptolide-binding protein. To further confirm that XPB is the 90-kDa triptolide-binding protein, we immunoprecipi-tated TFIIH complex after labeling the nuclear extract with [3H]-triptolide and then performed 5% SDS-PAGE in duplicate. One gel

TBP:a

b

c

dTFIID, TFIIA:TFIIE, TFIIH:

TFIIE, TFIIH:

TPL:

TPL (µM)WCE

TPL:

+ + + + + ++ + + +

0.1 0.3 1 3+ + + + +

+++++++

+ ++ +

++– –

– –– – –

–

–– –

–

– ––

– – ––

––Negatively supercoiled plasmidLinear DNA

1 2 3 4 5 6 7 8 9 10 11 12

––––

+ + ++

+++ +– – – –

– – – –

TFIIE, TFIIH:TPL:

++ +

++++– – –

+–

– – – –

+ + +

NERexcision

products

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 M triptolide. (b) Effect of triptolide (3.3 M) on in vitro transcription from a bubbled template. (c) Inhibition of TFIIH- and TFIIE-dependent enhancement of abortive initiation by triptolide (3.3 M). (d) Inhibition of nucleotide excision repair by triptolide. A representative sequencing gel is shown. See Supplementary Figure 7 for quantitation.

TFIIH

a b

c

10 M TPL

––– – – –

––– + +

No protein

No DNA

DMSO

++

TPL ( M)1.0 10.03.00.30.1

No protein

No DNA

DMSO TPL ( M)1.0 10.03.00.30.1

5 -3 (XPD)

3 -5 (XPB)

12 31 100 2022284667

7 19Relative ATPase activity (%)

Relative ATPase activity (%)

2332527410017

++

ATP

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 M starting concentration) for core TFIIH and 27.8% for holo TFIIH. Relative ATPase activity was quantified with a PhosphorImager.

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was stained with silver to visualize the XPB, XPD and p62 subunits of TFIIH whereas the second, identical gel, was subjected to fluo-rography. The 90-kDa band labeled by [3H]-triptolide had the same gel mobility as the XPB subunit (Fig. 4b). Lastly, we overexpressed and purified recombinant XPB from baculovirus-driven insect cells (Supplementary Fig. 12) and determined whether it is capable of binding to triptolide and whether its DNA-dependent ATPase activ-ity is inhibited by triptolide. The recombinant XPB also formed a covalent complex with [3H]-triptolide, and the binding was sensi-tive to competition by unlabeled triptolide (Fig. 4c), clearly dem-onstrating that XPB is the direct target of triptolide. Moreover, when we performed the ATPase assay, although the intrinsic DNA-dependent ATPase activity of purified XPB is much lower than that of the TFIIH complex, the residual ATPase activity was inhibited by triptolide in a dose-dependent manner (Fig. 4d and Supplementary Fig. 13). It took a much higher concentration of triptolide to inhibit the ATPase activity of purified XPB than that in the TFIIH complex, which suggests that other subunits of TFIIH may be involved in the binding of triptolide to XPB.

Correlating XPB ATPase and cell proliferation inhibitionTo further confirm that the antiproliferative effect of triptolide is indeed mediated by inhibition of XPB ATPase activity, we synthe-sized twelve analogs of triptolide with reported IC50 for inhibition of cell proliferation spanning three orders of magnitude34–37. We then determined their activities in the ATPase assay and HeLa cell proliferation assay, respectively (Table 1). There is a significant cor-relation (r = 0.98) between IC50 values of the analogs for inhibition of TFIIH ATPase activity and cell proliferation, offering further evidence that triptolide inhibits cell proliferation by blocking XPB DNA-dependent ATPase activity (Fig. 4e).

DISCUSSIONSince triptolide was first identified in 1972 (ref. 6), its mechanism of action has remained a mystery. In this study, we identified the XPB subunit of TFIIH as a key target of triptolide. The covalent binding of triptolide to XPB and the consequent inhibition of the ATPase activity of XPB offers a unified and coherent mechanism that can account for the majority of the known cellular and physiological activities of triptolide reported to date. Thus, the effect of triptolide on the activity of a number of transcription factors from NF- B to p53 and from AP-1 to HSF-1 at the transactivation step can be explained by the inhibition of XPB and TFIIH activity; inhibition of XPB and TFIIH blocks RNAPII-mediated transcription initia-tion and hence the transactivation by all those transcription factors.

This general inhibition of RNAPII-mediated transcription may underlie the inhibition of T cell activation and proliferation of nearly all cancer cell lines, thus explaining the anti-inflammatory, immu-nosuppressive and antiproliferative activity of triptolide. Given the essential role of RNAPII-mediated transcription in cell growth and survival, it is possible that inhibition of XPB ATPase activity by trip-tolide may also be an underlying cause of its toxicity.

The effects of triptolide on the ATPase activity of XPB and PolII-mediated transcription are consistent with previous find-ings on the inhibitory activity of the drug on the transactivation of several transcription factors16. More recently, effects of trip-tolide on RNA synthesis were further investigated using multiple approaches17. Triptolide was found to have an inhibitory effect on the majority of mRNA species, in agreement with an effect on PolII-mediated transcription. Using microarrays, it was shown that expression of over 4,400 genes was affected by triptolide, and the majority of these genes were downregulated. It was concluded that triptolide predominantly downregulated the transcription of short-lived mRNA17. As microarray only detects steady levels of RNA, the observed perturbation by triptolide resulted from a combination of effects on both de novo transcription and mRNA degradation. As such, inhibition of XPB and PolII-mediated tran-scriptional initiation can explain the inhibitory effect of triptolide on the expression levels of short-lived mRNA. Additional experi-ments to examine the effect of triptolide on the association of PolII and TFIIH with the different loci throughout the genome will be needed to further verify the relevance of its interaction with XPB to its cellular activity.

Two putative triptolide targets have been previously reported. One is a 90-kDa unknown nuclear protein that forms a covalent complex with [3H]-triptolide15, and the other is the calcium channel polycystin-2 (ref. 19). Our results indicate that XPB is the 90-kDa putative triptolide-binding protein. The possibility cannot be ruled out that triptolide also directly binds to polycystin-2, regulating its channel activity19. If that proves to be true, however, it would be extra ordinary that the relatively small triptolide is capable of binding to two structurally unrelated proteins, a DNA-dependent ATPase and a transmembrane ion channel. Although inhibition of XPB can account for most of the known biological activities of trip-tolide, the effect of triptolide on polycystin-2 seems to be unrelated to those activities. It will be interesting to determine whether the effect of triptolide on polycystin-2 is indirect and secondary to the inhibition of transcription.

We were surprised to observe that triptolide inhibited more than 80% of uridine incorporation in cells without inhibiting RNAPI

a b c d e1 µM [3H]-TPL

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Silver staining

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His-XPBBSA

p62Fluorography

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DMSO1 3 10 30 100

TPL (µM)

10 35 100 88 78 68 57 50Relative ATPase activity (%)

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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 M 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.

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activity in vitro, as it is known that more than 90% of RNA inside the cell is rRNA. Notably, two other RNAPII-specific inhibitors, fla-vopiridol and -amanitin (in permeabilized cells), have the same effect on uridine incorporation23. The existence of three different inhibitors of RNAPII machinery that inhibited the majority of uri-dine incorporation suggests that either RNAPII is responsible for the synthesis of the majority of RNA inside the cell or that RNAPI-mediated rRNA synthesis is also dependent on RNAPII activity, potentially through the regulated expression of some key compo-nent of RNAPI-mediated transcription.

As a key component of the multiprotein TFIIH complex, XPB has been shown to possess at least two distinct enzymatic activi-ties in vitro: ATP-dependent DNA helicase activity and DNA-dependent ATPase activity. To our surprise, we found that triptolide only inhibited the ATPase activity of XPB in the core- and holocom-plexes of TFIIH without affecting its conventional DNA helicase activity. Although the helicase activity of XPB has been thought

to be important in opening double-stranded DNA to facilitate transcription initiation by RNAPII, this notion was called into question when it was observed that TFIIH appeared to work as an ATP-dependent DNA-unwinding machine rather than a conven-tional DNA helicase that would promote promoter melting during transcription initiation38. The selective inhibition of the ATPase activity of XPB by triptolide offers new evidence in support of the latter model.

Triptolide is structurally distinct in that it contains multiple reac-tive functional groups, an , -unsaturated lactone, which can serve as a Michael acceptor, and three consecutive epoxide groups. It is thus not surprising that it forms a covalent complex with XPB that can survive the denaturing and reducing conditions of gel electro-phoresis. This is reminiscent of the natural products fumagillin and ovalicin, which specifically interact with MetAP2 despite the pres-ence of two potentially reactive epoxide groups4. It seems that nature has elaborated natural products containing reactive chemical groups

Table 1 | Bioactivities of triptolide (1) and its analogs

Entry Structure

IC50 s.e.m. (mM)

Entry Structure

IC50 s.e.m. (mM)

ATPase activity

HeLa proliferation

ATPase activity

HeLa proliferation

1

O

OH

O

O

O

OH

0.145 ( 0.01)

0.00459 ( 0.001)

7

O

OH

O

O

O

O

S N

N

1.44 ( 0.08)

0.017 ( 0.007)

2

O

OH

O

O

O

OH

1.32 ( 0.03)

0.0451 ( 0.01)

8

O

OH

O

O

O

F

0.475 ( 0.019)

0.0143 ( 0.004)

3

O

OOH

O

O

O

OH

2.9 ( 0.11)

0.0913 ( 0.021)

9

O

OH

O

O

O

O

2.07 ( 0.2)

0.0196 ( 0.0041)

4

O

OH

O

O

O

O

0.156 ( 0.024)

0.00545 ( 0.001)

10

O

OOH

O

O

O

O

51.4 ( 3.0)

0.935 ( 0.406)

5

O

OOH

O

O

O

O

6.32 ( 0.23)

0.175 ( 0.024)

11

O

OH

O

O

OH

O

Cl 4.19 ( 0.2)

0.0646 ( 0.0094)

6

O

OH

O

O

O

O

OH106

( 8)1.50

( 0.29)12

O

OH

OH

O

O

OH

>500 >10© 2

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in such a way that their presence does not compromise the binding specificity of natural products. In a model reaction with propanethiol in vitro, it was previously shown that the 9,11-epoxide was attacked by the thiol, assisted by the neighboring 14 -hydroxyl group, to form a covalent adduct39. It remains to be seen whether a cysteine resi-due in XPB is involved in the formation of the triptolide-XPB cova-lent complex. The presence of multiple epoxides in triptolide also raised the possibility that it may have nonspecific interactions with cysteine-containing proteins in general. However, only a single cova-lent complex was detected when the nuclear lysate was exposed to [3H]-triptolide, suggesting that triptolide is highly specific for XPB. In support of XPB as a physiologically relevant target of triptolide, we found a significant correlation between the antiproliferative activities and the potencies for inhibition of XPB ATPase activity by a num-ber of triptolide analogs with a wide range of activities. Moreover, we found that overexpression of XPB in 293T cells, but not XPD, conferred resistance to triptolide (Supplementary Fig. 14). Despite these lines of supportive evidence, however, we cannot completely rule out the possibility that triptolide may have additional targets with lower abundance but higher affinity that might have eluded detection under the current experimental conditions.

The potent inhibition of XPB and the accompanying RNAPII-mediated transcription makes triptolide a unique and useful molec-ular tool among the existing inhibitors of transcription: it has higher potency and greater cell permeability than -amanitin and greater specificity than either actinomycin D or DRB. A deeper understand-ing of the interaction between triptolide and XPB will facilitate the design of new inhibitors of XPB and other homologous DNA heli-cases as anticancer and antiproliferative drug leads. Moreover, our preliminary evidence also suggested a previously unknown activity for triptolide: inhibition of nucleotide excision repair. Although the effect of triptolide on nucleotide excision repair remains to be dem-onstrated in vivo, it is expected to be found, as the ATPase activity has been shown to be crucial for that activity of TFIIH. The abil-ity of triptolide to block DNA repair has important implications, as its impairment has been shown to be responsible for resistance of cancer cells to certain classes of anticancer drugs. For example, it has been shown that triptolide can enhance the anticancer activity of such DNA-damaging drugs as cisplatin and topoisomerase I inhibitors40,41. It is tempting to speculate that this unique inhibi-tory effect of triptolide on nucleotide excision repair in addition to transcription may be further exploited for the development of new anticancer modalities.

METHODSIn vitro transcription assay. The in vitro transcription assay was conducted as previously described42. HeLa cell nuclear extract was prepared as previously described43. Cytomegalovirus (CMV) promoter from Positive Control DNA (Promega), human rDNA promoter from pETS-RB digested with XhoI44 and adenovirus VAI promoter from pVATK digested with SacI45 were used for RNA polymerase II–, RNA polymerase I– and RNA polymerase III–directed transcrip-tion, respectively. pETS-RB and pVATK were gifts from B. Sollner-Webb.

RNA polymerase II activity on oligo(dC) tailed template. The tailed template assay for RNA polymerase II catalytic activity was performed as previously described26. Purified bovine RNA polymerase II was a gift from A. Gnatt.

In vitro transcription assay with purified or recombinant transcription factors. Recombinant (TBP, TFIIA, TFIIB, TFIIE and TFIIF) and native (TFIID, TFIIH and Pol II) human transcription factors were prepared as previously described46. In vitro transcription on the linear and negatively supercoiled templates containing the adenovirus major late promoter (−53 to +10) fused to a 380-bp G-less cassette was performed as previously described28,46. The heteroduplex template consisted of the adenovirus major late promoter with a mismatched region from −9 to +3 (ref. 29).

Abortive initiation assay. Abortive initiation was performed as previously described30.

In vitro nucleotide excision repair assay. A site-specific 1,3 GTG Pt lesion was placed in the center of a 150mer substrate as previously described47.

The preparation of HeLa whole-cell extracts and the NER excision assay were carried out as previously outlined in detail48.

ATPase assay. The DNA-dependent ATPase assay was performed as previously described49. Briefly, a 10- l reaction mixture contained 20 mM Tris (pH 7.9), 4 mM MgCl2, 1 M ATP, 1 Ci ATP (3,000 Ci mmol−1), 100 g ml−1 BSA, 50 ng RNA polymerase II promoter Positive Control DNA (Promega), 1–2 nM TFIIH or 100 nM his-XPB and indicated concentration of drugs. The reactions were started by either addition of TFIIH/his-XPB or ATP and incubated at 37 °C for 2 h. The reactions were stopped by addition of 2 l 0.5 M EDTA and dilution in up to 100 l with TE buffer. An aliquot of 1 l reaction mixture was spotted on PEI-cellulose, and the chromatogram was developed with 0.5 M LiCl and 1 M HCOOH. The percent of ATP hydrolysis was quantified using a PhosphorImager.

TFIIH helicase assay. The helicase assay was performed as previously described50. Briefly, a 10- l reaction mixture contained 20 mM Tris (pH 7.9), 4 mM MgCl2, 4 mM ATP, 100 g ml−1 BSA, 0.12 nM of M13mp18-based bidirectional helicase substrate and 0.4 nM TFIIH. The reactions were started by either addition of TFIIH/his-XPB or ATP and incubated at 37 °C for 2 h. The reactions were stopped by addition of 5 l of a quenching buffer (60 mM EDTA, 50% (v/v) glycerol, 0.75% (w/v) SDS). An aliq-uot of 10 l of the mixture was loaded on a 10% nondenaturing polyacrylamide gel containing 0.1% SDS and run at 200 V in 0.5x TBE buffer with 0.1% (w/v) SDS. The gel was dried and subjected to quantification using a PhosphorImager.

[3H]-triptolide binding to XPB in HeLa nuclear extract. HeLa nuclear extract ( containing 400 g of total proteins) was incubated with 1 M [3H]-triptolide with or without 50 M unlabeled triptolide in 50 l of 10 mM HEPES (pH 8.0), 50 mM KCl, 5 mM MgCl2, 10% (v/v) glycerol, 0.1 mM EDTA, 0.5 mM DTT and 50 M PMSF for 1 h at 30 °C. Three micrograms of affinity-purified rabbit anti-XPB antibody (A301-337A, Bethyl Laboratories) was added, and the mixture was incubated for an additional 30 min at 25 °C. The mixture was added to 50 l of Dynabeads Protein A (100.01D, Invitrogen, storage solution removed) and further mixed by rotation for 10 min at 25 °C. The supernatant was aspirated, and the beads were washed three times with PBS. The beads were resuspended in 40 l of sample buffer, boiled for 5 min and subjected to SDS-PAGE. After electrophoresis, the gel was soaked in En3hance solution (PerkinElmer) according to the manufacturer’s instructions and exposed to preflashed X-ray film for 2 weeks before autoradiography.

[3H]-triptolide binding to recombinant his-XPB. His-XPB (300 ng) was incubated with 1 M [3H]-triptolide with or without 50 M cold triptolide in 40 l binding buffer (20 mM Tris (pH 7.9), 4 mM MgCl2, 1 M ATP, 100 g ml−1 BSA) and 500 ng RNA polymerase II promoter Positive Control DNA (Promega) for 1 h at 30 °C. Samples were boiled in sample buffer and subjected to 12% SDS-PAGE. After electrophoresis, the gel was soaked in En3hance solution (PerkinElmer) according to manufacturer instructions and exposed to preflashed X-ray film for 2 weeks before development.

Other methods. For the remaining experimental procedures and a more detailed description of the above procedures, see Supplementary Methods.

Received 25 August 2010; accepted 6 January 2011;published online 30 January 2011

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13. Chang, W.T. et al. Triptolide and chemotherapy cooperate in tumor cell apoptosis. A role for the p53 pathway. J. Biol. Chem. 276, 2221–2227 (2001).

14. Westerheide, S.D., Kawahara, T.L., Orton, K. & Morimoto, R.I. Triptolide, an inhibitor of the human heat shock response that enhances stress-induced cell death. J. Biol. Chem. 281, 9616–9622 (2006).

15. McCallum, C. et al. Triptolide binds covalently to a 90 kDa nuclear protein. Role of epoxides in binding and activity. Immunobiology 212, 549–556 (2007).

16. McCallum, C. et al. In vitro versus in vivo effects of triptolide: the role of transcriptional inhibition. Therapy 2, 261–273 (2005).

17. Vispé, S. et al. Triptolide is an inhibitor of RNA polymerase I and II-dependent transcription leading predominantly to down-regulation of short-lived mRNA. Mol. Cancer Ther. 8, 2780–2790 (2009).

18. Pan, J. RNA polymerase—an important molecular target of triptolide in cancer cells. Cancer Lett. 292, 149–152 (2010).

19. Leuenroth, S.J. et al. Triptolide is a traditional Chinese medicine-derived inhibitor of polycystic kidney disease. Proc. Natl. Acad. Sci. USA 104, 4389–4394 (2007).

20. Lindell, T.J., Weinberg, F., Morris, P.W., Roeder, R.G. & Rutter, W.J. Specific inhibition of nuclear RNA polymerase II by alpha-amanitin. Science 170, 447–449 (1970).

21. Chao, S.H. et al. Flavopiridol inhibits P-TEFb and blocks HIV-1 replication. J. Biol. Chem. 275, 28345–28348 (2000).

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25. Leuenroth, S.J. & Crews, C.M. Triptolide-induced transcriptional arrest is associated with changes in nuclear substructure. Cancer Res. 68, 5257–5266 (2008).

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29. Gilman, B., Drullinger, L.F., Kugel, J.F. & Goodrich, J.A. TATA-binding protein and transcription factor IIB induce transcript slipping during early transcription by RNA polymerase II. J. Biol. Chem. 284, 9093–9098 (2009).

30. Kugel, J.F. & Goodrich, J.A. Translocation after synthesis of a four-nucleotide RNA commits RNA polymerase II to promoter escape. Mol. Cell. Biol. 22, 762–773 (2002).

31. Schaeffer, L. et al. DNA repair helicase: a component of BTF2 (TFIIH) basic transcription factor. Science 260, 58–63 (1993).

32. Takagi, Y. et al. Ubiquitin ligase activity of TFIIH and the transcriptional response to DNA damage. Mol. Cell 18, 237–243 (2005).

33. LeRoy, G., Drapkin, R., Weis, L. & Reinberg, D. Immunoaffinity purification of the human multisubunit transcription factor IIH. J. Biol. Chem. 273, 7134–7140 (1998).

34. Aoyagi, Y. et al. Semisynthesis of C-ring modified triptolide analogues and their cytotoxic activities. Bioorg. Med. Chem. Lett. 16, 1947–1949 (2006).

35. Aoyagi, Y. et al. Fluorination of triptolide and its analogues and their cytotoxicity. Bioorg. Med. Chem. Lett. 18, 2459–2463 (2008).

36. Ning, L. et al. Cytotoxic biotransformed products from triptonide by Aspergillus niger. Planta Med. 69, 804–808 (2003).

37. Li, Z. et al. Design and synthesis of novel C14-hydroxyl substituted triptolide derivatives as potential selective antitumor agents. J. Med. Chem. 52, 5115–5123 (2009).

38. Kim, T.K., Ebright, R.H. & Reinberg, D. Mechanism of ATP-dependent promoter melting by transcription factor IIH. Science 288, 1418–1422 (2000).

39. Kupchan, S.M. & Schubert, R.M. Selective alkylation: a biomimetic reaction of the antileukemic triptolides? Science 185, 791–793 (1974).

40. Matsui, Y. et al. Cancer-specific enhancement of cisplatin-induced cytotoxicity with triptolide through an interaction of inactivated glycogen synthase kinase-3beta with p53. Oncogene 27, 4603–4614 (2008).

41. Fidler, J.M. et al. PG490–88, a derivative of triptolide, causes tumor regression and sensitizes tumors to chemotherapy. Mol. Cancer Ther. 2, 855–862 (2003).

42. Dignam, J.D., Lebovitz, R.M. & Roeder, R.G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11, 1475–1489 (1983).

43. Pugh, B.F. Preparation of HeLa nuclear extracts. Methods Mol. Biol. 37, 349–357 (1995).

44. Miesfeld, R. & Arnheim, N. Identification of the in vivo and in vitro origin of transcription in human rDNA. Nucleic Acids Res. 10, 3933–3949 (1982).

45. Sisodia, S.S., Sollner-Webb, B. & Cleveland, D.W. Specificity of RNA maturation pathways: RNAs transcribed by RNA polymerase III are not substrates for splicing or polyadenylation. Mol. Cell. Biol. 7, 3602–3612 (1987).

46. Yakovchuk, P., Gilman, B., Goodrich, J.A. & Kugel, J.F. RNA polymerase II and TAFs undergo a slow isomerization after the polymerase is recruited to promoter-bound TFIID. J. Mol. Biol. 397, 57–68 (2010).

47. Mason, T.M., Smeaton, M.B., Cheung, J.C., Hanakahi, L.A. & Miller, P.S. End modification of a linear DNA duplex enhances NER-mediated excision of an internal Pt(II)-lesion. Bioconjug. Chem. 19, 1064–1070 (2008).

48. Smeaton, M.B., Miller, P.S., Ketner, G. & Hanakahi, L.A. Small-scale extracts for the study of nucleotide excision repair and non-homologous end joining. Nucleic Acids Res. 35, e152 (2007).

49. Conaway, R.C. & Conaway, J.W. An RNA polymerase II transcription factor has an associated DNA-dependent ATPase (dATPase) activity strongly stimulated by the TATA region of promoters. Proc. Natl. Acad. Sci. USA 86, 7356–7360 (1989).

50. Schaeffer, L. et al. The ERCC2/DNA repair protein is associated with the class II BTF2/TFIIH transcription factor. EMBO J. 13, 2388–2392 (1994).

AcknowledgmentsThis work was supported by discretionary funds (J.O.L.). We are grateful to A. Gnatt (University of Maryland) for a kind gift of purified RNAPII and B. Sollner-Webb (Johns Hopkins University) for plasmids. We thank D. Yang for earlier support of this project. We thank P. Cole, J. Corden, J. Stivers and members of the Liu lab for helpful suggestions.

Author contributionsD.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.

Competing financial interestsThe authors declare no competing financial interests.

Additional informationSupplementary information and chemical compound information is available online at http://www.nature.com/naturechemicalbiology/. Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/. Correspondence and requests for materials should be addressed to J.O.L.

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SUPPLEMENTARY INFORMATION

XPB, a Subunit of TFIIH, Is a Target of the Natural Product

Triptolide !

Denis!V.!Titov1,!Benjamin!Gilman2,!Qing8Li!He1,!Shridhar!Bhat1,!Woon8Kai!Low1,†!,Yongjun!Dang1,!Michael!Smeaton3,!Arnold!J.!Demain4,!Paul!S.!Miller3,!Jennifer!F.!Kugel2,!James!A.!

Goodrich2,!Jun!O.!Liu1,!5,*!!!1Department!of!Pharmacology!and!Molecular!Sciences,!5Department!of!Oncology,!The!Johns!Hopkins!University!School!of!Medicine,!Baltimore,!MD!2Department!of!Chemistry!and!Biochemistry,!University!of!Colorado,!Boulder,!CO!3Department!of!Biochemistry!and!Molecular!Biology,!Bloomberg!School!of!Public!Health,!Johns!Hopkins!University,!Baltimore,!MD!4Charles!A!Dana!Research!Institute!for!Scientists!Emeriti!(RISE),!Drew!University,!Madison,!NJ!!!!†Current!address:!Department!of!Pharmaceutical!Sciences,!College!of!Pharmacy,!St.!John’s!University,!Queens,!NY!!!!*Correspondence to: Jun O. Liu ([email protected])

Contents:

1. Supplementary Methods

2. Supplementary Results containing Figure S1-14

3. Original Gel Images of Figures

Nature Chemical Biology: doi:10.1038/nchembio.522

! 2!

Supplementary Methods !!Materials

Actinomycin D, α -amanitine and DRB were purchased from Sigma. Triptolide was purchased

from various commercial sources including Sigma. [3H]-triptolide was prepared by AmBios

Labs, Inc. with specific activity of 4.0 Ci/mmol. Anti-RNAPII antibody (sc-55493), anti-tubulin

antibody (sc-5286), anti-XPD antibody (sc-20696), anti-p52 antibody (sc-81403), anti-XPB

antibody (sc-293) and anti-cdk7 HRP (sc-7344 HRP) were purchased from Santa Cruz.

Methods

Cell proliferation assay. HeLa cells were seeded at 3000 cells per well in 96-well plates and

cultured in DMEM plus 10% FBS at 37 0C with 5% CO2. Twenty four hours after seeding, drugs

were added at indicated concentrations and incubation was continued for an additional 24 h

(unless indicated otherwise). An aliquot of 1 µCi of [3H]-thymidine (Perkin Elmer) was added

per well and incubation was continued for an additional 6 h (unless indicated otherwise). Cells

were harvested onto a Printed Filtermat A glass fiber filter (Perkin Elmer) using Tomtec

Harvester 96 Mach III and filters were immersed in Betaplate Scint (Perkin Elmer) scintillation

fluid, followed by scintillation counting on 1450 Microbeta JET (Perkin Elmer).

[3H]-uridine or [35S]-methionine incorporation assays. HeLa cells (20000 per well) were

seeded in 96-well plates. Twenty four hours after seeding, drugs were added at the indicated

concentrations and incubation was continued for 1 h. An aliquot of 1 µCi of [3H]-uridine or

[35S]-methionine (Perkin Elmer) was added per well and incubated for an additional 1 h. Cells


! 3!

were washed twice with 200 µl of cold PBS and lysed by addition of 100 µl lysis buffer [20 mM

Tris-HCl, pH 7.4, 1% SDS, 100 µg/ml yeast tRNA (for [3H]-uridine incorporation) or 25 mM

Tris-HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% deoxycholate, 0.1% SDS, 100 µg/ml BSA (for

[35S]-methionine incorporation)]. After a 20-minute incubation at 4 0C, 100 µl of 20%

trichloroacetic acid (TCA) was added and the resulting mixtures were transferred to Millipore

MSFCN6B10 filter plates. After 2 hours incubation at 4 0C, the mixtures were filtered under

vacuum, and the plates were washed twice with 200 µl 5% TCA and twice with 200 µl ethanol.

The plates were dried overnight, and 40 µl of Optiphase Supermix was added to each well and

incorporation of 3H or 35S was quantified by scintillation counting on 1450 Microbeta JET

instrument (Perkin Elmer).

In vitro transcription assay. HeLa cells nuclear extract was prepared as previously described 1.

A HeLa S3 cell pellet was purchased from National Cell Culture Center. Cytomegalovirus

(CMV) promoter from Positive Control DNA (Promega), human rDNA promoter from pETS-RB

digested with SalI 2 and adenovirus VAI promoter from pVATK digested with SacI 3 were used

for RNA polymerase II, RNA polymerase I and RNA polymerase III directed transcription,

respectively. pETS-RB and pVATK were gifts from Dr. Barbara Sollner-Webb (Johns Hopkins

University). The in vitro transcription assay was conducted as previously described 4. Briefly, a

25 µl reaction mixture containing 10 mM HEPES (pH 7.9), 50 mM KCl, 5 mM MgCl2, 0.1 mM

EDTA, 0.25 mM DTT, 10% Glycerol, 0.4 mM ATP, 0.4 mM rCTP, 0.4 mM UTP, 16 µM rGTP,

10 µCi rGTP (3000Ci/mmol), 70 µg HeLa nuclear extract proteins, 100-200 ng of RNA

polymerase promoters and indicated concentrations of drugs was preincubated for a 10-minute at

25 0C. The reaction was started by addition of ribonucleotides and allowed to proceed for 1 h at


! 4!

30 0C. The reaction was stopped and RNA was purified by extraction with phenol-chloroform-

isoamyl alcohol followed by ethanol precipitation. RNA samples were dissolved in 98%

formamide and electrophoresed on a 6% denaturing polyacrylamide gel. The gel was dried and

RNA products were quantified using a PhosphorImager.

RNA polymerase II activity on oligo(dC) tailed template. The tailed template assay for RNA

polymerase II catalytic activity was performed as previously described 5. Purified bovine RNA

polymerase II was a gift from Dr. Averell Gnatt (University of Maryland).

In vitro transcription assay with purified or recombinant transcription factors.

Recombinant (TBP, TFIIA, TFIIB, TFIIE, and TFIIF) and native (TFIID, TFIIH and Pol II)

human transcription factors were prepared as previously described 6. In vitro transcription on the

linear and negatively supercoiled templates containing the Adenovirus major late promoter (-53

to +10) fused to a 380 basepair G-less cassette was performed as previously described 6,7. The

heteroduplex template consisted of the Adenovirus major late promoter with a mismatched

region from -9 to +3 8.

Abortive initiation assay. Abortive initiation was performed as previously described 9.

In vitro Nucleotide Excision Repair assay. A site specific 1,3 GTG Pt lesion was placed in the

center of a 150mer substrate as previously described 10. The preparation of HeLa whole cell

extracts and the NER excision assay were carried out as previously outlined in detail 11.


! 5!

Immunoaffinity purification of core and holo TFIIH. Immunoaffinity purification of TFIIH

was based on a previously published procedure 12. A total of 2 mg HeLa nuclear extract proteins

was incubated with 25 µg of affinity purified rabbit anti-XPB antibody (A301-337A, Bethyl

Laboratories) in 200 µl of Buffer A [10 mM HEPES (pH 8.0), 50 mM KCl, 5 mM MgCl2, 10%

glycerol, 0.1 mM EDTA, 0.5 mM DTT and 50 µM PMSF] for 2 h at 4 0C. The mixture was

added to 50 µl bed volume of Protein A-Agarose (sc-2001, Santa Cruz Biotechnology, pre-

equilibrated with incubation buffer) and the beads were rotated for 2 h at 4 0C. The beads were

washed five times with 300 µl 0.4 M KCl in Buffer B [20 mM HEPES (pH 7.9), 0.2 mM EDTA,

1 mM DTT, 0.03% NP-40, 10% glycerol] followed by two washes with 300 µl of 0.1 M KCl in

Buffer B (for purification of core TFIIH, 1 M KCl in Buffer B was used instead of 0.4 M KCl

during washing). TFIIH was eluted by incubation with 100 µl of 0.4 M KCl in Buffer B

supplemented with 0.5 mg/ml of DDTVYMEYHSSRSKAPSKHVHPLFKRFRK derived from

the C-terminal 29 amino acids of human XPB for 2 h at 25 0C. The eluate was frozen in liquid

nitrogen in 10-µl aliquots and stored at -80 0C until use. The TFIIH concentration was estimated

by Western blotting using recombinant his-XPB as a standard. The typical yield is 150-300 ng of

XPB equivalent.

Purification of recombinant his-XPB. Purification of recombinant his-XPB was based on a

previously published procedure 13. High Five cells were grown in Grace’s Insect media plus 10%

FBS at 28 0C with 2% CO2. 20 x 5·106 High Five cells were seeded on 20 x 15 cm dishes.

Twenty four hours after seeding, the cells were infected with his-XPB-containing baculovirus.

Forty eight hours after infection, the cells were collected with a cell scraper and followed by

centrifugation at 600 x g for 10 min. Purification was conducted in a cold room at 4 0C. The cells


! 6!

were resuspended in 18 ml of lysis buffer [20 mM HEPES, pH 7.9, 500 mM KCl, 1 mM DTT,

20% glycerol], incubated for 15 min and lysed by 30 strokes in a Dounce Homogenizer (B type

pestle). The lysate was rotated for 45 min and then centrifuged at 20,000 x g for 1 h followed by

centrifugation at 100,000 x g for 1 h. The supernatant was added to 0.5 ml Ni-NTA Agarose

beads (QIAGEN) and the beads were rotated for 1 h. The resulting slurry was loaded onto a

column, washed with 30 ml of 10 mM imidazole lysis buffer and eluted with 250 mM imidazole

lysis buffer. The eluate was dialyzed with Buffer C [20 mM Tris, pH 8.3, 100 mM KCl, 1 mM

EDTA, 1 mM DTT, 20% glycerol] on a PD-10 column (GE Healthcare). The dialyzed eluate was

loaded on a 1-ml HiTrap Q column (GE healthcare) and eluted with 100 mM to 700 mM KCl

gradient. His-XPB containing fractions were combined and dialyzed into 20 mM HEPES, pH

7.9, 100 mM KCl, 1 mM DTT, 20% glycerol on a PD-10 column. The purified recombinant

XPB was frozen in small aliquots in liquid nitrogen and stored at -80 0C.

ATPase assay. The DNA-dependent ATPase assay was performed as previously described 14.

Briefly, a 10-µl reaction mixture contained 20 mM Tris (pH 7.9), 4 mM MgCl2, 1 µM ATP, 1

µCi ATP (3000 Ci/mmol), 100 µg/ml BSA, 50 ng RNA polymerase II promoter Positive Control

DNA (Promega), 1-2 nM TFIIH or 100 nM his-XPB and indicated concentration of drugs. The

reactions were started by either addition of TFIIH/his-XPB or ATP and incubated at 37 0C for 2

h. The reactions were stopped by addition of 2 µl 0.5 M EDTA and dilution in up to 100 µl with

TE buffer. An aliquot of 1 µl reaction mixture was spotted on PEI-cellulose and the

chromatogram was developed with 0.5 M LiCl and 1 M HCOOH. The percent of ATP

hydrolysis was quantified using a PhosphorImager.


! 7!

TFIIH helicase assay. The helicase assay was performed as previously described 15. Briefly, a

10-µl reaction mixture contained 20 mM Tris (pH 7.9), 4 mM MgCl2, 4 mM ATP, 100 µg/ml

BSA, 0.12 nM of M13mp18-based bidirectional helicase substrate, and 0.4 nM TFIIH. The

reactions were started by either addition of TFIIH/his-XPB or ATP and incubated at 37 0C for 2

h. The reactions were stopped by addition of 5 µl of a quenching buffer (60 mM EDTA, 50%

glycerol, 0.75% SDS). An aliquot of 10 µl of the mixture was loaded on a 10% non-denaturing

polyacrylamide gel containing 0.1% SDS and run at 200 V in 0.5x TBE buffer with 0.1% SDS.

The gel was dried and subjected to quantification using a PhosphorImager.

[3H]-triptolide binding to XPB in HeLa nuclear extract. HeLa nuclear extract (containing 400

µg of total proteins) was incubated with 1 µM [3H]-triptolide with or without 50 µM unlabeled

triptolide in 50 µl of 10 mM HEPES (pH 8.0), 50 mM KCl, 5 mM MgCl2, 10% glycerol, 0.1 mM

EDTA, 0.5 mM DTT and 50 µM PMSF for 1 h at 30 0C. Three micrograms of affinity purified

rabbit anti-XPB antibody (A301-337A, Bethyl Laboratories) was added and the mixture was

incubated for an additional 30 min at 25 0C. The mixture was added to 50 µl of Dynabeads

Protein A (100.01D, Invitrogen, storage solution removed) and further mixed by rotation for 10

min at 25 0C. The supernatant was aspirated and the beads were washed 3 times with PBS. The

beads were resuspended in 40 µl of sample buffer, boiled for 5 min and subjected to SDS-PAGE.

After electrophoresis, the gel was soaked in En3hance solution according to the manufacturer’s

instructions and exposed to pre-flashed x-ray film for 2 weeks prior to autoradiography.

[3H]-triptolide binding to recombinant his-XPB: His-XPB (300 ng) was incubated with 1 µM

[3H]-triptolide with or without 50 µM cold triptolide in 40 µl binding buffer [20 mM Tris (pH


! 8!

7.9), 4 mM MgCl2, 1 µM ATP, 100 µg/ml BSA], and 500 ng RNA polymerase II promoter

Positive Control DNA (Promega) for 1 h at 30 0C. Samples were boiled in sample buffer and

subjected to 12% SDS-PAGE. After electrophoresis, the gel was soaked in En3hance solution

(Perkin Elmer) according to manufacturer instructions and exposed to pre-flashed x-ray film for

2 weeks prior to development.

Over-expression of XPB and XPD in HEK293T. pLenti6m-STAP was made by inserting

streptavidin binding peptide (SBP) and the IgG binding domain of Protein A separated by TEV

cleavage site into the multiple cloning site of pLenti6 (Invitrogen). The full-length human XPB

and XPD coding sequences were PCR amplified using the following primers: XPB-forward:

TAT TCT AGA GCC ACC ATG GGC AAA AGA GAC CGA G, XPB-reverse: TAT ATA

CTC GAG TTT CCT AAA GCG CTT GAA GAG, XPD-forward: TAT TCT AGA GCC ACC

ATG AAG CTC AAC GTG GAC G, XPD-reverse: TATATA CTC GAG CTG CTG AGC AAT

CTG CTC. The PCR products were inserted into pLenti6m-STAP using XhoI and XbaI

restriction sites. The constructs were transfected into HEK293T cells with pSPAX2 and

pMD2G16 using Lipofectamine 2000 (Invitrogen) and viruses were harvested after 72 h.

HEK293T cells were infected with the corresponding lentivirus and cells stably expressing XPB

and XPD were selected with 20 µg/ml blasticidin for two weeks and maintained at the same

concentration of antibiotic for culture. These cell lines were used for cell proliferation assay.

Synthesis of triptolide analogs. Thin-layer chromatography (TLC) was performed on Analtech

precoated silica gel HLF UV254 Uniplates and was visualized using 254 nm UV light, or by

staining with iodine, ceric ammonium molybdate stain. Silica gel (60–200 mesh, from Acros)

was used for air-flashed chromatography. Reagents were purchased from Acros, TCI-America,

or Aldrich companies. NMR data were collected on a Varian Unity-400 (400 MHz ¹H, 100.6

MHz ¹³C) machine. ¹H NMR spectra were obtained in deuteriochloroform (CDCl₃) with

tetramethylsilane (TMS, δ = 0.00) or chloroform (δ = 7.24) as an internal reference. 13C NMR


! 9!

spectra were proton decoupled and were recorded in CDCl₃ with either TMS (δ = 0.0) or

chloroform (δ = 77.23) as an internal reference. Chemical shifts are reported in ppm (δ).

Multiplicities are indicated by s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd

(doublet of doublet), dt (doublet of triplet), br. (broad), app. (apparent) and exch. (exchangeable).

Coupling constants, J, are reported in Hertz (Hz); integration is provided; and assignments of

individual resonances are supported in most cases by the following NMR experiments: COSY,

DEPT, or HMBC and HMQC. Data are presented in the form: chemical shift (multiplicity,

coupling constants, integration and assignments where relevant). Low-resolution mass spectra

were obtained on a Voyager DE-STR, MALDI-TOF instrument at the AB Mass

Spectrometry/Proteomics Facility at the Johns Hopkins University. The MALDI-samples were

prepared by mixing droplets of the sample solutions in chloroform or methanol and 2,5-

dihydroxybenzoic acid solution in acetone, where the latter served as the matrix. At times, low-

resolution electrospray ionization mass spectra (ESI-MS) were recorded using Thermo Finnigan

LCQ ion-trap mass spectrometer.

!

Epitriptolide (2, 9:1 mixture of 2/1 based on ¹H NMR), triptonide (4), and the

imidazolylthiocarbamate 7 were prepared according to the procedure of Aoyagi et al. 17. A

diastereomeric mixture of 16/17-hydroxy triptonide (6) was isolated after subjecting triptonide

(2) to biotransformation following an earlier report 18. Triptolide was converted to 14-dexoy-

14α-fluorotriptolide (8) using the protocol described in the literature 19. The derivatives 9 and 11

were prepared according the reported procedure 20. The 5-hydrxoy derivative 10 was synthesized

using selenium dioxide (SeO2) oxidation as previously described 20; and the same procedure was

applied to making 5-hydroxy derivatives 3 and 5 starting from triptolide (1) and triptonide (4)

respectively. We obtained derivatives 3 and 5 in 21% and 55% yield respectively using the SeO2

oxidation, however, these two compounds have already been isolated and characterized in the

mixture of biotransformation products 18,21. The diol 12 was made as per the reported procedure

as well 22.


! 10!

A JASCO HPLC system consisting of PU-2089s pump and MD-2010 PDA detector was used

during the purification of triptolide analogs 2, 3, 6, and 11. Fractions were collected while

eluting off of an Alltech ECONOSIL C18 column (10 µm, 10×250 mm) using 68:32 water–

acetonitrile as the mobile phase (flow rate: 3 mL/min, monitored for UV absorption at 218 and

234 nm). The solvents used in reactions were reagent grade. The solvents used for extraction and

chromatography were technical grade. The reactions were performed in oven-dried glassware.

!

REFERENCES

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3! Sisodia,!S.!S.,!Sollner8Webb,!B.!&!Cleveland,!D.!W.!Mol(Cell(Biol!7,!3602!(1987).!

4! Dignam,!J.!D.,!Lebovitz,!R.!M.!&!Roeder,!R.!G.!Nucleic(Acids(Res!11,!1475!(1983).!

5! Kadesch,!T.!R.!&!Chamberlin,!M.!J.!!J.(Biol.(Chem.!257,!5286!(1982).!

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(2009).!

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Chem.!19,!1064!(2008).!

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(2007).!

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(1994).!

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! 12!

Supplementary Results


Figure'S1'

Figure'S1.'Effect'of'transcrip5on'inhibitors'on'HeLa'cell'prolifera5on'was'assessed'by'measuring'thymidine'incorpora5on'a?er'24Bhour'treatment'with'drugs'followed'by'6Bhour'[3H]thymidine'pulse.'Mean'values'±'SEM'(error'bars)'from'3'independent'experiments'are'shown.'IC50'values'are'listed'next'to'corresponding'curves'±'SEM.'

14.1'±'4.1pM' 4.57'±'0.9'nM' 1.87'±'0.28'μM' 14.3'±'1.9'μM'


Figure'S2'

Figure'S2.'Effect'of'transcrip5on'inhibitors'on'RNA'synthesis'was'assessed'by'measuring'uridine'incorpora5on'into'trichloroace5c'acid'(TCA)'insoluble'frac5on'of'HeLa'cells'a?er'1Bhour'drug'treatment'followed'by'1Bhour'[3H]uridine'pulse.'Mean'values'±'SEM'(error'bars)'from'3'independent'experiments'are'shown.'IC50'values'are'listed'next'to'corresponding'curves'±'SEM.'

73.4'±'16.8'nM'

65.3'±'8.4'nM'

109'±'8'nM'


Figure'S3'

Figure'S3.'Effect'of'transcrip5on'inhibitors'on'RNA'synthesis'was'assessed'by'measuring'uridine'incorpora5on'into'trichloroace5c'acid'(TCA)'insoluble'frac5on'of'HeLa'cells'a?er'3Bhour'drug'treatment'followed'by'1Bhour'[3H]uridine'pulse.'Mean'values'±'SEM'(error'bars)'from'3'independent'experiments'are'shown.'IC50'values'are'listed'next'to'corresponding'curves'±'SEM.'

21.5'±'4.0'nM' 69.6'±'7.2'nM'(Flavo)'

62.5'±'3.2'nM'(TPL)'


Figure'S4'

Figure'S4.'Effect'of'triptolide'on'thymidine'incorpora5on'into'HeLa'cells'a?er'24'hour,'6'hour,'3Bhour'and'1'hour'treatment'followed'by'1Bhour'[3H]thymidine'pulse'.'Mean'values'±'SEM'(error'bars)'from'5'independent'experiments'are'shown.'IC50'values'are'listed'next'to'corresponding'curves'±'SEM.'

18.2'±'3.1'nM'

103'±'7'nM'

40.6'±'28.3'nM'


Figure'S5'

Figure'S5.'Effect'of'transcrip5on'inhibitors'on'transla5on'was'assessed'by'measuring'methionine'incorpora5on'into'trichloroace5c'acid'(TCA)'insoluble'frac5on'of'HeLa'cells'a?er'1Bhour'drug'treatment'followed'by'1Bhour'[35S]methionine'pulse.'Mean'values'±'SEM'(error'bars)'from'3'independent'experiments'are'shown.'


Figure'S6'

Figure'S6.'Effect'of'transcrip5on'inhibitors'on'transla5on'was'assessed'by'measuring'methionine'incorpora5on'into'trichloroace5c'acid'(TCA)'insoluble'frac5on'of'HeLa'cells'a?er'3Bhour'drug'treatment'followed'by'1Bhour'[35S]methionine'pulse.'Mean'values'±'SEM'(error'bars)'from'3'independent'experiments'are'shown.'


Effect of Trytolide on NER excision in HeLa WCE

Concentration of Tryptolide (uM)

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

% e

xcis

ion

rela

tive

to c

on

trol

0

20

40

60

80

100

120

[TPL] (µM)"

NER

Pro

duct

(%)"

Figure'S7.'Quan5ta5on'of'the'inhibi5on'of'Nucleo5de'Excision'Repair'ac5vity'by'triptolide'(Figure'2D).'Average'values'from'3'independent'experiments'are'shown'±'SEM'(error'bars).'

Figure'S7'


Figure'S8'

Figure'S8.'Composi5on'of'immunoaffinity'purified'coreB'and'holoTFIIH'complexes'as'determined'by'SDSBPAGE'followed'by'silver'staining'(le?'panel)'or'Western'blot'analysis'(right'panel).'Note'that'XPD,'cdk7,'MAT1'and'cyclinH'are'absent'from'coreTFIIH.'

Core TFIIH"Holo TFIIH" Core TFIIH"Holo TFIIH"

50kDa"

100kDa"120kDa"

80kDa"

40kDa"

60kDa"

30kDa"

cdk7"

p52"

XPD "

XPB "

Silver staining! Western Blot!

XPB"XPD"p62"

p52 & p44"cdk7"MAT1, p34 & "CyclinH"


Figure'S9'

Figure'S9.'Quan5ta5on'of'the'inhibi5on'of'holoTFIIH'DNABdependent'ATPase'ac5vity'by'triptolide'(Figure'3b).'100%'corresponds'to'hydrolysis'of'27.8%'of'ATP.'Average'values'from'3'independent'experiments'are'shown'±'SD'(error'bars).'

6.6%'

17.0%'

100.0%'

74.4%'

52.4%'

31.6%'

23.4%'19.1%'

0%'

20%'

40%'

60%'

80%'

100%'

no'TFIIH' no'DNA' DMSO' 0.1μM'Triptolide'

0.3μM'Triptolide'

1μM'Triptolide'

3μM'Triptolide'

10μM'Triptolide'


Figure'S10'

Figure'S10.'Quan5ta5on'of'the'inhibi5on'of'coreTFIIH'DNABdependent'ATPase'ac5vity'by'triptolide'(Figure'3c).'100%'corresponds'to'hydrolysis'of'14.3%'of'ATP.'Average'values'from'3'independent'experiments'are'shown'±'SD'(error'bars).'

12.0%'

31.1%'

100.0%'

67.4%'

46.4%'

27.9%'22.1%' 19.7%'

0%'

20%'

40%'

60%'

80%'

100%'

no'TFIIH' no'DNA' DMSO' 0.1μM'Triptolide'

0.3μM'Triptolide'

1μM'Triptolide'

3μM'Triptolide'

10μM'Triptolide'


IgG" 3" 10" 3" 10"

Supe" IP"

IgG"

Figure'S11'

Figure'S11.'Quan5ta5ve'pullBdown'of'XPB'from'cell'lysate'using'an'αBXPB'an5body.''XPB'can'be'quan[ta[vely'pulled'down'from'400'μg'of'HeLa'nuclear'extract'with'3'μg'of'α]XPB'an[body.'Same'condi[ons'were'used'for'experiments'in'Figures'4a'and'4b.''


Frac[ons'from'PD]10'column'

50kDa'

100kDa'

80kDa'

40kDa'

60kDa'

30kDa'

Figure'S12'

Figure'S12.'SDSBPAGE'analysis'of'recombinant'XPB.''Coommassie'blue'staining'of'SDS]polyacrylamide'gel'with'recombinant'his]XPB'purified'from'baculovirus]driven'insect'cells'with'Ni'affinity'chromatography'followed'by'a'salt'gradient'on'HiTrap'Q'HR.''


Figure'S13'

Figure'S13.'Quan5ta5on'of'the'inhibi5on'of'recombinant'hisBXPB'DNABdependent'ATPase'ac5vity'by'triptolide'(same'as'Figure'3c).'100%'corresponds'to'hydrolysis'of'23%'of'ATP.'Average'values'from'3'independent'experiments'are'shown'±'SD'(error'bars).'

10.2%'

35.1%'

100.0%'

88.2%'

78.0%'

68.0%'

56.9%'

49.8%'

0%'

20%'

40%'

60%'

80%'

100%'

no'protein' no'DNA' DMSO' 1μM'Triptolide'

3μM'Triptolide'

10μM'Triptolide'

30μM'Triptolide'

100μM'Triptolide'

%,'Real[ve'ATP'hydrolysis'


Figure'S14'

' '''''''''293T ''''''''''XPD ''''''''''XPB'IC50'(nM)''''' '2.2'''''''''''''' '2.4''''''''''''''''''6.7'

a' b'

c'293T''''+XPD'''''+XPB'

Figure'S14.'Overexpression'of'XPB,'but'not'XPD,'confers'resistance'of'293T'cells'to'triptolide.'(a)'Dose]response'curves'of'293T'control'and'those'stably'transfected'with'vectors'carrying'XPB'or'XPD.'(b)'IC50'values'of'triptolide'in'the'three'different'cell'lines'in'(a).''(c)'Detec[on'of'exogenously'expressed'XPB'and'XPD.''Both'XPB'and'XPD'were'tagged'with'an'IgG]binding'pep[de'epitope'and'were'detected'using'IgG'as'primary'an[body.'


Original Gel Images of the Figures


Figure'1c'


Figure'1d'and'1e'


Figure'1f'and'2a'


Figure'2b'and'2c'


Figure'2d'and'3a'


Figure'3b'(lee)'and'3c'(right)'


Figure'4a'and'4b'


Figure'4c'and'4d'


xpb, a subunit of tfiih, is a target of the natural …...2011/01/30 · pressive activities....

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