Volume 21, Issue 3, Pages 398-407 (March 2014) A Small Molecule Screen Identifies an Inhibitor of DNA Repair Inducing the Degradation of TFIIH and the Chemosensitization of Tumor Cells to Platinum  Sergey Alekseev, Mériam Ayadi, Laurent Brino, Jean-Marc Egly, Annette K. Larsen, Frédéric Coin  Chemistry & Biology  Volume 21, Issue 3, Pages 398-407 (March 2014) DOI: 10.1016/j.chembiol.2013.12.014 Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 1 SP Inhibits the Repair of UV-Induced DNA Damage (A) Schematic representation of the protocol that was used for the identification of NER inhibitors. Following 12 hr incubation with the chemical library in 96-well plates, cells were UVC irradiated (60 J/m2), postincubated for 3 hr, and fixed. Subsequently (6-4)PP lesions were labeled using an anti-(6-4)PP antibody and detected by high throughput microscopy (HTM). (B) The protocol presented in (A) was validated using shCTL and shXPA HeLa cells. The levels of remaining (6-4)PP lesions was determined using HTM. One hundred percent corresponds to the (6-4)PP signal measured just after UV irradiation. (C) Results of the screening performed with the 1,200 small molecules from the Prestwick library tested at 10 μM following the protocol described in (A). The vertical axis represents the percent of (6-4)PP lesions relative to the level measured right after irradiation that is set to 100% in all experiments. (D) (6-4)PP repair inhibitory activity of different SP concentrations as determined by HTM. Antazoline (AN) did not induce any (6-4)PP repair inhibitory activity in our screening and thus was used as negative control. The vertical axis represents the percent of (6-4)PP lesions relative to the level measured just after irradiation that is set to 100% in all experiments. The values represent the averages (±SD) from three independent experiments. (E) Structure of Budesonide, Cortisone, Diflorasone, and Eplerenone, four compounds structurally close to SP (shown at the center of the figure). (F) The relative NER inhibitory activity of Budesonide (1), Cortisone (2), Diflorasone (3), and Eplerenone (4) was determined and compared to SP (5) following the protocol described in (A). The vertical axis represents the percent of (6-4)PP lesions relative to the level measured just after irradiation that is set to 100% in all experiments. The values represent averages (±SD) from three independent experiments. See also Figure S1. Chemistry & Biology 2014 21, 398-407DOI: (10.1016/j.chembiol.2013.12.014) Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 2 SP Increases the Sensitivity to UVC and Cisplatin Treatment (A and B) Quantitative UV (A) or cisplatin (B) survival analysis of HeLa cells treated with DMSO or SP (10 μM). Results are expressed as percent of cell survival relative to the nontreated controls that is set to 100%. The values represent averages (±SD) from three independent experiments; p value is indicated (∗∗p < 0.01; ∗∗∗p < 0.001). (C) Cytotoxic activity of SP(10 μM) or cisplatin (2.5 μM) in HeLa cells treated 24 hr with the indicated compound and stained with 7-AAD before flow cytometry analysis. Results are expressed as percent of dead cells (positive for 7-AAD staining) in the total population. The values represent averages (±SD) from three independent experiments; p value is indicated. (D) Upper panel: increasing amount (1 μg, 3 μg, and 6 μg) of nuclear extracts from DMSO (NEDMSO)-treated or SP (NESP)-treated HeLa cells were analyzed for dual incision. NEs were incubated with cis-platinum mono-damaged plasmid (Riedl et al., 2003). Incision products are indicated as radiolabeled 27 nt to 34 nt products. Several incisions products are observed that depend on the cutting site of XPG that varies from two to three nucleotides. As control, a reconstituted system (Rec) with recombinant proteins was used (Riedl et al., 2003). Lower panel: similar amount of NE as tested above was analyzed by western blotting with an anti-TBP antibody. (E) DMSO-treated or SP-treated HeLa cells were incubated in the presence (+) or absence (−) of 100 ng/ml NCS for 30 min. Cells were then cultured without NCS and harvested at 6 or 24 hr. Cells were lysed and analyzed by western blotting with anti-γH2AX or anti-TBP antibodies. When indicated, cells were treated with DNA-PK inhibitor NU7026 during the recovery time. Chemistry & Biology 2014 21, 398-407DOI: (10.1016/j.chembiol.2013.12.014) Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 3 SP-Induced NER Deficiency Is due to the Downregulation of TFIIH (A) Following incubation with DMSO or SP (10 μM) for 2 hr, HeLa cells were locally UV-irradiated (100 J/m2), fixed after 15 min, and costained with antibodies to XPC (a, b, e, f, i, and j) and to either TFIIH(p62) (c and d), XPA (g and h), or XPF (k and l). Arrows indicate locally irradiated areas. (B) HeLa cells were incubated with DMSO or SP (10 μM) for the indicated time before lysis. Extracts from DMSO-treated or SP-treated cells were resolved by SDS-PAGE and subsequently analyzed by immunoblotting. The different proteins are indicated and black arrows point to the corresponding bands. CTL, control. (C) HeLa cells were incubated with DMSO or SP (10 μM) for 2 hr. Increasing amounts (1 μg, 3 μg, and 6 μg) of nuclear extracts from DMSO (NEDMSO)-treated or SP (NESP)-treated HeLa cells were analyzed for dual incision as described in Figure 2C. When indicated, purified TFIIH (100 ng) from HeLa was added. Optical density was determined using a Typhoon PhosphorImager 8600 and expressed as relative activity (RA). (D) HeLa cells were treated with DMSO or SP (10 μM) for 2 hr, fixed and incubated with anti-XPC (a–c) and anti-XPB antibodies (b–d). (E) HeLa cells were transfected with XPB-GFP and treated with either DMSO or SP (10 μM). Fluorescence was determined by live imaging every 12 min during 120 min and plotted on the graph. The values are indicated relative to the expression level of XPB-GFP before treatment that is set to 100. Values represent averages (±SD) from three independent experiments. See also Figure S2 and Movie S1. Chemistry & Biology 2014 21, 398-407DOI: (10.1016/j.chembiol.2013.12.014) Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 4 SP Induces Ubiquitin-Activating Enzymes and Proteasome-Dependent Degradation of XPB (A) HeLa cells were treated with DMSO or SP (10 μM) for 2 hr. Total RNA was extracted and XPB mRNA was quantified by RT-qPCR. Values are normalized relative to GAPDH expression. Values represent averages (±SD) from three independent experiments. (B) HeLa cells were incubated in the presence (+) or absence (−) of the proteasome inhibitor MG132 (10 μM) for 1 hr before addition of DMSO or SP (10 μM) for 4 hr. Cells were lysed and the extracts were resolved by SDS-PAGE and analyzed by immunoblotting for XPB or TBP. (C) HeLa cells were incubated in the presence (+) or absence (−) of the ubiquitin-activating enzyme inhibitor PYR-41 (35 μM) for 2 hr before addition of DMSO or SP (10 μM) for 4 hr. Cells were lysed and the extracts were resolved by SDS-PAGE and analyzed by immunoblotting for XPB or TBP. (D) HeLa cells were transfected with Sug1-pool siRNA or Control (CTL) smart-pool siRNA (Dharmacon) for 36 hr following incubation with DMSO or SP (10 μM) for 4 hr. Cells were lysed and extracts were resolved by SDS-PAGE and analyzed by immunoblotting. The signals were quantified using Genetool (Syngene) and indicated below the immunoblot. Chemistry & Biology 2014 21, 398-407DOI: (10.1016/j.chembiol.2013.12.014) Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 5 SP-Induced Degradation of TFIIH Impairs Transcription (A and B) mRNA levels for Cyp26 (A) and RARβ2 (B) were analyzed in DMSO-treated or SP-treated cells through the time after the addition of tRA (1 μM). SP (10 μM) was added to the medium 2 hr before addition of tRA and kept during the time course. Expression was represented as the level of the Cyp26 or RARβ2 mRNA relative to GAPDH. Values represent averages (±SD) from three independent experiments. (C) HeLa cells were incubated with DMSO or SP (10 μM) for 2 hr. Increasing amounts (1 μg, 3 μg, and 6 μg) of NEDMSO or NESP were tested in an in vitro transcription assay using AdML promoter yielding a 309 nt transcript as indicated. When indicated, purified TFIIH (100 ng) from HeLa (Gerard et al., 1991) was added. The signals were quantified using Genetool (Syngene) and indicated below the autoradiogram. RA, relative activity. Chemistry & Biology 2014 21, 398-407DOI: (10.1016/j.chembiol.2013.12.014) Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 6 The SP-Induced Degradation of XPB Is Reversible (A) HeLa cells were pretreated with DMSO (lane 1) or SP (lanes 2–5) (10 μM) for 2 hr. SP was removed and fresh medium was added. Cells were allowed to recover for the indicated period of recovery times (RecTime). Extracts were resolved by SDS-PAGE and analyzed by immunoblotting for either XPB or TBP. The signals for XPB and TBP were quantified using Genetool (Syngene) and plotted on the graph relative to the signal in DMSO-treated cells that is set to 1. RA, relative amount. (B) HeLa cells were pretreated with DMSO (lanes 1–4) or SP (10 μM) (lanes 2, 3, 5, and 6) for 2 hr. SP was then removed and fresh medium was added with (lanes 4–6) or without (lanes 1–3) cycloheximide (CHX, 100 μM). Cells were allowed to recover for 6 hr before lysing. Extracts were resolved by SDS-PAGE and analyzed by immunoblotting for either XPB or TBP. (C) HeLa cells were treated with DMSO or SP (10 μM) for 2 hr. SP was removed and fresh medium was added. Cells were allowed to recover for different recovery times (RT) and subsequently UV-irradiated (60 J/m2), incubated for 3 hr (post-UV), and fixed. Subsequently (6-4)PP lesions were labeled using anti-(6-4)PP antibodies and detected by HTM. The vertical axis represents the percent of (6-4)PP lesions relative to the level of (6-4)PP lesions measured just after the irradiation that was set to 100 in all experiments; p value is indicated (∗∗p < 0.01; ∗∗∗p < 0.001). Chemistry & Biology 2014 21, 398-407DOI: (10.1016/j.chembiol.2013.12.014) Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 7 SP Sensitizes Human Carcinoma Cells to Platinum Derivatives (A and B) The MTT assay was used to measure cell viability of either A2780 (A) or HCT-116 (B) human carcinoma cells after treatment with cisplatin or oxaliplatin (0.1 to 50 μM), respectively, in the presence of AN (10 μM), SP (10 μM), or DMSO. Values represent averages (±SD) from three independent experiments. The p values (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001) and IC50 are indicated. One hundred percent corresponds to the amount of cells without any drugs. (C) A2780 and HCT-116 cells were incubated with DMSO or SP (10 μM) for 4 hr before lysis. Extracts were resolved by SDS−PAGE and subsequently analyzed by immunoblotting for XPB or TBP. Chemistry & Biology 2014 21, 398-407DOI: (10.1016/j.chembiol.2013.12.014) Copyright © 2014 Elsevier Ltd Terms and Conditions