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Irreversible TrxR1 inhibitors block STAT3 activity and induce cancer cell death

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Science Advances  20 Mar 2020:
Vol. 6, no. 12, eaax7945
DOI: 10.1126/sciadv.aax7945
  • Fig. 1 4,5-dichloropyridazinone compounds as inhibitors of STAT3-dependent transcription.

    (A) Top compounds from the HTS campaign having the 4,5-dichloropyridazinone core structure. Luciferase IC50 values are reported as an average of two experiments conducted in triplicate. (B) Summary of structure-activity relationship (SAR) study to explore the activity of top compounds. Modifications to the “pyridazinone” moiety (blue) were generally unfavorable, as these compounds lost STAT3 inhibitory capacity, whereas variations on the linker (purple) could increase the potency of the compounds, and several different linkers were tolerated. The tail moiety (green) was quite versatile and could incorporate a large range of functionalities. (C) Four of the most potent compounds from the SAR study, DG-4 to DG-7. (D) Bar graphs of IC50 values for the top four DG compounds for STAT3- and STAT1-driven luciferase assays. IFNγ, interferon γ. (E) Table describing the IC50 values shown in (D), together with fold selectivity for each compound. (F) Resazurin cell viability assays of top inhibitors against several cancer and noncancerous (CCD841 and BJ) cell lines. Compounds were incubated with cells for 72 hours at a concentration range of 0.78 to 100 μM (twofold dilutions); then, resazurin (0.02 mg/ml) was added, and resofurin fluorescence was measured after an additional 5 hours of incubation. Fluorescence values were normalized to DMSO (dimethyl sulfoxide) and media controls, and the resulting points were fit to a nonlinear variable slope curve (four parameters). HEK293, human embryonic kidney–293. (G) IC50 values from the dose-response cell viability experiments shown in (F).

  • Fig. 2 DG-8, a fluorescent 4,5-dichloropyridazinone probe for target identification and specificity.

    (A) The chemical structure of DG-8, which incorporates many characteristics of the top compounds from the SAR study. (B) STAT3-dependent luciferase assay data showing DG-8 is a potent inhibitor of STAT3-dependent transcription (IC50 = 0.98 μM). (C and D) DG-8 (0.5 to 50 μM) was incubated with 5 μg of two recombinant STAT3 protein truncations STAT3127–688 (C) and STAT3127–465 (D) for 30 min, then run on an SDS–polyacrylamide gel electrophoresis (SDS-PAGE) gel, and analyzed for dansyl fluorescence. The dye front is representative of the amount of DG-8 used in each sample. (E) DG-8 (0.5 to 50 μM) was incubated with A549 cell lysates for 30 min, and the protein content (30 μg) was analyzed by SDS-PAGE and dansyl fluorescence. A single fluorescent band was detected with a molecular weight (MW) of approximately 55 kDa (gray arrow). (F) DG-8 (0.1 to 10 μM) was incubated with A549 cells in culture for 30 min. Cells were then collected and lysed, and 30 μg of the resultant protein lysate was analyzed by SDS-PAGE and dansyl fluorescence. Again, a single fluorescent band was detected at approximately 55 kDa (gray arrow). (G) Recombinant TrxR1 protein (5 μg) was incubated with DG-8 (0.5 to 50 μM) in the presence or absence of NADPH (7.5 μg) as indicated. Following 30 min incubation, samples were analyzed by SDS-PAGE and dansyl fluorescence under reducing conditions. A ~55-kDa fluorescent band is detected only in the presence of NADPH, indicating that DG-8 reactivity with TrxR1 is dependent on NADPH, which is consistent with binding to the Sec residue of TrxR1 (23). (H) To assess whether the ~55-kDa band might be a Sec-containing protein, A549 cells were incubated for 72 hours with sodium selenite (25 to 100 nM) to promote Sec incorporation into cellular selenoproteins or SPO3 (0.25 to 1 mM) to induce Sec-to-Cys substitution (29). The cells were then lysed and treated with DG-8 (5 μM) for 30 min at room temperature. The resulting sample containing 30 μg of protein lysates was run on an SDS-PAGE gel (reducing conditions) and analyzed for dansyl fluorescence. Bands occurring at ~55 kDa were quantified and plotted as bar graphs (*P < 0.05, n = 2). Band intensities were normalized to the sample containing 25 nM selenite, as this was the concentration used throughout this work to ensure adequate selenium supplementation.

  • Fig. 3 DG-8 competition assays in A549 cell lysates.

    (A to C) To investigate whether top DG compounds shared the same target as DG-8, A549 protein lysates (30 μg) were incubated with DG compounds (0.5 to 50 μM) for 30 min and then with DG-8 (5 μM) for 30 min. Samples were then run on an SDS-PAGE gel, and DG-8 fluorescence was measured using a Gel Doc EZ Gel Documentation System with UV tray. Both DG-4 and DG-5 outcompeted DG-8 for binding at low micromolar concentrations, whereas DG-7 was much less potent. (D to G) Established TrxR1 inhibitors TRi-1, TRi-2, TRi-3, and auranofin were assessed in the DG-8 competition assay. TRi-3 is highly similar to our lead series of compounds and has a 4,5-dichloropyridazinone group, suggesting that it could have the same molecular target(s) as our top compounds. TRi-1 and auranofin inhibit TrxR1 through binding to its Sec residue, whereas TRi-2 is thought to function by a non-Sec binding mechanism. Hence, TRi-1, TRi-3, and auranofin could all compete with DG-8, suggesting that this band may correspond to the 55-kDa selenoprotein TrxR1 by covalently reacting with its Sec residue. (H and I) Two popular STAT3 inhibitors Stattic and BP1-102 were assessed for DG-8 competition. While both Stattic and BP1-102 claim to be direct binders of STAT3 protein in cells, Stattic showed competition with DG-8, indicating that it may share some common STAT3 inhibitory effects as our top DG compounds. BP1-102 was not able to compete with DG-8 for binding to the ~55-kDa band. All SDS-PAGE gels were run under reducing conditions, and the fluorescence of the dye front was used to ensure regular loading.

  • Fig. 4 Top DG compounds inhibit TrxR1 activity and contribute to TrxR1 inhibitory effects in cells.

    (A) Cellular TrxR1 activity was analyzed using the insulin end point assay (23) following incubation of the indicated compounds (1 μM) in cultured FaDu cells for 3 hours. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n = 3. (B) Inhibition of recombinant TrxR1 and TrxR2 proteins were assessed in vitro using an insulin reduction assay, where insulin was reduced by Trx1 and Trx2, respectively. (C) Inhibition of TrxR1 activity was assessed in vitro using an enzymatic DTNB assay after 90 min of incubation (23). In the absence of NADPH, the Sec residue of TrxR1 forms a Sec-cysteine (Cys) bond and is incapable of reacting with electrophilic compounds; therefore, no inhibitory activity is observed with any of the tested compounds without NADPH. Addition of NADPH reduces the Sec-Cys bond, releasing the Sec residue so that it can react with electrophilic compounds. Thus, when NADPH is present, strong inhibition of TrxR1 activity is detected. (D) Irreversible binding of the Sec residue of TrxR1 leads to the formation of selenium-compromised TrxR–derived apoptotic proteins (SecTRAPs), which can be measured by juglone reduction independent of the activity of the Sec residue. Redox cycling of juglone occurs at a distinct site from TrxR1 and will continue even in the absence of Sec redox activity. Under the same conditions that generated complete inhibition of TrxR1 activity in the DTNB assay, TrxR1 retained its ability to reduce juglone, indicating the formation of SecTRAPs. (E and F) Cellular H2O2 production was measured using the Amplex Red assay. Treatment of FaDu cells with top DG compounds led to a time-dependent increase (E) [compounds (0.5 μM)] and concentration-dependent (F) increase in cellular H2O2 levels. (G) Top DG compounds were assessed in mouse embryonic fibroblast (MEF) cells with altered mouse TrxR1 gene expression (Trxnd1). Resazurin cell viability was assessed following 72 hours of compound treatment and additional 5 hours of exposure to resazurin. TrxR1 knockout cells (−/−) were less sensitive to the top compounds compared to wild type (fl/fl). Overexpression of TrxR1 (498Sec) also increased their sensitivity to top compounds. (H) IC50 values for the viability curves shown in (G). (I) IC50 values for CellTiter-Glo cell viability of top DG compounds cultured without or with sodium selenite (100 nM) supplemented medium. Cell viability was assessed following 72 hours of compound treatment. Diminished compound activity by sodium selenite (100 nM) was only detected in noncancerous CCD841 cells, while for cancer cells, no changes were observed. (J) Known TrxR1 inhibitors TRi-1, TRi-2, TRi-3, and auranofin were analyzed in the STAT3- and STAT1-dependent luciferase assay. To measure STAT-dependent transcription, A4wt-SIE cells were stimulated with IL6 (50 ng/ml) and sIL6R (100 ng/ml) for 1 hour, while A4-SIE cells were stimulated with IFNγ (40 IU/ml) for 1 hour, and then, compounds were added for an additional 5 hours, followed by luciferase measurement. (K and L) IC50 values from the experiments described in (J) displayed as a bar graph (K) and table containing fold selectivity comparing STAT3 to STAT1 inhibition (L).

  • Fig. 5 Top DG compounds affect cellular redox balance.

    (A) Western blot analyses of HEK293 cells treated with the top DG compounds. Following a 30-min exposure to compounds, STAT3 and Prx2 were oxidized to form dimers in the absence of reducing agents such as DTT. (B) Similar to the experiment shown in (A), however, the addition of a reducing agent (DTT) during protein sample preparation reduces the inter- and intraprotein disulfide interactions, eliminating the bands corresponding to oligomeric STAT3 and Prx2. (C) Western blot analyses of pSTAT3/STAT3 and Prx2 expression in HEK293-shScramble and HEK293–shPrx1 + Prx2 cells. (D) IC50 values of indicated compounds for STAT-driven luciferase inhibition curves in HEK293-shScramble and HEK293–shPrx1 + Prx2 cells stimulated with IL6 (50 ng/ml) and sIL6R (100 ng/ml). Prx1 + Prx2 knockdown could rescue STAT-dependent transcription leading for our top DG compounds and for TRi-1 and Stattic. (E) Resazurin viability IC50 values for FaDu cells incubated with top DG compounds for 72 hours. Cells were preincubated with or without buthionine sulfoximine (BSO) (100 μM) for 24 hours before the addition of top DG compounds. (F) Cell viability curves for the data described in (E). (G) CellTiter-Glo viability IC50 values for FaDu cells incubated with top DG compounds for 72 hours. Cells were preincubated with or without catalase (100 U/ml) for 4 hours before the addition of top inhibitors. (H) Cell viability curves for the data described in (G).

  • Fig. 6 Proposed model of STAT3 inhibition mediated by TrxR1.

    Black arrows indicate predominantly occurring activities, whereas gray dashed arrows indicate infrequent or inhibited pathways. (A) A general model of the TrxR1 relay system: H2O2 levels are sensed and managed by Prx, which relays oxidative equivalents to STAT3, Trx, and/or other targets. High levels of reduced Trx are maintained by TrxR1, which passes reducing equivalents to Trx from NADPH. Upon cytokine stimulation (for example, with IL6), reduced STAT3 monomers or oligomers will be phosphorylated on Tyr705 to form the transcriptionally active pSTAT3 homodimer, which can interact with specific DNA sequences to promote STAT3-dependent gene expression. (B) Targeting the Sec residue of TrxR1 with electrophilic small-molecule inhibitors leads to the formation of SecTRAPs and disruption of the TrxR1 relay system. SecTRAP formation induces H2O2 production and consumption of NADPH by the second TrxR1 active site containing an FAD moiety and a redox active disulfide/dithiol motif. Increased cellular H2O2 levels cause oxidation of Prx, which relays oxidative equivalents to STAT3 and/or other downstream targets (including directly to Trx). This leads to the formation of disulfide-linked STAT3 dimers (and potentially other oxidized monomeric species) that are not transcriptionally active. SecTRAP formation also blocks the antioxidant function of TrxR1, which will no longer be able to supply reducing equivalents from NADPH to Trx, which prevents the subsequent reduction of Trx’s downstream targets.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/12/eaax7945/DC1

    Supplementary Material and Methods

    Fig. S1. Compounds inhibit STAT3-dependent luciferase and covalently interact with GSH.

    Fig. S2. Whole gel images of competition experiments of Fig. 3.

    Fig. S3. Whole gel images of competition experiments of Fig. 3.

    Fig. S4. SDS-PAGE fluorescent band matches TrxR1 protein on Western blot.

    Fig. S5. CellTiter-Glo cell viability with or without selenium supplementation.

    Fig. S6. Whole blot images of Western blot replicates.

    Fig. S7. STAT-driven luciferase inhibition in Prx1 + Prx2 knockdown cells.

    Fig. S8. Catalase decreases H2O2 production affecting cell cytotoxicity of top DG compounds.

    Fig. S9. Compound effects on alternative transcription factors.

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Material and Methods
    • Fig. S1. Compounds inhibit STAT3-dependent luciferase and covalently interact with GSH.
    • Fig. S2. Whole gel images of competition experiments of Fig. 3.
    • Fig. S3. Whole gel images of competition experiments of Fig. 3.
    • Fig. S4. SDS-PAGE fluorescent band matches TrxR1 protein on Western blot.
    • Fig. S5. CellTiter-Glo cell viability with or without selenium supplementation.
    • Fig. S6. Whole blot images of Western blot replicates.
    • Fig. S7. STAT-driven luciferase inhibition in Prx1 + Prx2 knockdown cells.
    • Fig. S8. Catalase decreases H2O2 production affecting cell cytotoxicity of top DG compounds.
    • Fig. S9. Compound effects on alternative transcription factors.

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