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. 2016 Oct 14;291(42):22231-22243.
doi: 10.1074/jbc.M116.734475. Epub 2016 Aug 30.

Sintokamide A Is a Novel Antagonist of Androgen Receptor That Uniquely Binds Activation Function-1 in Its Amino-terminal Domain

Carmen A Banuelos  1 , Iran Tavakoli  1 , Amy H Tien  1 , Daniel P Caley  1 , Nasrin R Mawji  1 , Zhenzhen Li  2 , Jun Wang  1 , Yu Chi Yang  1 , Yusuke Imamura  1 , Luping Yan  3 , Jian Guo Wen  4 , Raymond J Andersen  3 , Marianne D Sadar  5
Affiliations

Sintokamide A Is a Novel Antagonist of Androgen Receptor That Uniquely Binds Activation Function-1 in Its Amino-terminal Domain

Carmen A Banuelos et al. J Biol Chem. .

Abstract

Androgen receptor (AR) is a validated drug target for all stages of prostate cancer including metastatic castration-resistant prostate cancer (CRPC). All current hormone therapies for CRPC target the C-terminal ligand-binding domain of AR and ultimately all fail with resumed AR transcriptional activity. Within the AR N-terminal domain (NTD) is activation function-1 (AF-1) that is essential for AR transcriptional activity. Inhibitors of AR AF-1 would potentially block most AR mechanisms of resistance including constitutively active AR splice variants that lack the ligand-binding domain. Here we provide evidence that sintokamide A (SINT1) binds AR AF-1 region to specifically inhibit transactivation of AR NTD. Consistent with SINT1 targeting AR AF-1, it attenuated transcriptional activities of both full-length AR and constitutively active AR splice variants, which correlated with inhibition of growth of enzalutamide-resistant prostate cancer cells expressing AR splice variants. In vivo, SINT1 caused regression of CRPC xenografts and reduced expression of prostate-specific antigen, a gene transcriptionally regulated by AR. Inhibition of AR activity by SINT1 was additive to EPI-002, a known AR AF-1 inhibitor that is in clinical trials (NCT02606123). This implies that SINT1 binds to a site on AF-1 that is unique from EPI. Consistent with this suggestion, these two compounds showed differences in blocking AR interaction with STAT3. This work provides evidence that the intrinsically disordered NTD of AR is druggable and that SINT1 analogs may provide a novel scaffold for drug development for the treatment of prostate cancer or other diseases of the AR axis.

Keywords: androgen receptor; drug development; intrinsically disordered protein; prostate cancer; steroid hormone receptor; transcription factor.

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Figures

FIGURE 1.
FIGURE 1.
SINT1 specifically inhibits AR transcriptional activity. A, LNCaP cells were transfected with PSA (6.1 kb)-luciferase, PRE-luciferase, or GRE-luciferase reporters and expression vector for PRβ or GRα. Cells were pretreated with 10 μm SINT1, 10 μm bicalutamide, or vehicle before exposure to the corresponding ligand (1 nm R1881, 10 nm 4-pregnene-3,20-dione, or 10 nm dexamethasone). Error bars represent mean percentage of vehicle activity ±S.E. of at least three independent experiments with triplicate wells. SINT1, 10 μm; bicalutamide (BIC), 10 μm. *, p < 0.05; ***, p < 0.001. B, representative competition binding curve showing displacement of fluorescently labeled ligand (Fluoromone) from recombinant AR LBD by increasing concentrations of bicalutamide, EPI-002, enzalutamide (ENZA), SINT1, or agonist R1881. Error bars represents mean ± S.E. of three technical replicates. C, in the absence of androgen, nuclear translocation of YFP-AR in LNCaP cells treated for 1 h with SINT1, bicalutamide, enzalutamide, or vehicle (DMSO). D, in the presence of androgen (R1881 at 1 nm), nuclear translocation of YFP-AR in LNCaP cells treated with SINT1, bicalutamide, enzalutamide, or vehicle. DAPI staining indicates the location of the nucleus. Scale bars, 20 μm.
FIGURE 2.
FIGURE 2.
SINT1 binds endogenous AR in living cells to inhibit transactivation of AR NTD. A, chemical structures of EPI-002, EPI-053, LPY19, LPY30, LPY31, and SINT1 (left). The effect of SINT1 probes in AR transcriptional activity was tested in LNCaP cells transiently transfected with PSA (6.1 kb)-luciferase and exposed for 1 h to 40 μm LPY19 and 20 μm LPY30 and LPY31 before induction with 1 nm R881 for 48 h. Error bars represent the mean ± S.E. of n = 3 independent experiments with triplicate wells (right). B, binding of SINT1 click chemistry analogs to FL-AR in cells. LNCaP cells were exposed to modified SINT1 (LPY19, -30, and -31), EPI-053, or DMSO vehicle overnight prior to preparing whole cell lysates for click chemistry reactions. Biotinylated SINT1 or EPI probes bound to proteins were captured with streptavidin-agarose resin prior to separation by 10% SDS-PAGE and subjected to Western blotting analysis with antibodies directed to either biotin (left) or AR (middle). Red boxes highlight where AR migrates on the gel. Right, schema of experiment. C, purified recombinant AF1-His tag protein was incubated with LPY19 (inactive), LPY30 and LPY31 (both active), or vehicle (DMSO) prior to click chemistry for biotin labeling, SDS-PAGE, and detection of biotin-labeled probes covalently bound to AF1-His tag (left). Western blotting analysis of the same membrane using an antibody to His tag revealed equal loading of AF1-His tag protein in each lane (middle). Right, schema of experiment.
FIGURE 3.
FIGURE 3.
EPI-002 and SINT1 potentially target different regions of AR AF-1. A, transactivation assay of AR NTD in LNCaP cells co-transfected with Gal4UAS-TATA-luciferase and AR(1–558)-Gal4 DBD treated for 1 h with EPI-002, SINT1, or DMSO vehicle control followed by 24 h of induction of transactivation by incubation with forskolin (FSK) (left) or IL-6 (right). Luciferase activities were normalized to protein concentration and are presented as percentage of vehicle control. Error bars represent the mean ± S.E. of n = 4 independent experiments with triplicate wells. One-way ANOVA Dunnett's multiple comparison test was used for statistical analyses. ****, p < 0.0001. B, LNCaP cells were serum-starved and exposed to IL-6 for 6 h. Whole cell lysates were precleared with mouse IgG, immunoprecipitated with anti-STAT3 antibodies (IP), and then analyzed by immunoblotting (IB). Relative densities of AR in immunoprecipitated samples were normalized to STAT3 levels. Error bars are mean ± S.E. of n = 3 independent experiments with triplicate wells. One-way ANOVA Bonferroni's multiple comparison test was used for statistical analyses comparing the treatment groups with each other. *, p < 0.05. C, combination treatments in LNCaP cells co-transfected with PSA-Luc, PB-Luc, and ARR3-Luc. Cells were treated with SINT1, EPI-002, or a combination at a 1:1.3 ratio followed by R1881 treatment. Error bars represent the mean percentage inhibition ±S.E. of n = 3 independent experiments.
FIGURE 4.
FIGURE 4.
SINT1 inhibits AR splice variant and FL-AR transcriptional activities. The effect of SINT1 on AR splice variant AR-v567es is shown. A, whole cell protein lysates from cells treated in B were analyzed by Western blotting to reveal levels of forced expression of AR-V567es compared with endogenous FL-AR in LNCaP cells. B, COS-1 cells co-transfected with PB-Luc and an expression vector for AR-V567es prior to treatment with EPI-002, EPI-014 (inactive), SINT1, enzalutamide (ENZA), or DMSO vehicle control. Luciferase activities were normalized to protein concentrations of the samples. Error bars represent the mean ± S.E. of n = 3 separate experiments with triplicate wells. The effect of SINT1 on proliferation of cells expressing FL-AR (C) and constitutively active AR splice variant (D) is shown. LNCaP cells treated with 0.1 nm R1881 (C) and LNCaP95 cells (D) were exposed to EPI-002, bicalutamide (BIC), enzalutamide, SINT1, or DMSO vehicle control for 72 or 48 h, respectively. Error bars represent the mean percentage of vehicle control ±S.E. of n = 3 independent experiments with six technical replicates. One-way ANOVA Dunnett's multiple comparison test was used for statistical analyses. ****, p < 0.0001.
FIGURE 5.
FIGURE 5.
SINT1 decreases levels of expression of both FL-AR- and AR-V7-regulated genes. LNCaP95 cells were treated with vehicle (DMSO), bicalutamide (10 μm), enzalutamide (5 μm) or SINT1 (10 μm) prior addition of R1881 for 48 h. A, Transcript levels of FL-AR and its regulated genes KLK3/PSA, KLK2, TMPRSS2, NKX3.1, FKBP5, RHUO, and SLC1A1. B, transcript levels of AR-V7 and its regulated genes cyclin A2, CDC20, UBE2C, AKT1, and CDK1. Levels of transcripts were normalized to RPL13, a housekeeping gene. Data are presented as percentage of controls in the presence of androgen. Error bars represent mean ± S.E. of n = 4 independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. One-way ANOVA Tukey's multiple comparison test was used for statistical analyses.
FIGURE 6.
FIGURE 6.
In vivo, SINT1 inhibits the growth of CRPC expressing solely FL-AR or both FL-AR and AR splice variants. Tumor growth (A, LNCaP cells expressing FL-AR; B, LNCaP95 expressing FL-AR and AR splice variants) in castrated mice that received intratumoral doses of SINT1 (30 mg/kg of body weight) for a total of five doses or matching vehicle (DMSO) every 3 days is shown. Error bars represent the mean ± S.E. of n = 8. C, individual animal tumor volume change of the LNCaP tumors from A at the duration of the experiment on day 15. D, day 15 serum PSA percent change from the day of first dose. The dotted gray line indicates a 50% drop in serum PSA levels. E, photograph of harvested representative LNCaP tumors from animals that received SINT1 or vehicle (DMSO) treatment. F, effect of SINT1 on expression levels of AR-FL, AR-V7, ENO2, and SYP in LNCaP95 xenographs from B. Unpaired t test was used for statistical analyses. Error bars represent the mean ± S.E. of n = 9. *, p < 0.05; **, p < 0.01; ns, not significant.
FIGURE 7.
FIGURE 7.
SINT1 decreases proliferation and expression of PSA in CRPC xenografts. A, immunohistochemistry of sections of LNCaP xenografts harvested at the duration of the experiment and stained for Ki67 and hematoxylin and eosin (H&E). B, quantification of tissue samples stained for proliferation marker Ki67. Percentages of Ki67-positive cells were counted in sections from three xenografts for each treatment. At least 1100 cells per xenograft were counted. The total numbers of cells counted were 4323 (DMSO) and 5000 (SINT1). Error bars represent the mean ± S.E. of n = 3. Unpaired t test was used for statistical analyses. *, p < 0.05. C, staining for AR in three representative harvested xenografts from vehicle control-treated animals or SINT1-treated animals. D, PSA staining of four representative xenografts from vehicle control-treated animals or SINT1-treated animals. Scale bar, 20 μm.
FIGURE 8.
FIGURE 8.
Proposed binding model of SINT1. EPI binds Tau5 (36), whereas SINT1 may bind more toward the N terminus, perhaps overlapping into Tau1 (Figs. 2 and 3). STAT3 binds within the region encoding residues 234–558 (14). Brd4 binds within residues 120–160 (35). H, hinge region.
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