Figure 1.
Niphatenone B enantiomers block transactivation of AR NTD.
(A) Chemical structures of sintokamide, EPI-001, and niphatenone B enantiomers. (B) Transactivation assay of the AR NTD performed in LNCaP cells that were co-transfected with Gal4UAS-TATA-luciferase and AR-(1-558)-Gal4 DBD prior pre-treatment for 1 hour with 25 µM EPI-002, 7.0 µM niphatenones or DMSO vehicle control. Transactivation of AR NTD was induced by incubation with IL-6 (50 ng/ml) for 24 hours. Luciferase activity was normalized to protein concentration. (C) Niphatenones inhibit the constitutively active AR V567es splice variant. Cos-1 cells co-transfected with PB-luciferase and an expression vector for ARvar567 were treated with each niphatenone B (1 µM) or DMSO vehicle control. After 24 hours of exposure, cells were harvested and luciferase activities were normalized to protein concentrations of the samples. Data represent the mean ± SEM of n = 3 separate experiments with triplicate wells. Student's t test: **P<0.01; ***P<0.001.
Figure 2.
Niphatenone B inhibits AR activity.
Effect of (S)-niphatenone B, or bicalutamide (BIC) on R1881 induced endogenous AR transcriptional activity (A), 4-pregnene-3,20-dione (Prog, 10 nM) induced PR transcriptional activity (B), or dexamethasone (Dex, 10 nM) induced GR transcriptional activity (C) in LNCaP cells that were transiently transfected with PSA(6.1 kb)-luciferase reporter, PRE-luciferase or GRE-Luciferase reporters and expression vector for PR-β or GR. Luciferase activities were represented as percentage of vehicle activity. Data is presented as the mean ± SEM (n = 3). Representative competition binding curve showing displacement of 1 nM fluorescently labeled ligand from recombinant AR (D), PR (E), or GR (F) LBDs (25 nM) by bicalutamide (BIC), agonist R1881, progesterone or dexamethasone, and (S)-niphatenone B.
Figure 3.
Niphatenones block N/C interaction and inhibit expression of AR regulated genes.
(A) Mammalian two-hydrid assay using CV1 cells transfected with GAL4-AR DBD and/or VP16-AR TAD and Gal4-luciferase reporter. Cells were pretreated for 1 hour with bicalutamide or (S)-niphatenone B before addition of R1881 for 24 hours. Data represent the mean ± SEM of n = 3 separate experiments with triplicate wells. Student's t test: ***P<0.001. (B) PSA/KLK3 or (C) KLK2 mRNAs were inhibited by (S)-niphatenone B. LNCaP cells were pre-treated for 1 hour with (S)-niphatenone B, bicalutamide or vehicle prior to 16 hours incubation with R1881. Data represent the mean normalized expression (MNE) of PSA or KLK2 transcripts normalized to levels of GAPDH transcripts. Representative data of n = 3 separate experiments. *P<0.05, **P<0.01, *** P <0.001 (Student's t-test). (D) (S)-niphatenone B does not decrease endogenous AR levels in LNCaP cells. Cells were pre-treated for 1 hour with (S)-niphatenone B, bicalutamide or vehicle prior to the addition of R1881 for an additional 16 hours incubation before harvesting and Western blot analysis for AR and β-actin protein levels. Samples were run on the same gel but were not contiguous (black Lines) Error bars represent the mean ± SD of n = 3 separate experiments. Student's t test: ***p<0.001. (E) Nuclear translocation of YFP-AR in LNCaP cells transfected with an expression plasmid encoding an AR-YFP fusion protein in serum-free conditions for 24 hours prior to pre-treatment with (S)-niphatenone, bicalutamide, vehicle (DMSO), MDV3100, or R1881 for 2 hours. DAPI staining indicates the location of the nucleus. Scale bar, 20 µm.
Figure 4.
Niphatenone B covalently binds both AR AF-1 and GR AF-1.
(A) AR AF-1 and GR AF-1 peptides were incubated with DMSO (vehicle), niphatenone B 44 (structure shown at bottom of the) or EPI-054 for 45 minutes prior to Click-chemistry to add a fluorescein moiety to the probe. Fluorescein-labeled probes covalently bound to the AF-1 peptides were detected with reducing SDS-PAGE. Total protein in each of the lanes was shown by Coomassie blue staining of the gel. (B) Niphatenone alkylates glutathione. A mixture of glutathione (0.07 mmol), (S)-niphatenone (0.014 mmol), EPI-002 (0.014 mmol) was monitored by NMR after 1, 2, 4, 8, 24, 72 and 99 hours. Proton 8 and 9 integral values were used as a reference for niphatenone and carbon 21 integral value was used a reference for EPI-002. See Tables 1 and 2.
Table 1.
Integral Value of (S)-, (R)-Niphatenone B, and EPI-002.
Table 2.
Relative Intensity of (S)-, (R)-Niphatenone B, and EPI-002.