Characterisation of a Tip60 Specific Inhibitor, NU9056, in Prostate Cancer

<div><p>Tip60 (KAT5) is a histone acetyltransferase (HAT enzyme) involved in multiple cellular processes including transcriptional regulation, DNA damage repair and cell signalling. In prostate cancer, aggressive cases over-express Tip60 which functions as an androgen receptor co-activator via direct acetylation of lysine residues within the KLKK motif of the receptor hinge region. The purpose of this study was to identify and characterise a Tip60 acetylase inhibitor. High-throughput screening revealed an isothiazole that inhibited both Tip60 and p300 HAT activity. This substance (initially identified as 4-methyl-5-bromoisothiazole) and other isothiazoles were synthesised and assayed against Tip60. Although an authentic sample of 4-methyl-5-bromoisothiazole was inactive against Tip60, in an <em>in vitro</em> HAT assay, 1,2-bis(isothiazol-5-yl)disulfane (NU9056) was identified as a relatively potent inhibitor (IC₅₀ 2 µM). Cellular activity was confirmed by analysis of acetylation of histone and non-histone proteins in a prostate cancer cell line model. NU9056 treatment inhibited cellular proliferation in a panel of prostate cancer cell lines (50% growth inhibition, 8–27 µM) and induced apoptosis via activation of caspase 3 and caspase 9 in a concentration- and time-dependent manner. Also, decreased androgen receptor, prostate specific antigen, p53 and p21 protein levels were demonstrated in response to treatment with NU9056. Furthermore, pre-treatment with NU9056 inhibited both ATM phosphorylation and Tip60 stabilization in response to ionising radiation. Based on the activity of NU9056 and the specificity of the compound towards Tip60 relative to other HAT enzymes, these chemical biology studies have identified Tip60 as a potential therapeutic target for the treatment of prostate cancer.</p> </div

NU9056 reduces PSA and p53 protein levels.

<p>To confirm the effects of Tip60 on androgen receptor activity we used 2.5 nM siRNA specifically targeted against Tip60 in LNCaP cells, or non-silencing control. Knockdown was achieved after 48 hours in steroid depleted medium after which time 10 nM DHT was applied to induce androgen receptor activity and PSA expression. RNA was collected after 24 hours DHT stimulation, reverse transcription and real-time PCR performed. Expression of (A) PSA and (B) Tip60 was normalised relative to HPRT1 expression. (C) LNCaP cells were treated with 24 µM NU9056 over 48 hours and protein samples were collected in SDS sample buffer. Protein analysis was carried out by SDS PAGE and Western blotting for p53, p21, AR, PSA and alpha tubulin. (D) Densitometry was performed on Western blots. All experiments were performed twice and the mean ± standard deviation is shown.</p

Chemical synthesis of Tip60 inhibitors.

<p>Part 1 - Synthesis of compounds <b>4–7</b>. Part 2 - Synthesis of Compounds <b>1</b> and <b>11</b>.</p

NU9056 reduces LNCaP cell survival by inducing apoptosis.

<p>(A) LNCaP cells were seeded onto 6 well plates for 24 hours, then increasing doses of NU9056 were applied for (i) 24 hours, (ii) 96 hours or (iii) GI₂₅ (17 µM) or (iv) GI₅₀ (24 µM) was applied over 4 days. All cells were collected and fixed with cytofix/cytoperm (BD) then caspase 3 and caspase 9 assay kits (BD) were used to assess their activity by flow cytometry. Fluorescence was detected on the FL-1 channel of the FACSCAN. (B) Analysis of the SubG1 population was performed on these same cells using propidium iodide to stain cellular DNA. LNCaP cells were seeded onto 6 well plates for 24 hours, then NU9056 was applied for (C) 1 or (D) 4 days. (E) LNCaP, LNCaP-AI and LNCaP-CdxR cells were seeded out onto 6 well plates and NU9056 was applied for 24 hours. Analysis of SubG1 was performed as described above. All cells were collected and fixed with cytofix/cytoperm (BD) then cell cycle analysis was performed using propidium iodide to stain cellular DNA. All FACS data was analysed using WinMDI. All experiments were performed 3 times and the mean ± standard error is shown. *p-value <0.05; **p-value <0.005; ***p-value <0.001.</p

NU9056 inhibits Tip60 accumulation and ATM phosphorylation in response to ionising radiation.

<p>To demonstrate the inhibition of Tip60 activity by NU9056, levels of pATM, Tip60 and γH2AX were investigated in response to IR in the presence and absence of drug. LNCaP cells were treated with 24 µM NU9056 or vehicle control for 1 hour prior to 0.5 Gy IR. Protein lysates were collected at various time points post-IR and analysed by Western blotting for (A) pATM and (B) Tip60 levels. (C) 293T, (D) LNCaP and (E) LNCaP-CdxR cells were pretreated with NU9056 (24 µM) or vehicle control for 1 hour prior to 2 Gy IR. Cells were fixed and stained for γH2AX foci over time and foci determined by immunofluorescence for 293T cells and flow cytometry for LNCaP and LNCaP-CdxR. Experiments were repeated 3 times with representative images shown and quantified data shown as mean % cells stained for γH2AX ± standard deviation.</p

NU9056 inhibits protein acetylation in prostate cancer cell lines.

<p>LNCaP cells were treated with increasing concentrations of NU9056 or the control compound, 1,2 bis(4-pyridyl)-ethane for 24 hours. (A) Levels of Tip60 were assessed by Western blotting. (B) LNCaP cells were treated with 2 µM TSA for 6 hours and levels of histone H4 acetylated-lysine 16, histone H4 acetylated-lysine 8 and histone H3 acetylated lysine 14 were assessed by Western blotting. LNCaP cells were treated with increasing concentrations of NU9056 or the control compound for 2 hours, then treated with the HDAC inhibitor TSA (2 µM) for a further 4 hours. (C) Levels of histone H4 acetylated-lysine 16, histone H4 acetylated-lysine 8 and histone H3 acetylated lysine 14 were assessed by Western blotting. (D) LNCaP cells were treated with 24 µM NU9056 over 4 days and the levels of acetylated tubulin were assessed by Western blotting. (E) LNCaP cells were treated with increasing concentrations of NU9056 or the control compound for 2 hours, then treated with the HDAC inhibitor TSA (2 µM) for a further 4 hours. Levels of acetylated tubulin were assessed by Western blotting. Alpha-tubulin was used as a loading control. Representative blots are shown for duplicate experiments.</p

IC₅₀ values of 1,2-bis(isothiazol-5-yl)disulfane (7) and related compounds towards HATs.

<p>To assess the activity against Tip60 HAT, <i>in vitro</i> HAT assays using ³H acetyl-CoA were carried out using histones as substrates. Assays were performed in quadruplicate and repeated twice. Individual IC₅₀ values are presented.</p

Table of Tip60 IC₅₀ values of isothiazoles and related compounds.

<p>To assess the activity against Tip60 HAT, <i>in vitro</i> HAT assays using ³H acetyl-CoA were carried out using histones as substrates. Assays were performed in quadruplicate and repeated twice. For compounds producing >50% inhibition at 100 μM, IC₅₀ values were calculated. Individual IC₅₀ values are presented. For other compounds the % inhibition at 100 μM is presented.</p

Knockdown of Tip60 reduces proliferation in LNCaP cells.

<p>(A) To confirm that inhibition of Tip60 can reduce cellular proliferation 2.5 nM siRNA specifically targeted against Tip60 in LNCaP cells or a non-silencing control was used. Proliferation was determined by sulforhodamine B (SRB) assays at 3 control proliferation doubling times after siRNA transfection in normal growth media. To confirm Tip60 knockdown, RNA was collected at 96 hours from a parallel experiment and assessed for Tip60 expression using (B) real-time PCR and (C) Western blotting. (D) Tip60 levels in prostate cancer cell lines were assessed by Western blotting. (E) Prostate cancer cell survival was assessed by treating LNCaP cells with 24 µM NU9056 for 24 hours then plating at varying cell densities (3×10³, 1.6×10⁴ and 3×10⁴) and allowing colonies to form over 2 weeks. Colonies were then fixed with Carnoy’s fixative and stained with crystal violet. Colonies were then counted and colony forming efficiency calculated. The mean of 3 experiments ± standard deviation is shown on bar charts. *p-value <0.05.</p