Citation: Coffey K, Blackburn TJ, Cook S, Golding BT, Griffin RJ, et al. (2012) Characterisation of a Tip60 Specific Inhibitor, NU9056, in Prostate Cancer. PLoS ONE 7(10): e45539. doi:10.1371/journal.pone.0045539 Editor: Zoran Culig, Innsbruck Medical University, Austria Received April 18, 2012; Accepted August 21, 2012; Published October 8, 2012 Copyright: ?2012 Coffey et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding was provided by Cancer Research UK (CRUK) (C240/A7409; C29821/A10348) (www.cancerresearchuk.org) and Medical Research Council (MRC) PROMPT (G0100100/64424) (www.mrc.ac.uk). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: KH was employed by OSI Pharmaceuticals, Inc. Compounds discussed in this article are not patented, in development or being marketed by this company. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.
Introduction
Histone acetylation and deacetylation are key events in the regulation of chromatin structure. Histone acetyltransferases (HATs) catalyze the addition of acetyl groups to the e-amino terminus of lysine residues within histones. Acetylation results in an open chromatin structure by removing positive charges from histones, thus inducing protein conformational changes, which allows transcriptional machinery to access the DNA and promote transcriptional activity. Histone deacetylases (HDAC) oppose this process by promoting a closed chromatin structure, which is transcriptionally repressed. Furthermore, histone acetylation marks can function as docking sites for other proteins to interpret the `histone code’; for example, the tripartite motif containing 24 (TRIM24) was recently described as a `reader’ protein, which recognises both unmodified histone H3 at lysine 4 and histone H3 acetylated at lysine 23 on the same histone tail resulting in increased gene expression [1]. In addition, non-histone proteins such as p53 [2,3], ataxia telangiectasia mutated (ATM) [4] andandrogen receptor (AR) [5,6] can also be acetylated resulting in altered protein activity. Hence, protein acetylation and deacetylation can have significant effects on cell function, and for cells to maintain normal growth and differentiation it is important that these two functions maintain equilibrium. In support of this concept, HDAC inhibitors have been found to have wide ranging cellular effects and clinical activity in leukaemia [7,8], with Vorinostat (SAHA) being approved for clinical use in this disease. Modulation of histone acetylation clearly has therapeutic potential. Tip60, recently renamed KAT5, is a member of the MYST family of HAT enzymes first identified in 1996 [9]. Since then many cellular functions have been found to use this protein. Loss of Tip60 results in impaired DNA repair, as this HAT is activated in response to ionising radiation (IR), causing acetylation of histones and activation of p53 and ATM [4]. Inhibition of Tip60 should therefore sensitise cells to DNA damaging agents used as cancer therapeutics. Tip60 also functions in the NF-kB pathway,via interactions with B-cell CLL/lymphoma 3 (BCL-3) [10] and cAMP-dependent signalling [11]. Furthermore, Tip60 can function as a co-activator for a number of steroid hormone receptors including the AR, which is involved in the development and progression of prostate cancer (CaP). Studies have shown that AR can be acetylated by a number of HAT enzymes, including p300, p300/CBP-associated factor (PCAF) and Tip60, to increase its transcriptional activity [6,12]. AR acetylation is thought to regulate the recruitment of co-activators to the transcriptional machinery of androgen responsive genes [13]. Additionally, Tip60 is functionally up-regulated in clinical CaP specimens and expression correlates with disease progression [14]. In contrast, one report suggested that Tip60 is required to express the tumour metastasis suppressor KAI1 in CaP cell lines, suggesting that Tip60 is a tumour suppressor [15]. Similarly, a Tip60 gene knockout study proposed Tip60 as a haplo-insufficient tumour suppressor at pre and early-tumoral stages of lymphoma, breast and head and neck cancers [16]. However, studies on clinical prostate specimens contradict this suggestion and support Tip60 as an oncogene in CaP [13,17]. Thus, targeting the acetylase activity of Tip60 could be a useful therapeutic strategy in CaP. A small number of HAT inhibitors have been reported. Coupling a histone H3 peptide to CoA to form a bisubstrate inhibitor of HAT activity has been described; however, the compound has poor cell membrane permeability [18]. The natural products anacardic acid and garcinol are HAT inhibitors that are cell permeable; they sensitise cells to IR, which could be useful as a combination therapy for cancer treatment. Other inhibitors of HAT function include a-methylene butyrolactones [19], benzylidene acetones [20] and alkylidene malonates [21]. More recently, isothiazolones, which covalently bind to the HAT active site thiol, have been described as an effective starting point for molecular modelling-based approaches for generating more potent and specific inhibitors [22?4]. In the current study we employed a high throughput screening approach to identify selective inhibitors of Tip60. Based on the lead molecule, structurally related compounds were generated and tested for HAT inhibition and Tip60 specificity in order to identify a molecular tool for studies in cell line models of CaP.
Specificity of NU9056 for Tip60 Acetyltransferase
NU9056 (7), as well as compounds 5 and 6, were tested for in vitro activity against a panel of recombinant HAT enzymes, including p300, PCAF and GCN5, to determine whether they show greater specificity towards Tip60 than compound 1. ICR CCT129182, which has been shown to have inhibitory activity against p300 and PCAF, was also tested [24]. A number of compounds were found to inhibit the activity of Tip60 at low micromolar concentrations (Figure 1; Supplemental Figure S1). However, specificity towards Tip60 over other HAT enzymes tested was found to be greatest with compound 7 (NU9056) as shown in Table 1 (16.5-, 29- and .50-fold for selectivity for Tip60 over PCAF, p300 and GCN5, respectively).
NU9056 Inhibits Protein Acetylation in Prostate Cancer Cell Lines
Tip60 acetylates histone proteins, specifically histones H4 and H2A, in a nucleosomal context [25]. Furthermore, in vitro studies have demonstrated that Tip60 can also acetylate core histones H2A (Lys 5), H3 (Lys 14) and H4 (Lys 5, Lys 8, Lys 12 and Lys 16) [26,27]. To test whether NU9056 could inhibit acetylation of endogenous proteins targeted by Tip60, LNCaP cells were used as these are a representative model of androgen dependent CaP. The acetylation status of histone H4 at lysine 8 (H4K8) and 16 (H4K16) and histone H3 at lysine 14 (H3K14) was investigated in these cells by Western blotting.