In addition, the level of Tip60 was assessed following treatment with NU9056 and the control compound, 1,2-bis(4-pyridyl)ethane (Figure 3A) demonstrating that Tip60 levels themselves are unaffected. As the basal levels of these acetylated histone marks are quite low, the HDAC inhibitor trichostatin A (TSA) was introduced to remove the influence of HDAC activity on acetylation in the cellular assay. In the presence of TSA alone, acetylation was increased at H4K8, H4K16 and H3K14 (Figure 3B), consistent with previous reports [28?0]. Increasing concentrations of NU9056 resulted in decreased levels of acetylated histone H4K16, H3K14 and H4K8, targets for Tip60-mediated acetylation (Figure 3C). Furthermore, the control compound, 1,2-bis(4-pyridyl)ethane, did not affect any of the histone modifications studied. To define further the HAT inhibitory activity of NU9056, acetylation of the non-histone protein a-tubulin was also investigated. When LNCaP cells were incubated with NU9056 (24 mM) for 24 hours a decrease in acetylated tubulin was observed. However, by 72 hours acetylation had returned to basal levels (Figure 3D). To confirm that this effect was due to NU9056, acetylated tubulin was monitored but no change in levels was observed within 6 hours unlike alterations to histone modifications (Figure 3E). Densitometry for all Western blots is shown in Supplemental Figure S2.
Results High Throughput Screen (HTS) and Hit Validation
A high throughput screening campaign for Tip60 inhibitors was conducted at OSI Pharmaceuticals Ltd. Assays based on the ALPHATM screen and DELFIATM formats were developed and used to screen a structurally diverse compound collection (,80,000 members). A number of hits were identified from the primary ALPHATM screen. However, most of these did not show significant activity in the secondary screen. A single compound OXA-10 (initially identified as 4-methyl-5-bromoisothiazole, 1), was identified as a hit in both screens and the activity was replicated with repurchased material. Repurchased OXA-10 showed activity against Tip60 (IC50 1.1 mM – Figure 1) and p300 (IC50 2.7 mM) (Table 1), but not other histone acetyltransferases, e.g. PCAF and GCN5 (IC50.100 mM). LC-MS analysis of the repurchased sample of OXA-10 indicated the presence of ,80% 4-methyl-5-bromoisothiazole 1, but showed that it contained ,20% of an unknown impurity. In order to validate 1 as the active inhibitor of Tip60 in OXA-10, the synthesis of 1 was undertaken, along with analogues, in order to develop structure activity relationships. Details of the chemical synthesis (Figure 2) can be found in Supplementary Information.
NU9056 Inhibits Cell Growth
We observed that knockdown of Tip60 in LNCaP cells resulted in an inhibition of cell proliferation by approximately 33% (pvalue = 0.0019) (Figure 4A). Efficient knockdown of Tip60 mRNA in these cells was confirmed by real-time PCR (Figure 4B, 70% knockdown; p-value = 0.0313) and Western blotting (Figure 4C). If NU9056 action was mediated through inhibiting Tip60 enzymatic activity, inhibition of cell proliferation by NU9056 would be expected. Indeed, proliferation was found to be reduced in the presence of NU9056 in all CaP cell lines tested as measured by sulforhodamine B (SRB) assay (Table 2, Supplementary Figure S3, S4). Interestingly LNCaP cells, which are androgen responsive and express a mutated but functional AR as well as wild type p53, and the bone metastasis derived PC3 cells, which do not express aFigure 1. Table of Tip60 IC50 values of isothiazoles and related compounds. To assess the activity against Tip60 HAT, in vitro 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 mM, IC50 values were calculated. Individual IC50 values are presented. For other compounds the % inhibition at 100 mM is presented.
functional AR and are p53 null, displayed similar GI50 concentrations (24 mM 62 and 27 mM 62 for LNCaP and PC3 cells, respectively). However, LNCaP-AI cells, a sub-line of LNCaP cells serially maintained in steroid-depleted media, which still express a functional AR and are a model of castrate resistant CaP post androgen ablation therapy, have a lower GI50 (24 mM 62 and 16 mM 61 for LNCaP and LNCaP-AI, respectively). Other cell line models of androgen independence, e.g. LNCaP-CdxR, which is serially maintained in the presence of bicalutamide and CWR22rv1, show significantly greater sensitivity to NU9056 than the parentalLNCaP cell line (GI50 values of 12 mM 62.5 and 7.5 mM 60.2 (p,0.05 and p,0.0005, respectively)). Tip60 levels were measured by Western blotting in these cell lines (Figure 4D) to reveal that the most sensitive cell line, CWR22rv1, actually expresses the most Tip60. To test whether NU9056 can reduce the survival of CaP cells, colony forming ability was evaluated following exposure of LNCaP cells to 24 mM (GI50) NU9056 for 24 hours. Colony forming ability was reduced following NU9056 exposure, which was statistically significant (p,0.05) (Figure 4E).assessed by Western blotting. In addition, we investigated the levels of p53 and its target gene p21, as p53 is also a target for Tip60. We discovered that the levels of both AR and p53 were reduced after 24 hours by 2- and 3-fold respectively, and that the products of the target genes, p21 and PSA, were also reduced by 3and 2-fold respectively (Figure 6C,D). This result suggests that indeed Tip60 is involved in AR and p53 signalling in this cell line and that NU9056 may, by modulating Tip60 acetylation activity, affect these important downstream targets.NU9056 Inhibits the DNA Damage Response in Prostate Cancer Cells
Tip60 is known to play a role in the DNA damage response [25]. Upon exposure to IR Tip60 is activated, which results in acetylation of histone proteins and activation of ATM and p53 [4]. To test whether NU9056 could inhibit the DNA damage response via inhibition of Tip60 acetylase activity, the activation of ATM was assessed by Western blotting in response to IR after pretreatment with NU9056.
