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

Abstract

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 in vitro 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.

Lysine Acetyltransferase GCN5 Potentiates the Growth of Non-small Cell Lung Cancer via Promotion of E2F1, Cyclin D1, and Cyclin E1 Expression

“…Our findings provide new insights into the previously under-appreciated roles of histone hyper-acetylation during cancer development. Recently, small molecule KAT inhibitors have demonstrated relevance in oncology (17,47), thereby providing an additional strategy for combating cancer through the manipulation of histone acetylation. Given the diverse cellular roles of KATs and their working mechanism within transcription factor-containing complexes, KAT inhibitors face the same challenge of a lack of specificity that hampers their therapeutic application in cancer cases.…”

“…Aggressive cases of prostate cancers have been found to exhibit overexpression of the KAT Tip60, the extent of which correlates with disease progression (17,18). In contrast, another report has suggested that Tip60 is required for the expression of the tumor metastasis suppressor KAI1 in prostate cancer cell lines, suggesting that Tip60 is a tumor suppressor (19).…”

Deubiquitinating Enzyme Usp12 Is a Novel Co-activator of the Androgen Receptor

“…To measure colony-forming ability cells were reverse transfected with siRNA for 72 h, followed by reseeding at varying cell densities and incubated for 14 days to allow colony formation and stained with crystal violet. Colonies were counted and the surviving fraction calculated (31).…”

Epigenetic Regulation of Cytosolic Phospholipase A2 in SH-SY5Y Human Neuroblastoma Cells

“…This suggests that the p300/CBP and GCN5/PCAF classes of HATs were minimally or not involved in the suppression of cPLA 2 α upregulation. In contrast, treatment of cells with theTip60-specific HAT inhibitor NU9056 [77] led to significant reduction of TSA-induced upregulation of cPLA 2 α mRNA expression, suggesting a role of Tip60 in cPLA 2 α expression. We further showed that NU9056 inhibited MS-275-induced upregulation of cPLA 2 α mRNA, indicating that the class I HDACs and Tip60 HAT are specific enzymes involved in epigenetic regulation of cPLA 2 .…”

“…We further showed that NU9056 inhibited MS-275-induced upregulation of cPLA 2 α mRNA, indicating that the class I HDACs and Tip60 HAT are specific enzymes involved in epigenetic regulation of cPLA 2 . HDAC inhibitors TSA and MS-275 induce acetylation of H3K9, H3K14, and trimethylation of H3K4 [65,[77][78][79][80] (Table 1). Inhibition of the Tip60 HAT by the synthetic compound, NU9056, has been reported to cause reduction in acetylation levels of H3K14, H4K8, and H4K16 [77] (Table 1).…”