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

Kelly Coffey,Claudia A Ryan-Munden,Anna Watson,Celine Roche,Ian R Hardcastle,Bernard T Golding,David R Newell,Lorraine Hewitt,Kety Huberman,Roger J Griffin,Susan Cook,Timothy J Blackburn,Hesta V Mcneill,Craig N Robson,Zoran Culig

doi:10.1371/journal.pone.0045539

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.

Highlights

Histone acetylation and deacetylation are key events in the regulation of chromatin structure
A number of hits were identified from the primary ALPHATM screen
Inhibitors against Histone deacetylases (HDAC) have been found to be successful in clinical trials; Histone acetyltransferases (HATs) inhibitors are at an earlier stage of development

Summary

Introduction

Histone acetylation and deacetylation are key events in the regulation of chromatin structure. Acetylation results in an open chromatin structure by removing positive charges from histones, inducing protein conformational changes, which allows transcriptional machinery to access the DNA and promote transcriptional activity. 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.

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