Screen for TIP60 Inhibitors

描述(由申请人提供)：根据最近的初步数据，建议Tip60乙酰转移酶抑制剂将用作细胞DNA损伤反应的调节剂和化疗或放射治疗的敏感剂。用从细胞中分离的Tip60复合体和细菌产生的重组Tip60可以在体外测定乙酰基转移酶的活性。AIM 1将开发一种Tip60活性的体外测试方法，并对其进行优化，用于96孔板，用于高通量筛选Tip60抑制剂。AIM 2将演示该筛查在识别已知的(相对非特异性的)Tip60抑制物方面的作用。这种抑制剂将通过二次筛选，测试该抑制剂对细胞对DNA损伤的反应、对DNA损伤后细胞存活的影响以及对细胞Tip60复合体的乙酰转移酶活性的影响。综合起来，这些目标将建立Tip60抑制剂的高通量检测，并证明该检测已准备好用于高通量的化学抑制剂筛选。建立的初级高通量筛选和生物二级筛选将有助于筛选具有生物活性的化学物质，以抑制细胞中的Tip60。由于Tip60参与了DNA损伤反应，因此这些抑制剂有望成为化疗或放射治疗的增敏剂。此外，由于Tip60调节许多癌症相关蛋白的转录程序，如雄激素受体、P53和Myc，Tip60抑制剂可以作为致癌或应激诱导途径的特异性抑制剂。

DESCRIPTION (provided by applicant): Based on recent preliminary data, it is proposed that inhibitors of TIP60 acetyltransferase will be useful as modulators of a cell's DNA damage response and as sensitizers to chemo- or radiotherapy. The acetyltransferase activity can be measured in vitro with TIP60 complex isolated from cells and with bacterially produced recombinant TIP60. Aim 1 will develop an in vitro assay for TIP60 activity and optimize it for use in 96-well plates for a high throughput screen for TIP60 inhibitors. Aim 2 will demonstrate the utility of the screen to identify a known (relatively non-specific) inhibitor of TIP60. This inhibitor will be taken through the secondary screens that will test the effect of the inhibitor on a cell's response to DNA damage, on cell survival after DNA damage and on the acetyltransferase activity of the cell's TIP60 complex. Together these aims will establish a high throughput assay of TIP60 inhibitors and demonstrate the readiness of the assay for a high throughput screen for chemical inhibitors. The primary high throughput screen and the biological secondary screens established will help screen for chemicals that are biologically active for inhibiting TIP60 in cells. Because TIP60 is involved in the DNA damage response the inhibitors are expected to be useful as sensitizers to chemo- or radio-therapy. In addition, because TIP60 regulates the transcriptional program of many cancer related proteins like the androgen receptor, p53 and Myc, the TIP60 inhibitors could function as specific inhibitors of oncogenic or stress-induced pathways.