A high throughput scintillation proximity imaging assay for protein methyltransferases.
A high throughput scintillation proximity imaging assay for protein methyltransferases.
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DOI：
10.2174/138620712800194468
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发表时间：
2012-06-01
影响因子：
1.8
通讯作者：
Djaballah H
中科院分区：
文献类型：
--
作者：
Ibanez G;Shum D;Blum G;Bhinder B;Radu C;Antczak C;Luo M;Djaballah H
蛋白质甲基转移酶（pmt）通过翻译后各种蛋白质底物（包括组蛋白）的甲基化来协调表观遗传修饰。由于这一过程的失调与许多癌症广泛相关，因此筛选pmt抑制剂具有相关的兴趣，因为它们为发现具有潜在化疗用途的小分子提供了新的靶向性机会。因此，我们开发了一种酶筛选策略，可以适应闪烁接近成像测定（SPIA）格式，以识别这些抑制剂。我们利用s -腺苷基- l- [3H-甲基]-蛋氨酸的可用性，对生物素化肽底物赖氨酸残基上的酶催化[3H]-甲基加成进行了监测。放射性标记的肽随后被链霉亲和素包被的SPA成像PS珠捕获。我们将该策略应用于4个pmt: SET7/9、SET8、SETD2和EuHMTase1，并优化了检测条件，使Z '值在0.48至0.91之间。该SPIA对四种pmt的稳定性能在大约7,000种化合物的中试筛选中得到验证。我们确定了四个目标的80次累计命中。NF279是一种苏拉明类似物，可特异性抑制SET7/9和SETD2， IC50值分别为1.9 μM和1.1 μM。另一种已鉴定的化合物，Merbromin，一种局部防腐剂，被归类为四种pmt的泛活性抑制剂。这些发现表明，我们提出的SPIA策略对多种pmt是通用的，可以成功地用于识别pmt的新型和特异性抑制剂。特异性PMT抑制剂可能构成一类新的抗增殖药物，具有潜在的治疗用途。
Protein methyltransferases (PMTs) orchestrate epigenetic modifications through post-translational methylation of various protein substrates including histones. Since dysregulation of this process is widely implicated in many cancers, it is of pertinent interest to screen inhibitors of PMTs, as they offer novel target-based opportunities to discover small molecules with potential chemotherapeutic use. We have thus developed an enzymatic screening strategy, which can be adapted to scintillation proximity imaging assay (SPIA) format, to identify these inhibitors. We took advantage of S-adenosyl-L-[3H-methyl]-methionine availability and monitored the enzymatically catalyzed [3H]-methyl addition on lysine residues of biotinylated peptide substrates. The radiolabeled peptides were subsequently captured by streptavidin coated SPA imaging PS beads. We applied this strategy to four PMTs: SET7/9, SET8, SETD2, and EuHMTase1, and optimized assay conditions to achieve Z′ values ranging from 0.48 to 0.91. The robust performance of this SPIA for the four PMTs was validated in a pilot screen of approximately 7,000 compounds. We identified 80 cumulative hits across the four targets. NF279, a suramin analogue found to specifically inhibit SET7/9 and SETD2 with IC50 values of 1.9 and 1.1 μM, respectively. Another identified compound, Merbromin, a topical antiseptic, was classified as a pan-active inhibitor of the four PMTs. These findings demonstrate that our proposed SPIA strategy is generic for multiple PMTs and can be successfully implemented to identify novel and specific inhibitors of PMTs. The specific PMT inhibitors may constitute a new class of anti-proliferative agents for potential therapeutic use.
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影响因子：
2.9
作者：
Suh-Lailam, Brenda B.;Hevel, Joan M.
通讯作者：
Hevel, Joan M.
影响因子：
16
作者：
Kubicek, Stefan;O'Sullivan, Roderick J.;Jenuwein, Thomas
通讯作者：
Jenuwein, Thomas
影响因子：
11.4
作者：
Chevillard-Briet, M;Trouche, D;Vandel, L
通讯作者：
Vandel, L
影响因子：
7.3
作者：
Feng, You;Li, Mingyong;Zheng, Yujun George
通讯作者：
Zheng, Yujun George
影响因子：
1.8
作者：
Shelton, Christopher C.;Tian, Yuan;Li, Yue-Ming
通讯作者：
Li, Yue-Ming