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首页> 外文期刊>Combinatorial Chemistry & High Throughput Screening >Screening for Inhibitors of Histone Deacetylase by Incorporating a Spraying Method to Micro-Arrayed Compound Screening
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Screening for Inhibitors of Histone Deacetylase by Incorporating a Spraying Method to Micro-Arrayed Compound Screening

机译：通过将喷雾方法结合到微阵列化合物筛选中筛选组蛋白脱乙酰基酶抑制剂

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摘要

We have developed a method of spraying assay reagents onto a target gel in the Micro-Arrayed Compound Screening (µARCS) format. After application of target gels to compound sheets, subsequent reagents can be applied by spraying onto the target gel. The spraying method conserves on assay reagents by up to 10-fold, eliminates the need for casting additional agarose gels, and increases the throughput of a screen by 3-fold. To demonstrate the efficacy of applying the spraying method to µARCS, we screened over 600,000 compounds for inhibitors of histone deacetylase (HDAC). Commercially available HDAC substrate and reaction developer were sprayed directly onto the gel to initiate the reaction and to amplify the signal, respectively. Picks in the primary screen were retested at a density of 384 compounds per sheet in the µARCS format. IC50 values for active compounds were confirmed in a 96-well plate assay. The screen identified several small molecule inhibitors of the enzyme, including members of several classes of known HDAC inhibitors. The combination of the high-density format of µARCS, the efficiency of the spraying method, and a timed sequence of adding assay reagents permitted a screening throughput of 200,000 tests an hour. We present the details of the screening format and the analysis of the hits from the screen.
机译：我们已经开发了一种以微阵列化合物筛选（µARCS）格式将测定试剂喷雾到目标凝胶上的方法。在将目标凝胶施加到化合物片材上之后，可以通过喷涂到目标凝胶上来施加随后的试剂。喷涂方法可节省多达10倍的分析试剂，无需浇铸额外的琼脂糖凝胶，并使筛分的通量提高3倍。为了证明将喷雾方法应用于µARCS的功效，我们筛选了600,000种化合物作为组蛋白脱乙酰基酶（HDAC）抑制剂。将市售的HDAC底物和反应显影剂分别直接喷涂到凝胶上以引发反应并放大信号。以µARCS格式以每张384个化合物的密度重新测试主筛中的选料。活性化合物的IC50值在96孔平板测定中得到确认。筛选确定了该酶的几种小分子抑制剂，包括几种已知HDAC抑制剂的成员。 µARCS的高密度形式，喷涂方法的效率以及定时添加测定试剂的顺序相结合，每小时筛选产量为200,000个测试。我们介绍了筛选格式的详细信息以及来自屏幕的点击量分析。

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