Sabet R S, Marcotte P, Glaser K, Burns D J, Warrior U, Groebe D R
Abbott Laboratories, Biological Screening, R4PN, 200 Abbott Park Rd, Abbott Park IL 60064, USA.
Comb Chem High Throughput Screen. 2004 Mar;7(2):93-100. doi: 10.2174/138620704773120784.
We have developed a method of spraying assay reagents onto a target gel in the Micro-Arrayed Compound Screening ( micro ARCS) 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 micro ARCS, 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 micro ARCS format. IC(50) 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 micro ARCS, 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.
我们开发了一种将检测试剂以微阵列化合物筛选（micro ARCS）形式喷覆到目标凝胶上的方法。将目标凝胶铺到化合物片上后，后续试剂可通过喷覆到目标凝胶上施加。这种喷覆方法可使检测试剂节省多达10倍，无需再浇铸琼脂糖凝胶，并使筛选通量提高3倍。为证明将喷覆方法应用于micro ARCS的效果，我们针对组蛋白去乙酰化酶（HDAC）抑制剂筛选了超过600,000种化合物。将市售的HDAC底物和反应显色剂分别直接喷覆到凝胶上以启动反应和放大信号。初筛中的挑选物以micro ARCS形式在每张片384种化合物的密度下重新检测。活性化合物的半数抑制浓度（IC50）值在96孔板检测中得到确认。该筛选鉴定出了几种该酶的小分子抑制剂，包括几类已知HDAC抑制剂的成员。micro ARCS的高密度形式、喷覆方法的效率以及添加检测试剂的定时顺序相结合，使得筛选通量达到每小时200,000次检测。我们展示了筛选形式的细节以及对筛选命中物的分析。
