Groebe Duncan R, Maus Mary L, Pederson Terry, Clampit Jill, Djuric Stevan, Trevillyan James, Lin Chun W, Burns David J, Warrior Usha
Biological Screening, Abbott Laboratories, Abbott Park, IL, USA.
J Biomol Screen. 2003 Dec;8(6):668-75. doi: 10.1177/1087057103258587.
In micro-arrayed compound screening (microARCS), an agarose gel is used as a reaction vessel that maintains humidity and compound location as well as being a handling system for reagent addition. Two or more agarose gels may be used to bring test compounds, targets, and reagents together, relying on the pore size of the gel matrix to regulate diffusion of reactants. It is in the microenvironment of the agarose matrix that all the components of an enzymatic reaction interact and result in inhibitable catalytic activity. In an effort to increase the throughput of microARCS-based screens, reduce the effort involved in manipulating agarose gels, and reduce costs, blotter paper was used rather than a second agarose gel to introduce a substrate to a gel containing a target enzyme. In this assay, the matrix of the blotter paper did not prevent the substrate from diffusing into the enzyme gel. The compound density of the microARCS format, the ease of manipulating sheets of paper for reagent addition, and a scheduled protocol for running multiple gels allowed for a throughput capacity of more than 200,000 tests per hour. A protease assay was developed and run in the microARCS format at a rate of 200,000 tests per hour using blotter paper to introduce the substrate. Picks in the primary screen were retested in the microARCS format at a density of 384 compounds per sheet. IC(50) values were confirmed in a 96-well plate format. The screen identified several small molecule inhibitors of the enzyme. The details of the screening format and the analysis of the hits from the screen are presented.
在微阵列化合物筛选（microARCS）中，琼脂糖凝胶用作反应容器，它既能保持湿度和化合物位置，又可作为添加试剂的操作体系。可使用两块或更多琼脂糖凝胶将测试化合物、靶点和试剂混合在一起，依靠凝胶基质的孔径调节反应物的扩散。正是在琼脂糖基质的微环境中，酶促反应的所有组分相互作用并产生可被抑制的催化活性。为了提高基于microARCS筛选的通量，减少操作琼脂糖凝胶的工作量并降低成本，使用吸水纸而非第二块琼脂糖凝胶向含有目标酶的凝胶中引入底物。在该检测中，吸水纸基质并未阻止底物扩散到酶凝胶中。microARCS形式的化合物密度、添加试剂时操作纸片的便利性以及运行多块凝胶的预定方案使得每小时的通量能力超过200,000次检测。开发了一种蛋白酶检测方法，并以每小时200,000次检测的速率采用microARCS形式运行，使用吸水纸引入底物。在初筛中挑选的样本以每片384种化合物的密度在microARCS形式中重新检测。半数抑制浓度（IC50）值在96孔板形式中得到确认。该筛选鉴定出了几种该酶的小分子抑制剂。本文介绍了筛选形式的细节以及对筛选得到的命中结果的分析。
