Department of Chemistry and Biochemistry, University of Colorado, Campus Box 215, Boulder, Colorado 80309, USA.
Biochemistry. 2010 Mar 23;49(11):2551-62. doi: 10.1021/bi9020764.
Typically, biochemical screens that employ pure macromolecular components focus on single targets or a small number of interacting components. Researches rely on whole cell screens for more complex systems. Bacterial DNA replicases contain multiple subunits that change interactions with each stage of a complex reaction. Thus, the actual number of targets is a multiple of the proteins involved. It is estimated that the overall replication reaction includes up to 100 essential targets, many suitable for discovery of antibacterial inhibitors. We have developed an assay, using purified protein components, in which inhibitors of any of the essential targets can be detected through a common readout. Use of purified components allows each protein to be set within the linear range where the readout is proportional to the extent of inhibition of the target. By performing assays against replicases from model Gram-negative and Gram-positive bacteria in parallel, we show that it is possible to distinguish compounds that inhibit only a single bacterial replicase from those that exhibit broad spectrum potential.
通常，采用纯大分子成分的生化筛选集中于单个靶标或少数相互作用的成分。研究人员依赖于全细胞筛选来研究更复杂的系统。细菌 DNA 复制酶包含多个亚基，这些亚基与复杂反应的每个阶段的相互作用发生变化。因此，实际的靶标数量是涉及的蛋白质数量的倍数。据估计，整个复制反应包括多达 100 个必需的靶标，其中许多靶标适合发现抗菌抑制剂。我们已经开发了一种使用纯化蛋白成分的测定法，其中任何必需靶标的抑制剂都可以通过共同的读出结果来检测。使用纯化的成分可以使每个蛋白都处于线性范围内，其中读出结果与靶标的抑制程度成正比。通过对模型革兰氏阴性菌和革兰氏阳性菌的复制酶进行平行测定，我们表明有可能区分仅抑制单个细菌复制酶的化合物和具有广谱潜力的化合物。
