Brandish Philip E, Chiu Chi-Sung, Schneeweis Jonathan, Brandon Nicholas J, Leech Clare L, Kornienko Oleg, Scolnick Edward M, Strulovici Berta, Zheng Wei
Department of Neurobiology, West Point, PA, USA.
J Biomol Screen. 2006 Aug;11(5):481-7. doi: 10.1177/1087057106288181. Epub 2006 Jun 7.
Enzymes are often considered less "druggable" targets than ligand-regulated proteins such as G-protein-coupled receptors, ion channels, or other hormone receptors. Reasons for this include cellular location (intracellular vs. cell surface), typically lower affinities for the binding of small molecules compared to ligand-specific receptors, and binding (catalytic) sites that are often charged or highly polar. A practical drawback to the discovery of compounds targeting enzymes is that screening of compound libraries is typically carried out in cell-free activity assays using purified protein in an inherently artificial environment. Cell-based assays, although often arduous to design for enzyme targets, are the preferred discovery tool for the screening of large compound libraries. The authors have recently described a novel cell-based approach to screening for inhibitors of a phosphatase enzyme and now report on the development and implementation of a homogeneous 3456-well plate assay for D-amino acid oxidase (DAO). Human DAO was stably expressed in Chinese hamster ovary (CHO) cells, and its activity was measured as the amount of hydrogen peroxide detected in the growth medium following feeding the cells with D-serine. In less than 12 weeks, the authors proved the concept in 96-and then 384-well formats, miniaturized the assay to the 3456-well (nanoplate) scale, and screened a library containing more than 1 million compounds. They have identified several cell-permeable inhibitors of DAO from this cell-based high-throughput screening, which provided the discovery program with a few novel and attractive lead structures.
与配体调节蛋白（如G蛋白偶联受体、离子通道或其他激素受体）相比，酶通常被认为是较难“成药”的靶点。原因包括细胞定位（细胞内与细胞表面）、与配体特异性受体相比，小分子结合的亲和力通常较低，以及结合（催化）位点通常带电荷或极性很高。发现靶向酶的化合物的一个实际缺点是，化合物库的筛选通常在无细胞活性测定中进行，使用纯化的蛋白质，处于本质上人工的环境中。基于细胞的测定，尽管针对酶靶点设计通常很艰巨，但却是筛选大型化合物库的首选发现工具。作者最近描述了一种基于细胞的新方法来筛选磷酸酶抑制剂，现在报告一种用于D-氨基酸氧化酶（DAO）的均相3456孔板测定的开发和实施。人DAO在中国仓鼠卵巢（CHO）细胞中稳定表达，其活性通过在用D-丝氨酸喂养细胞后在生长培养基中检测到的过氧化氢量来测量。在不到12周的时间里，作者在96孔然后384孔板形式中验证了该概念，将测定小型化到3456孔（纳米板）规模，并筛选了一个包含超过100万种化合物的文库。他们从这种基于细胞的高通量筛选中鉴定出了几种DAO的细胞可渗透抑制剂，为发现计划提供了一些新颖且有吸引力的先导结构。
