A cell-based ultra-high-throughput screening assay for identifying inhibitors of D-amino acid oxidase
A cell-based ultra-high-throughput screening assay for identifying inhibitors of D-amino acid oxidase
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DOI：
10.1177/1087057106288181
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发表时间：
2006-08-01
影响因子：
--
通讯作者：
Zheng, Wei
中科院分区：
文献类型：
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作者：
Brandish, Philip E.;Chiu, Chi-Sung;Zheng, Wei
酶通常被认为是比配体调节的蛋白质（如G蛋白偶联受体、离子通道或其他激素受体）更不“可药用”的靶标。其原因包括细胞位置（细胞内与细胞表面），与配体特异性受体相比，小分子的结合亲和力通常较低，以及通常带电荷或高极性的结合（催化）位点。发现靶向酶的化合物的实际缺点是化合物文库的筛选通常在固有的人工环境中使用纯化的蛋白质在无细胞活性测定中进行。基于细胞的分析，虽然往往很难设计酶的目标，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新的基于细胞的方法来筛选磷酸酶的抑制剂，现在报告了D-氨基酸氧化酶（DAO）的均匀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，并且其活性测量为用D-丝氨酸喂养细胞后在生长培养基中检测到的过氧化氢的量。在不到12周的时间里，作者在96孔和384孔的格式中证明了这一概念，将检测方法小型化到3456孔（纳米板）规模，并筛选了包含超过100万种化合物的库。他们已经从这种基于细胞的高通量筛选中确定了几种细胞可渗透的DAO抑制剂，这为发现计划提供了一些新颖且有吸引力的先导结构。
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 toot 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 I 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.