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Off-DNA DNA-Encoded Library Affinity Screening.
ACS Combinatorial Science Pub Date : 2019-12-12 , DOI: 10.1021/acscombsci.9b00153
Amber L Hackler , Forrest G FitzGerald , Vuong Q Dang , Alexander L Satz 1 , Brian M Paegel
Affiliation  

DNA-encoded library (DEL) technology is emerging as a key element of the small molecule discovery toolbox. Conventional DEL screens (i.e., on-DNA screening) interrogate large combinatorial libraries via affinity selection of DNA-tagged library members that are ligands of a purified and immobilized protein target. In these selections, the DNA tags can materially and undesirably influence target binding and, therefore, the experiment outcome. Here, we use a solid-phase DEL and droplet-based microfluidic screening to separate the DEL member from its DNA tag (i.e., off-DNA screening), for subsequent in-droplet laser-induced fluorescence polarization (FP) detection of target binding, obviating DNA tag interference. Using the receptor tyrosine kinase (RTK) discoidin domain receptor 1 (DDR1) as a proof-of-concept target in a droplet-scale competition-binding assay, we screened a 67 100-member solid-phase DEL of drug-like small molecules for competitive ligands of DDR1 and identified several known RTK inhibitor pharmacophores, including azaindole- and quinazolinone-containing monomers. Off-DNA DEL affinity screening with FP detection is potentially amenable to a wide array of target classes, including nucleic acid binding proteins, proteins that are difficult to overexpress and purify, or targets with no known activity assay.

中文翻译:

非DNA DNA编码文库亲和力筛选。

DNA编码文库(DEL)技术正在成为小分子发现工具箱的关键要素。常规的DEL筛选(即DNA上筛选)通过亲和选择DNA标记的文库成员来查询大型组合文库,这些成员是纯化并固定的蛋白质靶标的配体。在这些选择中,DNA标签会实质性和不希望地影响靶标结合,从而影响实验结果。在这里,我们使用固相DEL和基于液滴的微流体筛选将DEL成员与其DNA标签分离(即脱DNA筛选),用于随后的液滴内激光诱导的荧光偏振(FP)检测靶标结合,避免了DNA标签的干扰。使用受体酪氨酸激酶(RTK)盘状蛋白结构域受体1(DDR1)作为液滴规模竞争结合测定中的概念证明目标,我们筛选了67 100个成员的药物样小分子固相DEL用于DDR1的竞争性配体,并鉴定了几种已知的RTK抑制剂药效基团,包括含氮吲哚和喹唑啉酮的单体。带有FP检测的DNA外DEL亲和力筛选可能适用于各种各样的目标类别,包括核酸结合蛋白,难以过表达和纯化的蛋白质或没有已知活性测定的目标。
更新日期:2019-12-31
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