Deploying Fluorescent Nucleoside Analogues for High-Throughput Inhibitor Screening
Deploying Fluorescent Nucleoside Analogues for High-Throughput Inhibitor Screening
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DOI：
10.1002/cbic.201900671
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发表时间：
2019-12-03
期刊：
影响因子：
3.2
通讯作者：
Imperiali, Barbara
中科院分区：
文献类型：
--
作者：
Seebald, Leah;Madec, Amael G. E.;Imperiali, Barbara
药物发现过程中的高通量小分子筛选通常依赖于基于荧光的方法，包括荧光偏振和荧光/福斯特共振能量转移。这些技术使用高度可访问的仪器；然而，它们可能受到高假阴性率和背景信号的影响，或者可能涉及引入荧光团对的复杂方案。在这里，我们介绍了荧光核苷类似物的合成和应用，作为竞争性结合分析的直接方法的基础。一般方法描述了选择性荧光环境敏感（ES）核苷类似物，可适应于作用于核苷基底物的各种酶。我们展示了一组尿苷类似物的筛选，并开发了一种利用TcdB糖基转移酶（GT）发现基于片段的铅的检测方法，TcdB糖基转移酶是一种与艰难梭菌毒力相关的酶。用于该高通量筛选的基于尿嘧啶的探针与TcdB GT的K-D值为7.2 μ m，结合后荧光强度增加了约30倍。基于es的探针检测与其他两种筛选方法比较。
High-throughput small-molecule screening in drug discovery processes commonly rely on fluorescence-based methods including fluorescent polarization and fluorescence/Forster resonance energy transfer. These techniques use highly accessible instrumentation; however, they can suffer from high false-negative rates and background signals, or might involve complex schemes for the introduction of fluorophore pairs. Herein we present the synthesis and application of fluorescent nucleoside analogues as the foundation for directed approaches for competitive binding analyses. The general approach describes selective fluorescent environment-sensitive (ES) nucleoside analogues that are adaptable to diverse enzymes that act on nucleoside-based substrates. We demonstrate screening a set of uridine analogues and development of an assay for fragment-based lead discovery with the TcdB glycosyltransferase (GT), an enzyme associated with virulence in Clostridium difficile. The uridine-based probe used for this high-throughput screen has a K-D value of 7.2 mu m with the TcdB GT and shows a >30-fold increase in fluorescence intensity upon binding. The ES-based probe assay is benchmarked against two other screening approaches.