Quantitative analyses of aggregation, autofluorescence, and reactivity artifacts in a screen for inhibitors of a thiol protease.
Quantitative analyses of aggregation, autofluorescence, and reactivity artifacts in a screen for inhibitors of a thiol protease.
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DOI：
10.1021/jm901070c
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发表时间：
2010-01-14
影响因子：
7.3
通讯作者：
Simeonov A
中科院分区：
文献类型：
--
作者：
Jadhav A;Ferreira RS;Klumpp C;Mott BT;Austin CP;Inglese J;Thomas CJ;Maloney DJ;Shoichet BK;Simeonov A
高通量筛查中误报的感知和实际负担受到了相当多的关注；然而，关于非特异性效应的不同机制（例如化学反应性、测定信号干扰和胶体聚集）的贡献的研究很少。在这里，我们分析了针对半胱氨酸蛋白酶 cruzain 的浓度响应形式筛选 197,861 种不同化合物的结果，该靶标预计对反应性化合物特别敏感，并使用在通常会遇到显着化合物自发荧光的短波长区域进行光检测的测定形式。大约 1.9% 的筛选化合物是去垢剂敏感抑制剂。自发荧光和带有反应性官能团的化合物的贡献要低得多：在所有命中中，只有 1.8% 是自发荧光，1.48% 包含反应性或不需要的官能团。各个库源中误报的分布相对稳定。在测定缓冲液中加入去污剂的简单步骤抑制了约 93% 的原始命中的非特异性效应。
The perceived and actual burden of false positives in high-throughput screening has received considerable attention; however, few studies exist on the contributions of distinct mechanisms of non-specific effects like chemical reactivity, assay signal interference, and colloidal aggregation. Here, we analyze the outcome of a screen of 197,861 diverse compounds in a concentration-response format against the cysteine protease cruzain, a target expected to be particularly sensitive to reactive compounds and using an assay format with light detection in the short-wavelength region where significant compound autofluorescence is typically encountered. Approximately 1.9% of all compounds screened were detergent-sensitive inhibitors. The contribution from autofluorescence and compounds bearing reactive functionalities was dramatically lower: of all hits, only 1.8% were autofluorescent and 1.48% contained reactive or undesired functional groups. The distribution of false positives was relatively constant across library sources. The simple step of including detergent in the assay buffer suppressed the nonspecific effect of approximately 93% of the original hits.
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