A specific mechanism for nonspecific activation in reporter-gene assays.
A specific mechanism for nonspecific activation in reporter-gene assays.
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DOI：
10.1021/cb8000793
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发表时间：
2008-08-15
影响因子：
4
通讯作者：
Inglese, James
中科院分区：
文献类型：
--
作者：
Auld, Douglas S.;Thorne, Natasha;Nguyen, Dac-Trung;Inglese, James
生物发光在实现广泛的高通量筛查（HTS）测定格式方面的重要性证明了在行业和学术界的广泛使用。因此，了解可以通过小分子调节报告者酶活性的机制对于解释HTS数据至关重要。从这个角度来看，我们为基于细胞的荧光素酶报告基因测定法中抑制剂稳定荧光素酶的证据，从而导致信号激活的违反直觉现象。这些数据来自我们对荧光素酶抑制剂化合物结构的分析及其在分子文库中使用100个HTS实验的分子库中的流行率。因此，我们发现荧光素酶报道基因激活测定法中荧光素酶抑制剂的富集，但在使用其他记者的测定中没有。此外，对于几种荧光素酶抑制剂化学型，我们测量了报告基因稳定和信号激活的细胞，该细胞与使用纯化的荧光素酶确定的抑制作用并行，以对这些对比效应提供进一步的实验支持。
The importance of bioluminescence in enabling a broad range of high-throughput screening (HTS) assay formats is evidenced by widespread use in industry and academia. Therefore, understanding the mechanisms by which reporter enzyme activity can be modulated by small molecules is critical to the interpretation of HTS data. In this Perspective, we provide evidence for stabilization of luciferase by inhibitors in cell-based luciferase reporter-gene assays resulting in the counterintuitive phenomenon of signal activation. These data were derived from our analysis of luciferase inhibitor compound structures and their prevalence in the Molecular Libraries Small Molecule Repository using 100 HTS experiments available in PubChem. Accordingly, we found an enrichment of luciferase inhibitors in luciferase reporter-gene activation assays but not in assays using other reporters. In addition, for several luciferase inhibitor chemotypes, we measured reporter stabilization and signal activation in cells that paralleled the inhibition determined using purified luciferase to provide further experimental support for these contrasting effects.
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影响因子：
2.7
作者：
Leclerc, GM;Boockfor, FR;Frawley, LS
通讯作者：
Frawley, LS
影响因子：
3.5
作者：
THOMPSON, JF;HAYES, LS;LLOYD, DB
通讯作者：
LLOYD, DB
影响因子：
1.8
作者：
Thompson, Peggy A.;Wang, Shaohui;Appleman, James R.
通讯作者：
Appleman, James R.
影响因子：
7.3
作者：
Auld, Douglas S.;Southall, Noel T.;Inglese, James
通讯作者：
Inglese, James
DOI：
10.1073/pnas.0604348103
发表时间：
2006-08-01
影响因子：
11.1
作者：
Inglese, James;Auld, Douglas S.;Austin, Christopher P.
通讯作者：
Austin, Christopher P.