Reporter Enzyme Inhibitor Study To Aid Assembly of Orthogonal Reporter Gene Assays
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- 作者：Pei-i Ho ; Kimberley Yue ; Pramod Pandey ; Lyne Breault ; Fred Harbinski ; Aaron J. McBride ; Brian Webb ; Janaki Narahari ; Natasha Karassina ; Keith V. Wood ; Adam Hill ; Douglas S. Auld
- 刊名：ACS Chemical Biology
- 出版年：2013
- 出版时间：May 17, 2013
- 年：2013
- 卷：8
- 期：5
- 页码：1009-1017
- 全文大小：506K
- 年卷期：v.8,no.5(May 17, 2013)
- ISSN：1554-8937
文摘
Reporter gene assays (RGAs) are commonly used to measure biological pathway modulation by small molecules. Understanding how such compounds interact with the reporter enzyme is critical to accurately interpret RGA results. To improve our understanding of reporter enzymes and to develop optimal RGA systems, we investigated eight reporter enzymes differing in brightness, emission spectrum, stability, and substrate requirements. These included common reporter enzymes such as firefly luciferase (Photinus pyralis), Renilla reniformis luciferase, and 尾-lactamase, as well as mutated forms of R. reniformis luciferase emitting either blue- or green-shifted luminescence, a red-light emitting form of Luciola cruciata firefly luciferase, a mutated form of Gaussia princeps luciferase, and a proprietary luciferase termed 鈥淣anoLuc鈥?derived from the luminescent sea shrimp Oplophorus gracilirostris. To determine hit rates and structure鈥揳ctivity relationships, we screened a collection of 42,460 PubChem compounds at 10 渭M using purified enzyme preparations. We then compared hit rates and chemotypes of actives for each enzyme. The hit rates ranged from <0.1% for 尾-lactamase to as high as 10% for mutated forms of Renilla luciferase. Related luciferases such as Renilla luciferase mutants showed high degrees of inhibitor overlap (40鈥?0%), while unrelated luciferases such as firefly luciferases, Gaussia luciferase, and NanoLuc showed <10% overlap. Examination of representative inhibitors in cell-based assays revealed that inhibitor-based enzyme stabilization can lead to increases in bioluminescent signal for firefly luciferase, Renilla luciferase, and NanoLuc, with shorter half-life reporters showing increased activation responses. From this study we suggest strategies to improve the construction and interpretation of assays employing these reporter enzymes.