当前位置： SCI文献检索 > ACS Chemical Biology期刊下所有文献 > Reporter enzyme inhibitor study to aid assembly of orthogonal reporter gene assays.

Reporter enzyme inhibitor study to aid assembly of orthogonal reporter gene assays.

Abstract：

: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 β-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 "NanoLuc" derived from the luminescent sea shrimp Oplophorus gracilirostris. To determine hit rates and structure-activity relationships, we screened a collection of 42,460 PubChem compounds at 10 μM using purified enzyme preparations. We then compared hit rates and chemotypes of actives for each enzyme. The hit rates ranged from <0.1% for β-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 (40-70%), 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.

journal_name

ACS Chem Biol

journal_title

ACS chemical biology

authors

Ho PI,Yue K,Pandey P,Breault L,Harbinski F,McBride AJ,Webb B,Narahari J,Karassina N,Wood KV,Hill A,Auld DS

doi

10.1021/cb3007264

subject

Has Abstract

pub_date

2013-05-17 00:00:00

pages

1009-17

issue

eissn

1554-8929

issn

1554-8937

journal_volume

pub_type

杂志文章

在线工具

Reporter enzyme inhibitor study to aid assembly of orthogonal reporter gene assays.