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

Auld, Douglas, Breault, Lyne, Pandey, Pramod, Ho, Pei-I, Harbinski, Fred and Hill, William (2013) Reporter enzyme inhibitor study to aid assembly of orthogonal reporter gene assays. ACS Chemical Biology, 8 (5). pp. 1009-1017. ISSN 1554-8929


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 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 propriety 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 10µM using purified enzyme preparations. We then compared hit rates and chemotypes of confirmed 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 ranging from 40-70%, while unrelated luciferases such as firefly luciferases, GLuc, and NanoLuc showed <10% overlap. Examination of representative inhibitors in cell-based assays revealed that inhibitor-based-enzyme stabilization can lead to rises 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 enzymes.

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Date Deposited: 13 Oct 2015 13:13
Last Modified: 04 Jul 2016 23:46


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