Evaluation of Firefly and Renilla Luciferase Inhibition in Reporter-Gene Assays: A Case of Isoflavonoids

Abstract

Firefly luciferase is susceptible to inhibition and stabilization by compounds under investigation for biological activity and toxicity. This can lead to false-positive results in in vitro cell-based assays. However, firefly luciferase remains one of the most commonly used reporter genes. Here, we evaluated isoflavonoids for inhibition of firefly luciferase. These natural compounds are often studied using luciferase reporter-gene assays. We used a quantitative structure–activity relationship (QSAR) model to compare the results of in silico predictions with a newly developed in vitro assay that enables concomitant detection of inhibition of firefly and Renilla luciferases. The QSAR model predicted a moderate to high likelihood of firefly luciferase inhibition for all of the 11 isoflavonoids investigated, and the in vitro assays confirmed this for seven of them: daidzein, genistein, glycitein, prunetin, biochanin A, calycosin, and formononetin. In contrast, none of the 11 isoflavonoids inhibited Renilla luciferase. Molecular docking calculations indicated that isoflavonoids interact favorably with the D-luciferin binding pocket of firefly luciferase. These data demonstrate the importance of reporter-enzyme inhibition when studying the effects of such compounds and suggest that this in vitro assay can be used to exclude false-positives due to firefly or Renilla luciferase inhibition, and to thus define the most appropriate reporter gene.

High sensitivity and low-cost flavin luciferase (FLUXVc)-based reporter gene for mammalian cell expression

“…Polyphenol derivatives are among the bioactive compounds commonly investigated for their therapeutic use because of their wide biological activities ( 63 ) and due to their common availability from various higher plants including vegetables, fruits, cereals, legumes, herbs, and spices ( 64 , 65 ). As it is known that some of these compounds can affect luciferase-based reporter assays ( 13 , 66 ), we thus explored whether the Lux activity can be affected by these compounds. The effects of polyphenol compounds including resveratrol, daidzein, and formononetin ( Fig.…”

“…The data suggest that these compounds, particularly resveratrol, are strong inhibitors of Fluc possibly due to their ability to bind to the enzyme because of their structural similarity to the native D-luciferin ( Fig. S12 ) ( 12 , 13 ). The ability of FLUX Vc to withstand artifacts influenced by these compounds suggest that the FLUX Vc reporter gene should be useful for screening the biological activities of plant polyphenols in the future.…”

“…S1 B ). However, this reporter system has limitations in screening plant-derived compounds especially polyphenols or isoflavonoids because these compounds interfere with Fluc activities ( 12 , 13 ). Although the coelenterazine system is simpler than other systems because it only requires one substrate plus oxygen ( Fig.…”

A Multiplex Molecular Cell-Based Sensor to Detect Ligands of PPARs: An Optimized Tool for Drug Discovery in Cyanobacteria

“…Reduced Fluc values were previously referred, during the screening of low molecular weight compound libraries, and related to either toxicity or interference with Fluc activity [ 36 ]. For example, flavonoids are identified as one group of compounds that can inhibit Fluc [ 62 ] and are present in cyanobacteria [ 55 ]. However, in our case, reduced raw values were observed in both enzymes used in the constitutive vectors of the two sensor systems, Fluc and Rluc, when exposed to rosiglitazone concentrations above 10–100 nM.…”

Internal Light Sources‐Mediated Photodynamic Therapy Nanoplatforms: Hope for the Resolution of the Traditional Penetration Problem

“…The FLuc-luciferin bioluminescence system is the earliest and most widely used system. [57,58] With the participation of adenosine triphosphate (ATP), Mg 2+ , and O 2 , the FLuc-luciferin luminescence process involves two steps: in the first step, the substrate luciferin reacts with ATP to form the adenyl intermediate, while in the second step, the intermediate is oxidized by 3 O 2 to form firefly dioxetanone, which then decomposes and emits a yellow-green light with a peak at 560 nm. [59,60] By matching the emission of bioluminescence with the absorption of the PS, the PS can be effectively stimulated to generate ROS for cancer therapy.…”