Quenching the firefly bioluminescence by various ions

Zhang, Huateng; Bai, Haixiu; Jiang, Tianyu; Ma, Zhao; Cheng, Ye; Zhou, Yubin; Du, Lüpei; Li, Minyong

doi:10.1039/c5pp00432b

Cited by 10 publications

(7 citation statements)

References 36 publications

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“…Although the bioluminescence measurement shows potential for the continuous monitoring of cytotoxicity in 3D cultures, in some cases, we may need to pay careful attention because some compounds may inhibit the luciferin-luciferase reaction [26, 27] or stabilize luciferase protein in cells by interacting with it [28], even though the toxicants used in this study do not affect D-luciferin and ELuc reaction (data not shown). This issue may be resolved by the combined use of multiple luciferases [6], such as the dual luciferase reporter assay that harnesses firefly and Renilla luciferases by utilizing substrate specificity, or a multicolor luciferase assay that uses multiple luciferases that produce different color emissions by reacting with D-luciferin.…”

Section: Discussionmentioning

confidence: 99%

Continuous long-term cytotoxicity monitoring in 3D spheroids of beetle luciferase-expressing hepatocytes by nondestructive bioluminescence measurement

et al. 2017

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BackgroundThree-dimensional (3D) spheroids are frequently used in toxicological study because their morphology and function closely resemble those of tissue. As these properties are maintained over a long term, repeated treatment of the spheroids with a test object is possible. Generally, in the repeated treatment test to assess cytotoxicity in the spheroids, ATP assay, colorimetric measurement using pigments or high-content imaging analysis is performed. However, continuous assessment of cytotoxicity in the same spheroids using the above assays or analysis is impossible because the spheroids must be disrupted or killed. To overcome this technical limitation, we constructed a simple monitoring system in which cytotoxicity in the spheroids can be continuously monitored by nondestructive bioluminescence measurement.ResultsMouse primary hepatocytes were isolated from transchromosomic (Tc) mice harboring a mouse artificial chromosome (MAC) vector expressing beetle luciferase Emerald Luc (ELuc) under the control of cytomegalovirus immediate early enhancer/chicken β-actin promoter/rabbit β-globin intron II (CAG) promoter, and used in 3D cultures. We confirmed that both luminescence and albumin secretion from the spheroids seeded in the 96-well format Cell-ableTM were maintained for approximately 1 month. Finally, we repetitively treated the luminescent 3D spheroids with representative hepatotoxicants for approximately 1 month, and continuously and nondestructively measured bioluminescence every day. We successfully obtained daily changes of the dose-response bioluminescence curves for the respective toxicants.ConclusionsIn this study, we constructed a monitoring system in which cytotoxicity in the same 3D spheroids was continuously and sensitively monitored over a long term. Because this system can be easily applied to other cells, such as human primary cells or stem cells, it is expected to serve as the preferred platform for simple and cost-effective long-term monitoring of cellular events, including cytotoxicity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-017-0374-1) contains supplementary material, which is available to authorized users.

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Section: Discussionmentioning

confidence: 99%

Continuous long-term cytotoxicity monitoring in 3D spheroids of beetle luciferase-expressing hepatocytes by nondestructive bioluminescence measurement

et al. 2017

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“…15,16 Rui Fontes et al indicated that the bioluminescence of luciferin could be inhibited by inorganic pyrophosphate and tripolyphosphate due to their reaction with L-AMP. 17 So far, many compounds containing different structures have been reported that could inhibit firefly luciferase, such as ions, 18 (E)-2-fluoro-4′methoxystilbene, 19 N-pyridin-2-ylbenzamides, 20 2-phenylnaphthalenes, 21 aryltriazoles, 22 2-phenylnaphthalenes, 23 5-benzyl-3phenyl-4,5-dihydroisoxazoles, and 5-benzyl-3-phenyl-1,4,2-dioxazoles. 11 In the current study, we found that some chalcone derivatives could potently inhibit the firefly luciferase activity.…”

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confidence: 99%

“…Auld et al reported PTC124 (3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid) as a potent Fluc inhibitor with an IC 50 of 7 nM. , Rui Fontes et al indicated that the bioluminescence of luciferin could be inhibited by inorganic pyrophosphate and tripolyphosphate due to their reaction with L-AMP . So far, many compounds containing different structures have been reported that could inhibit firefly luciferase, such as ions, (E)-2-fluoro-4′-methoxystilbene, N-pyridin-2-ylbenzamides, 2-phenylnaphthalenes, aryltriazoles, 2-phenylnaphthalenes, 5-benzyl-3-phenyl-4,5-dihydroisoxazoles, and 5-benzyl-3-phenyl-1,4,2-dioxazoles …”

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confidence: 99%

Inhibiting Firefly Bioluminescence by Chalcones

Zhang

Lin

et al. 2017

Anal. Chem.

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Chalcone refers to an aromatic ketone and an enone that constitutes the central core for various important biological compounds in drug discovery. Moreover, the firefly luciferase (Fluc) as the bioluminescent reporter has been widely used in life science research and high-throughput screening (HTS). However, Fluc might suffer from direct inhibition by HTS compounds resulting in the occurrence of "false positives." In the current research, we discovered a series of chalcone compounds as Fluc inhibitors with favorable potency both in vitro and in vivo. Moreover, our compound 3i showed remarkable systemic inhibition in transgenic mice. Both enzymatic kinetics study and cocrystal structure demonstrated that compound 3i is competitive for substrate aminoluciferin, while noncompetitive for ATP. Besides, compound 3i exhibited excellent selectivity as a promising quenching agent in a simulated dual-luciferase reporter assay. We believed that our research would contribute to improving scientists' awareness of the Fluc inhibitors, pay attention to the bias results, and even expand the utilization of bioluminescence in life science research.

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“…Control of variables affecting the bioluminescence reaction is essential, including the composition of the reading medium. Several ions have been reported to quench the bioluminescence reaction of the firefly luciferase ( 13 ). Cations should be strictly controlled while performing the assay, not only because of the potential quenching effect of the medium, but also due to the requirement of magnesium for the activity of this enzyme ( 11 , 14 ).…”

Section: Discussionmentioning

confidence: 99%