Evaluation of firefly and Renilla luciferase inhibition in reporter-gene assays : a case of isoflavonoids

Kenda, Maša (author), Vegelj, Jan (author), Herlah, Barbara (author), Perdih, Andrej (author), Mladěnka, Přemysl (author), Sollner Dolenc, Marija (author)

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.

• Language: English
• Keywords: isoflavonoids, firefly luciferase, Renilla luciferase, inhibition, quantitative structure–activity relationships, molecular docking, reporter-gene assay, OECD test guidelines, alternative methods
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: FFA - Faculty of Pharmacy
• Publication status: Published
• Publication version: Version of Record
• Year: 2021
• Number of pages: 14 str.
• Numbering: Vol. 22, iss. 13, art. 6927
• PID: 20.500.12556/RUL-135754
• UDC: 547.972:615.4.:54
• ISSN on article: 1422-0067
• DOI: 10.3390/ijms22136927
• COBISS.SI-ID: 72671747
• Publication date in RUL: 30.03.2022

Record is a part of a journal

• Title: International journal of molecular sciences
• Shortened title: Int. j. mol. sci.
• Publisher: MDPI
• ISSN: 1422-0067
• COBISS.SI-ID: 2779162

Licences

• License: CC BY 4.0, Creative Commons Attribution 4.0 International
• Link: http://creativecommons.org/licenses/by/4.0/
• Description: This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.

Secondary language

• Language: Slovenian
• Keywords: izoflavonoidi, zaviralci, alternativne metode, molekularno mapiranje, farmacevtska kemija, flavonoidi