An optimized protocol with a stepwise approach to identify specific nuclear receptor ligands from cultured mammalian cells

Abstract

Summary Here, we describe an optimized protocol to identify specific nuclear receptor ligands. First, to rule out any compound interference with luciferase activity per se, we describe an in vitro assay assessing potential inhibition or activation of luciferase enzymatic activity. Second, to comply with EMA and FDA guidelines to mitigate drug-drug interactions, we detail assays assessing constitutive androstane receptor (CAR) and pregnane X receptor (PXR) activation ability. Finally, to minimize off-target detection effects, we describe the use of mammalian one- (or two-) hybrid systems. For complete details on the use and execution of this protocol, please refer to Hering et al. (2018) .

The Molecular Circadian Clock Is a Target of Anti-cancer Translation Inhibitors

“…For this purpose, BMAL1 and PER2 promoters hooked to a luciferase gene were transfected in AC16 cells, from which luciferase activity was continuously recorded for several days. Prior to running these assays, we first assessed whether HHT interferes directly with luciferase enzymatic activity, as this is a major source for false positive detection in luciferase-based screening procedures (Berthier et al, 2021). The activity of purified recombinant luciferase enzyme was determined in the presence of varying concentrations of HHT or of a specific luciferase inhibitor as a positive control.…”

“…The purified recombinant luciferase (Promega, E2940) activity was determined in vitro as previously described (Berthier et al, 2021). The bioluminescence signal was quantified using a Victor Light Luminometer (PerkinElmer) in the presence of 0.1, 1, or 10 µM Luciferase Inhibitor II (Calbiochem, 119114) or of 0.1, 1, or 10 µM HHT.…”