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

STAR Protoc. 2021 Jul 7;2(3):100658. doi: 10.1016/j.xpro.2021.100658. eCollection 2021 Sep 17.

Abstract

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).

Keywords: Cell Biology; Cell-based Assays; High Throughput Screening; Molecular Biology.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cells, Cultured
  • Constitutive Androstane Receptor
  • Cytological Techniques / methods*
  • Drug Discovery / methods*
  • Ligands*
  • Pregnane X Receptor
  • Receptors, Cytoplasmic and Nuclear* / agonists
  • Receptors, Cytoplasmic and Nuclear* / antagonists & inhibitors
  • Receptors, Cytoplasmic and Nuclear* / chemistry
  • Receptors, Cytoplasmic and Nuclear* / metabolism

Substances

  • Constitutive Androstane Receptor
  • Ligands
  • Pregnane X Receptor
  • Receptors, Cytoplasmic and Nuclear