Related Experiment Video

Updated: Aug 30, 2026

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators
07:41

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

Published on: February 20, 2018

Comparison of assay technologies for a nuclear receptor assay screen reveals differences in the sets of identified

Xiang Wu1, J Fraser Glickman, Benjamin R Bowen

  • 1Exelixis, South San Francisco, CA 94083-0511, USA. swu@exelixis.com

Journal of Biomolecular Screening
|October 22, 2003
PubMed

Insights

Different high-throughput screening assay technologies yield varying results for nuclear receptor antagonist discovery. AlphaScreen identified the most functional antagonists compared to time-resolved fluorescence (TRF) and time-resolved fluorescence resonance energy transfer (TR-FRET).

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Drug Discovery

Background:

  • High-throughput screening (HTS) relies on diverse assay technologies for drug discovery.
  • Previous studies suggest assay technology choice impacts hit identification.
  • Assay format selection is critical for identifying biologically relevant compounds.

Purpose of the Study:

  • To compare the performance of three assay technologies: AlphaScreen, time-resolved fluorescence (TRF), and time-resolved fluorescence resonance energy transfer (TR-FRET).
  • To evaluate the hit identification consistency across different assay formats for nuclear receptor antagonists.
  • To determine which assay technology identifies the most functionally active compounds in a cell-based assay.

Main Methods:

  • Developed a nuclear receptor antagonist assay using AlphaScreen, TRF, and TR-FRET formats.
  • Screened approximately 42,000 compounds from the Novartis library across all three formats.
  • Validated hits through dose-response experiments and a subsequent cell-based functional assay.

Main Results:

  • AlphaScreen, TRF, and TR-FRET identified 104, 23, and 57 active compounds, respectively, with only 18 common hits.
  • Of 128 compounds tested in a cell-based assay, 35 showed activity.
  • AlphaScreen yielded the highest number of validated functional antagonists (34 hits).

Conclusions:

  • Assay technology significantly influences hit identification in high-throughput screening.
  • AlphaScreen demonstrated superior performance in identifying functional nuclear receptor antagonists compared to TRF and TR-FRET.
  • Results underscore the importance of selecting appropriate assay technologies for effective drug discovery.

More Related Videos

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists10:51

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

Published on: November 15, 2013

Related Concept Videos

Drug-Receptor Interactions01:29

Drug-Receptor Interactions

Drug-receptor interaction describes the binding of receptors by drugs, but not all drug-receptor interactions result in activation and tissue response. For instance, the binding of agonists activates the receptor to generate a cellular reaction, while antagonists bind to receptors without causing their activation.
Several parameters, such as the drug's affinity for its receptor and its efficacy, which is its ability to activate the receptor, determine the drug's effect on the tissue.
Transducer Mechanism: Nuclear Receptors01:31

Transducer Mechanism: Nuclear Receptors

Nuclear receptors, or NRs, are unique transcription factors that regulate gene transcription and affect the cellular pathways involved in reproduction, development, or metabolism. Their ability to be stimulated by small lipophilic ligands and control vital cellular processes makes them ideal drug targets. Nearly 10-15% of currently prescribed drugs target these receptors.
About 48 different soluble family members of nuclear receptors are identified that can be divided into two main classes:
Drug-Receptor Interaction: Antagonist01:28

Drug-Receptor Interaction: Antagonist

An antagonist is a drug that binds strongly to a receptor without activating it. An antagonist prevents other molecules, such as neurotransmitters or hormones, from binding to the receptor and triggering a cellular response. Such interaction effectively hinders the normal physiological processes mediated by the receptor, resulting in various pharmacological effects depending on the specific receptor targeted.
Antagonists can be classified as competitive or noncompetitive based on their...
The Two-State Receptor Model01:29

The Two-State Receptor Model

The two-state receptor model explains a drug's interaction with receptors, such as G protein-coupled receptors and ligand-gated ion channels, to induce or inhibit a biological response. When no natural ligands are present, a receptor exists in an equilibrium of inactive (Ri) and active (Ra) conformations. The inactive form does not produce a response, while the active form generates a basal effect known as constitutive activity.
The binding affinity of a drug determines its interaction with one...
Drug-Receptor Interaction: Agonist01:25

Drug-Receptor Interaction: Agonist

Agonists are drugs that interact with specific receptors in the body to produce a biological response. When an agonist binds to a receptor, it activates or enhances the receptor's function, leading to physiological effects. The interaction between agonist drugs and receptors is crucial for their therapeutic action in various medical treatments.
Agonists can bind to receptors in different ways. Some agonists bind directly to the receptor's active site, mimicking the endogenous ligand's action.
Spare Receptors01:30

Spare Receptors

Some receptors remain unoccupied even when an agonist produces a maximal response. Such empty ones are called spare receptors. In presence of spare receptors the maximum effect of an agonist drug is achieved with fewer than 100% of the receptors being occupied. To determine the presence of spare receptors, scientists often compare the concentration of the drug needed to produce 50% of the maximum effect (EC50) with the concentration of the drug needed to occupy 50% of the receptors (Kd). If the...