Related Experiment Video
Updated: Jul 2, 2026
10:51
Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists
Published on: November 15, 2013
Characterization of nuclear receptor ligands by multiplexed peptide interactions
1GlaxoSmithKline, Research Triangle Park, North Carolina, USA.
Current Protocols in Cytometry
|September 5, 2008
Summary
This study presents a novel microsphere-based system to assess how small molecules affect nuclear receptor protein and peptide binding. This method enables rapid, sensitive analysis of molecular interactions and protein conformation changes.
Area of Science:
- Biochemistry
- Molecular Biology
- Assay Development
Background:
- Nuclear receptors are crucial drug targets, but characterizing their interactions with ligands is complex.
- Existing methods for studying nuclear receptor-ligand interactions can be time-consuming and low-throughput.
- Understanding these interactions is key to developing novel therapeutics.
Purpose of the Study:
- To develop a multiplexed, high-throughput assay for evaluating small molecule effects on nuclear receptor-peptide binding.
- To characterize nuclear receptor ligands based on their impact on protein-peptide interaction profiles.
- To demonstrate the adaptability of the assay for diverse molecular interaction studies.
Main Methods:
- Utilized a multiplexed microsphere-based system with uniquely fluorescent, peptide-coupled microspheres.
- Employed flow cytometric analysis to identify and quantify interactions in a 96-well microtiter plate format.
- Developed a no-wash assay format for efficient analysis of molecular binding.
Main Results:
- Successfully analyzed up to 100 different peptide-nuclear receptor interactions in a single assay well.
- Demonstrated rapid and sensitive characterization of nuclear receptor ligands.
- Showcased the ability to evaluate ligands that induce unique protein conformations.
Conclusions:
- The microsphere-based system offers a sensitive, moderately high-throughput method for analyzing nuclear receptor-ligand interactions.
- This approach allows for the rapid evaluation of small molecules impacting protein structure and binding dynamics.
- The assay is versatile and applicable to various molecular interaction studies beyond nuclear receptors.
Related Concept Videos
01:31Transducer 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:
About 48 different soluble family members of nuclear receptors are identified that can be divided into two main classes:
01:29The 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...
The binding affinity of a drug determines its interaction with one...
00:49Ligand Binding and Linkage
Allosteric proteins have more than one ligand binding site; the binding of a ligand to any of these sites influences the binding of ligands to the other sites. When a protein is allosteric, its binding sites are called coupled or linked. In the case of enzymes, the site that binds to the substrate is known as the active site and the other site is known as the regulatory site. When a ligand binds to the regulatory site, this leads to conformational changes in the protein that can influence the...
00:49Ligand Binding and Linkage
Allosteric proteins have more than one ligand binding site; the binding of a ligand to any of these sites influences the binding of ligands to the other sites. When a protein is allosteric, its binding sites are called coupled or linked. In the case of enzymes, the site that binds to the substrate is known as the active site and the other site is known as the regulatory site. When a ligand binds to the regulatory site, this leads to conformational changes in the protein that can influence the...
01:29Drug-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.
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.
02:40Ligand Binding Sites
Proteins are dynamic macromolecules that carry out a wide variety of essential processes; however, the activities of most proteins depend on their interactions with other molecules or ions, known as ligands.
Protein-ligand interactions are quite specific; even though numerous potential ligands surround a cellular protein at any given time, only a particular ligand can bind to that protein. Moreover, a ligand binds only to a dedicated area on the surface of the protein, known as the...
Protein-ligand interactions are quite specific; even though numerous potential ligands surround a cellular protein at any given time, only a particular ligand can bind to that protein. Moreover, a ligand binds only to a dedicated area on the surface of the protein, known as the...