Nuclear Receptor Assays for Xenobiotic Response
PXR (SXR) and CAR – modulating the xenobiotic response
PXR (SXR) and CAR have been reported to mediate the expression of drug metabolizing enzymes, notably certain members of the cytochrome P450 family and other proteins relevant to ADME studies, such as drug transporters. Invitrogen has developed LanthaScreen™ TR-FRET assays designed to quickly assess test compounds for interactions with these specialized nuclear receptors.
LanthaScreen™ PXR (SXR) Competitive Binding Assay
Our LanthaScreen™ PXR (SXR) Competitive Binding Assay contains all the needed reagents you need to conduct compound affinity testing:
- A proprietary fluorescent Fluormone™ ligand
- An optimized buffer system
- Terbium-labeled anti-GST antibody
- Purified PXR (SXR)-LBD protein
The PXR protein is indirectly labeled with terbium via the anti-GST antibody bound to the GST tag on the receptor. When this labeled protein binds to the Fluormone™ ligand, a high TR-FRET ratio will result. If a test compound that binds to the PXR protein is added to the reaction, the test compound will compete with the Fluormone™ ligand, resulting in a lower TR-FRET ratio. Since the change in TR-FRET ratio only occurs in the presence of a competitor, it can be used as a convenient and accurate indicator of the relative affinity of the test compound for PXR (Figure 1 below).
LanthaScreen CAR Coactivator Assay
Ligand binding to nuclear receptors causes conformational changes in the receptor, resulting in a cascade of events including dissociation of repressor proteins, association of coactivator proteins, and assembly of pol II and other transcriptional factors for activation of target genes. A coactivator interaction assay has been developed for the study of CAR-LBD protein and the effects of test compound binding (Figure 2). The CAR assay relies on the conformational change in the receptor that takes place upon ligand binding, whereupon the constitutive association of CAR with a fluorescein-labeled coactivator peptide is either disrupted (by inverse agonists) or the interaction between the receptor and fluorescein-labeled coactivator peptide is enhanced (by agonists).
For your in-house profiling efforts, Life Technologies has developed a diverse panel of human nuclear receptor proteins and assays designed to allow easy profiling of these targets.
The PXR (SXR) protein is available from Life Technologies under an exclusive license from Puracyp, Inc.