29 human nuclear receptors – the worldwide largest collection of cellular and biochemical nuclear receptor assays
Phenex has assembled the NR Resource - a worldwide unmatched collection of clones, expression constructs and assays to enable Nuclear Receptor drug discovery & research.
This collection comprises over 3,000 recombinant Nuclear Receptor and Cofactor constructs in various assay and expression formats, i.e. including all 48 human Nuclear Receptors. Taking the NR resource as a basis, Phenex has developed assays for most of the Nuclear Receptors in different formats.
The following list of Gal4-reporter assays (so-called "Mammalian-1-Hybrid" or "Mammalian-2-Hybrid" assays) can be employed for general
or selectivity screening purposes. The quality and standardisation parameters for each assay are available upon request.
List of available Cellular assays
The Mammalian-1-Hybrid is derived from the Mammalian-2-Hybrid setup. Whereas in the latter, the binding partners are defined in their role as interaction, transactivation and DNA-binding components, the Mammalian-1-Hybrid makes use of the fact that all mammalian cell types express a set of Nuclear Receptors and their associated cofactors. Formally correct, the M1H constructs are Gal-DNA binding domain (Gal4-DBD) fusion constructs with a Nuclear Receptor Ligand binding domain (NR-LBD). The reporter construct is a Gal4 minimal promoter driven luciferase reporter. The difference, however is, that the Gal4-DBD - NR-LBD fusion protein by itself cannot initiate transcription and hence is dependent on the presence of other cofactors which are not defined in this system. Therefore, different mammalian cell lines which are employed for this system can yield different results, depending on the cofactor environment.
Since all M1H constructs from Phenex use homologous LBDs from different NRs, the same Gal4-DBD and the same reporter in the same cell line, Phenex has chosen this assay type for comparative binding analysis, e.g. for selectivity screening against a nuclear receptor panel.
To have an alternative for the Y2H system and to verify cofactor (CoF) interactions identified in yeast, Phenex uses a classical Mammalian-2-Hybrid system for a more quantitative analysis. In this setup, the NR is usually expressed as a NFkappaB activation domain fused LBD. The cofactor construct is usually expressed as a Gal4-DNA binding domain fusion. Both are transiently transfected into a mammalian cell line and assayed for NR-CoF interaction by a Gal4 promoter driven reporter construct.
The following list of FRET assays can be employed for general or selectivity screening purposes.
List of available Biochemicals assays
FRET Assay System
Phenex uses Nuclear Receptor Ligand Binding Domains, fused to GST as one binding partner and biotinylated peptides with sequences around the Cofactors LXXLL motifs as the other binding partner.
The FRET assay principle is the energy transfer, when Eu-chelate as fluorescent donor and Allophycocyanin as fluorescent acceptor come close to each other. If the titrated ligand modulates the interaction between Nuclear Receptor and peptide, the fluorescent signal is modulated in a ligand dose dependent manner.