Biochemical Cofactor peptide assay

This assay represents a special subtype of the classical FRET interaction assays that are used by Phenex as standard screening techniques.

Once there is a priority list of Cofactor constructs identified that interact with the NR of interest in a ligand dose dependant manner, this interaction can be precisely quantified in this biochemical cell-free assay.

Most canonical coactivators and corepressors contain different interaction domains (IDs) that usually follow a certain consensus sequence, e.g. LXXLL in the case of coactivators. This native interaction in vivo can be recapitulated in vitro to some extent by using synthetic peptides that resemble the sequence around the canonical interaction motif.

It is possible to analyze the ligand dependency of Cofactor recruitment in a precise and quantitative fashion by determining the EC50 for interacting peptides as a function of the ligand used. In this so-called peptide gradient mode, the test compound is kept constant at saturating concentrations and the EC50 for each Cofactor peptide can be determined as function of the peptide concentration.

By these means, Phenex has been able to determine compound-specific differences in cofactor recruitment which is a basis for a binning of NR ligands into different groups. Representatives for each CoF recruitment pattern can then be tested either in gene expression assays such as microarrays or qRT-PCR or in more complex animal disease models with regard to their differential pharmacological profile.

Application examples for the Biochemical CoF peptide assay system:

- LXRß: FRET-based compound-CoF peptide profiling, peptide gradient
- LXRα/ß: FRET-based compound-CoF peptide profiling