Classification of compounds according to their cofactor recruitment and selective gene induction capabilities

Selective Cofactor recruitment by a ligand-stimulated Nuclear Receptor is the major reason for compound- and tissue-selective gene regulation or pharmacological effects.

Phenex is the pioneer in performing SNuRM® analyses in the sense of determining compound-dependent differences in Cofactor recruitment in different assays formats. Phenex has established the following modules for the qualitative and quantitative assessment of Cofactor selectivity:

Quantitative differences in Cofactor recruitment which can be determined using our SNuRM® platform translate in vivo into differential gene response and selective pharmacological effects. It is the goal of a SNuRM® project to
a. bin the existing panel of ligands at a given Nuclear Receptor into different response groups, and
b. ideally identify those "surrogate marker" Cofactors whose recruitment is predictive for a certain gene or even pharmacological response

Phenex SNuRM® platform

If such a correlation b. can be successfully established, these "indicative" Cofactors can be used in respective assays (Mammalian-2-Hybrid or FRET) to guide high content screening or lead optimisation towards more selective Nuclear Receptor ligands with more desired properties.

The Yeast-2-Hybrid profiling system

Phenex has cloned a comprehensive list of human Nuclear Receptors Ligand Binding Domains (LBDs) and a huge panel of Cofactors (CoF) that are described in the literature. This panel is used by Phenex to determine ligand-dependent Cofactor recruitment in the yeast system. For this purpose, Phenex employs the direct mating of respective Nuclear Receptor and CoF interaction vectors harboured by a yeast a and alpha type, respectively. Together with an robotized all-fluid phase dilution scheme, Phenex is able to assay for ligand dependency of Cofactor interactions in a dose response manner.

The Yeast-2-Hybrid direct mating system is ideal to obtain an overview which Cofactor (constructs) are recruited to a certain Nuclear Receptor in a ligand-dependent fashion. Each Nuclear Receptor has its own distinct CoF binding pattern upon administration of its cognate native ligand. However, subtle differences between classical full agonists and partial agonists can be detected in this system. This makes it a good starting point to analyze SNuRM-effects of a set of Nuclear Receptor binding drug candidates.

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 Nuclear Receptor of interest in a ligand dose dependent 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 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 the basis for binning of Nuclear Receptor 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: