A quantitative high-throughput screen identifies novel inhibitors of the interaction of thyroid receptor beta with a peptide of steroid receptor coactivator 2.

The thyroid hormone receptors (TR) are members of the nuclear hormone receptor (NHR) superfamily that regulate development, growth, and metabolism. Upon ligand binding, TR releases bound corepressors and recruits coactivators to modulate target gene expression. Steroid receptor coactivator 2 (SRC2) is an important coregulator that interacts with TRß to activate gene transcription. To identify novel inhibitors of the TRß and SRC2 interaction, the authors performed a quantitative high-throughput screen (qHTS) of a TRß-SRC2 fluorescence polarization assay against more than 290 000 small molecules. The qHTS assayed compounds at 6 concentrations up to 92 µM to generate titration-response curves and determine the potency and efficacy of all compounds. The qHTS data set enabled the characterization of actives for structure-activity relationships as well as for potential artifacts such as fluorescence interference. Selected qHTS actives were tested in the screening assay using fluoroprobes labeled with Texas Red or fluorescein. The retest identified 19 series and 4 singletons as active in both assays with 40% or greater efficacy, free of compound interference, and not toxic to mammalian cells. Selected compounds were tested as independent samples, and a methylsulfonylnitrobenzoate series inhibited the TRß-SRC2 interaction with 5 µM IC(50). This series represents a new class of thyroid hormone receptor-coactivator modulators.

Methylsulfonylnitrobenzoates, a new class of irreversible inhibitors of the interaction of the thyroid hormone receptor and its obligate coactivators that functionally antagonizes thyroid hormone.

Thyroid hormone receptors (TRs) are members of the nuclear hormone receptor (NR) superfamily and regulate development, growth, and metabolism. Upon binding thyroid hormone, TR undergoes a conformational change that allows the release of corepressors and the recruitment of coactivators, which in turn regulate target gene transcription. Although a number of TR antagonists have been developed, most are analogs of the endogenous hormone that inhibit ligand binding. In a screen for inhibitors that block the association of TRß with steroid receptor coactivator 2 (SRC2), we identified a novel methylsulfonylnitrobenzoate (MSNB)-containing series that blocks this interaction at micromolar concentrations. Here we have studied a series of MSNB analogs and characterized their structure activity relationships. MSNB members do not displace thyroid hormone T3 but instead act by direct displacement of SRC2. MSNB series members are selective for the TR over the androgen, vitamin D, and PPARγ NR members, and they antagonize thyroid hormone-activated transcription action in cells. The methylsulfonylnitro group is essential for TRß antagonism. Side-chain alkylamine substituents showed better inhibitory activity than arylamine substituents. Mass spectrum analysis suggested that MSNB inhibitors bind irreversibly to Cys-298 within the AF-2 cleft of TRß to disrupt SRC2 association.

Quantification of the vitamin D receptor-coregulator interaction.

The vitamin D receptor (VDR) regulates a diverse set of genes that control processes including bone mineral homeostasis, immune function, and hair follicle cycling. Upon binding to its natural ligand, 1alpha,25(OH)(2)D(3), the VDR undergoes a conformational change that allows the release of corepressor proteins and the binding of coactivator proteins necessary for gene transcription. We report the first comprehensive evaluation of the interaction of the VDR with a library of coregulator binding motifs in the presence of two ligands, the natural ligand 1alpha,25(OH)(2)D(3) and a synthetic, nonsecosteroidal agonist LG190178. We show that the VDR has relatively high affinity for the second and third LxxLL motifs of SRC1, SRC2, and SRC3 and second LxxLL motif of DRIP205. This pattern is distinct in comparison to other nuclear receptors. The pattern of VDR-coregulator binding affinities was very similar for the two agonists investigated, suggesting that the biologic functions of LG190178 and 1alpha,25(OH)(2)D(3) are similar. Hairless binds the VDR in the presence of ligand through a LxxLL motif (Hr-1), repressing transcription in the presence and absence of ligand. The VDR binding patterns identified in this study may be used to predict functional differences among different tissues expressing different sets of coregulators, thus facilitating the goal of developing tissue- and gene-specific vitamin D response modulators.