Discovery of Small Molecule Inhibitors of the Interaction of the Thyroid Hormone Receptor with Transcriptional Coregulators

Abstract

Thyroid hormone (3,5,3-triiodo-L-thyronine, T3) is an endocrine hormone that exerts homeostatic regulation of basal metabolic rate, heart rate and contractility, fat deposition, and other phenomena (1, 2). T3 binds to the thyroid hormone receptors (TRs) and controls their regulation of transcription of target genes. The binding of TRs to thyroid hormone induces a conformational change in TRs that regulates the composition of the transcriptional regulatory complex. Recruitment of the correct coregulators (CoR) is important for successful gene regulation. In principle, inhibition of the TR-CoR interaction can have a direct influence on gene transcription in the presence of thyroid hormones. Herein we report a high throughput screen for small molecules capable of inhibiting TR coactivator interactions. One class of inhibitors identified in this screen was aromatic ␤-aminoketones, which exhibited IC 50 values of ϳ2 M. These compounds can undergo a deamination, generating unsaturated ketones capable of reacting with nucleophilic amino acids. Several experiments confirm the hypothesis that these inhibitors are covalently bound to TR. Optimization of these compounds produced leads that inhibited the TR-CoR interaction in vitro with potency of ϳ0.6 M and thyroid signaling in cellular systems. These are the first small molecules irreversibly inhibiting the coactivator binding of a nuclear receptor and suppressing its transcriptional activity. Thyroid hormone receptors (TRs)3 regulate development, growth, and metabolism (1, 2). The TRs are nuclear receptors (NR), part of a superfamily whose members function as hormone-activated transcription factors (3). The majority of thyroid hormone responses are induced by regulation of transcription by the thyroid hormone T3 (4). Two genes, THRA and THRB encode the two protein isoforms TR␣ and TR␤, which yield four distinct subtypes by alternative splicing (5). Several functional domains of TRs have been identified: a ligand-independent transactivation domain (AF-1) on the amino terminus, a central DNA binding domain, a ligand binding domain (LBD), and a carboxylterminal ligand dependent activation function (AF-2) (6). TR binds specific sequences of DNA in the 5Ј-flanking regions of T3-responsive genes, known as thyroid response elements, most often as a heterodimer with the retinoid X receptor (7). Both unliganded and liganded TRs can bind thyroid response elements and regulate genes under their control. The unliganded TR complex can recruit a nuclear receptor corepressor (NCoR) or a silencing mediator of retinoic acid to silence basal transcription (8). In the presence of T3, TRs undergo a conformational change with the result that the composition of the coregulator complex can change with strong effects on transcriptional regulation. Several coactivator proteins have been identified (9). The best studied group of coactivators is the p160 or steroid receptor coactivator (SRC) proteins (7) including SRC1 (10), SRC2 (11,12), and SRC3 (13). Another group of ligand-dependent-interactin...

Inhibitors of the Interaction of a Thyroid Hormone Receptor and Coactivators: Preliminary Structure−Activity Relationships

“…Point mutation analysis showed significantly less binding affinity for the TR C309A mutant than for the wild type, as determined by two independent assays. 19 We observed a ridged binding site for the electrophilic head group and a possible activation of a carbonyl functionality by K306. The distance between the carbonyl oxygen and the K306 amide hydrogen was 3.3 Ǻ.…”

“…19 The most potent molecules identified by the screen were substituted β-aminophenylketones. An initial SAR for this series was established using the relative response of other β-aminophenylketones present in the screening collection.…”

“…17,18 The first small-molecule inhibitors were discovered in a high-throughput screen using fluorescence polarization. 19 The most promising hit from this screen was an aromatic β-aminoketone, which, after in situ elimination to form an enone, bound covalently to the TR and inhibited its interaction with SRCs. This compound class, known as Mannich bases, was systematically studied by Carl Mannich in the beginning of the 20th century.…”

A New Small Molecule Inhibitor of Estrogen Receptor α Binding to Estrogen Response Elements Blocks Estrogen-dependent Growth of Cancer Cells

“…High Throughput Screening Using FAMA-Previous microplate-based fluorescence polarization/anisotropy assays used 20-to 30-l volumes (19,27,30). To minimize protein use and to identify the appropriate concentrations of ER␣ to use in HTS, we carried out ER␣ binding studies in 10 and 20 l. As we recently reported for RNA-binding proteins (29), the use of small volumes results in only a slight decline in FA signal with no change in K d or loss of reproducibility (Fig.…”

“…␤-Aminoketones were identified using HTS as inhibitors that covalently react with the thyroid hormone receptor and inhibit coactivator binding (19). Their specificity for the thyroid hormone receptor compared with other nuclear receptors has not been reported.…”