Design of thyroid hormone receptor antagonists from first principles

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Thyroid hormone (TH) actions are mediated by nuclear receptors (TRs ␣ and ␤) that bind triiodothyronine (T 3 , 3,5,3-triiodo-L-thyronine) with high affinity, and its precursor thyroxine (T 4 , 3,5,3,5-tetraiodo-L-thyronine) with lower affinity. T 4 contains a bulky 5 iodine group absent from T 3 . Because T 3 is buried in the core of the ligand binding domain (LBD), we have predicted that TH analogues with 5 substituents should fit poorly into the ligand binding pocket and perhaps behave as antagonists. We therefore examined how T 4 affects TR activity and conformation. We obtained several lines of evidence (ligand dissociation kinetics, migration on hydrophobic interaction columns, and non-denaturing gels) that TR-T 4 complexes adopt a conformation that differs from TR-T 3 complexes in solution. Nonetheless, T 4 behaves as an agonist in vitro (in effects on coregulator and DNA binding) and in cells, when conversion to T 3 does not contribute to agonist activity. We determined x-ray crystal structures of the TR␤ LBD in complex with T 3 and T 4 at 2.5-Å and 3.1-Å resolution. Comparison of the structures reveals that TR␤ accommodates T 4 through subtle alterations in the loop connecting helices 11 and 12 and amino acid side chains in the pocket, which, together, enlarge a niche that permits helix 12 to pack over the 5 iodine and complete the coactivator binding surface. While T 3 is the major active TH, our results suggest that T 4 could activate nuclear TRs at appropriate concentrations. The ability of TR to adapt to the 5 extension should be considered in TR ligand design. Thyroid hormone (TH)1 plays important regulatory roles in metabolism, homeostasis, and development by binding and altering the transcriptional regulatory properties of two related nuclear receptors (NRs), the thyroid hormone receptors (TRs) ␣ and ␤ (1, 2). Most TH produced in the thyroid gland is secreted in the form of thyroxine (T 4 ; 3,5,3Ј,5Ј-tetraiodo-L-thyronine) (2, 3). The thyroid gland also produces smaller amounts of triiodothyronine (T 3 ; 3,5,3Ј-triiodo-L-thyronine) and reverse T 3 (rT 3 ; 3,3Ј,5Ј-triiodo-L-thyronine), and 80% of T 4 is converted to T 3 and rT 3 in peripheral tissues by two selenium deiodinases, which are tissue-specific (4). Current beliefs are that T 3 is the dominant active form of TH; T 3 binds the TRs with an affinity about 20 -30 times higher than that of T 4 (5-9), and some studies suggest that T 3 is present at higher concentrations in the nucleus than T 4 (10, 11). Nonetheless, the question of whether T 4 is simply a prohormone or an active TH species is not completely resolved. T 4 exerts rapid nongenomic effects at several loci distinct from TRs (12). Moreover, saturating levels of T 4 activate transcription of TH-responsive genes in cell culture (see for example Ref. 5). Whereas it is possible that at least some of this activity is due to T 3 generated from T 4 in the cell, these results suggest that T 4 may act as a TR agonist. Normal concentrations of plasma-free T 4 are about 4 -6-fold higher than th...

Section: Discussionmentioning

confidence: 99%

Thyroxine-Thyroid Hormone Receptor Interactions

Sandler

Webb

Apriletti

et al. 2004

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“…Although a number of small molecule modulators of TR have been developed recently, including agonists such as GC-1, TRIAC, reverse T 3 , and KB-141 and also antagonists such as NH-3 (28,29), most modulates target the ligand binding pocket in the LBD. We have previously reported the discovery and characterization of a ␤-aminoketone series that disrupts the TR-coactivator interaction without affecting T 3 binding (30 -32).…”

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confidence: 99%

Methylsulfonylnitrobenzoates, a New Class of Irreversible Inhibitors of the Interaction of the Thyroid Hormone Receptor and Its Obligate Coactivators That Functionally Antagonizes Thyroid Hormone

Hwang

Huang

Arnold

et al. 2011

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 TR␤ 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 T 3 but instead act by direct displacement of SRC2. MSNB series members are selective for the TR over the androgen, vitamin D, and PPAR␥ NR members, and they antagonize thyroid hormone-activated transcription action in cells. The methylsulfonylnitro group is essential for TR␤ 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 TR␤ to disrupt SRC2 association.

“…These small molecule derivatives show selectivity toward different isoforms of TR resulting in tissue specific activities (34). GC-1, a TR␤ selective agonist shows interesting properties in vivo and could be crystallized with TR LBD (35)(36)(37)(38)(39)(40).…”

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confidence: 99%

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

Arnold¹,

Estébanez‐Perpiñá²,

Togashi

et al. 2005

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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...