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Endocrinology Vol. 143, No. 2 517-524
Copyright © 2002 by The Endocrine Society

TRH-TSH-THYROID

Structure-Based Design and Synthesis of a Thyroid Hormone Receptor (TR) Antagonist

John D. Baxter, Patrick Goede, James W. Apriletti, Brian L. West, Weijun Feng, Karin Mellstrom, Robert J. Fletterick, Richard L. Wagner, Peter J. Kushner, Ralff C. J. Ribeiro, Paul Webb, Thomas S. Scanlan and Stefan Nilsson

Departments of Medicine (J.D.B., P.J.K., R.C.J.R.), Biochemistry and Biophysics (R.J.F., R.L.W.), Pharmaceutical Chemistry (T.S.S.), and Cellular and Molecular Pharmacology (R.J.F., T.S.S.), Metabolic Research Unit (J.D.B., J.W.A., B.L.W., W.F., P.W.), and the Diabetes Center (J.D.B, J.W.A., B.L.W., W.F., P.W.), University of California, San Francisco, California 94143; and Karo Bio AB (P.G., K.M., S.N.), Novum, 141 57 Huddinge, Sweden

Address all correspondence and requests for reprints to: John D. Baxter, Metabolic Research Unit, Box 0540, University of California, San Francisco, California 94143-0540. E-mail: jbaxter918{at}aol.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Antagonists have been developed for several nuclear receptors but not for others, including TRs. TR antagonists may have significant clinical utility for treating hormone excess states and other conditions. A structure derived "extension hypothesis" was applied to synthesize a TR antagonist. The principal design feature was to attach an extension group to a TR agonist whose structure would perturb formation of the TR coactivator-binding surface. The compound, 3,5-dibromo-4-(3',5'-diisopropyl-4'-hydroxyphenoxy)benzoic acid, has no (TR{alpha}) or very weak partial (TRß) TR agonist activity and blocks TR binding of T3, formation of the coactivator-binding surface, and both a positive T3 response on a thyroid hormone response element and a negative T3 response on the TSHß promoter in cultured cells. The results suggest that 3,5-dibromo-4-(3',5'-diisopropyl-4'-hydroxyphenoxy)benzoic acid is a TR antagonist for thyroid hormone response element-mediated responses, this approach can be used more generally to generate nuclear receptor antagonists, and this compound or analogues may have medical and research utility.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NUCLEAR RECEPTORS ARE ligand-dependent transcription factors that regulate a variety of important physiological processes (1). Antagonists that act through several nuclear receptors have been described (2, 3, 4, 5). These include the ER "mixed" antagonists tamoxifen and raloxifene (3, 5), PR antagonist RU-486 ( 4, 6), RAR antagonist Ro 41-5253 (7), mineralocorticoid and AR antagonist spironolactone (8, 9), and mineralocorticoid antagonist eplerenone (8). These compounds are used extensively in medical therapy and research. However, antagonists for many nuclear receptors have not been reported.

Antagonists to TRs would be useful for medical therapy and research. Thyroid hormone excess results in serious sequelae, including cardiac arrhythmias, heart failure, weakness, and nervousness (10). A TR antagonist might ameliorate some of these manifestations. Current therapies for hyperthyroidism employ blockade of release of thyroid hormones by the gland, peripheral conversion of T4 to the major active thyroid hormone form, T3, or ß-adrenergic receptors to ameliorate some of the symptoms (10). None of these approaches is ideal. The half-life of T4 in the circulation is around 8 d (10). Thus, blockade of thyroid hormone release usually requires weeks before hormone levels are low enough for symptoms to be ameliorated. Blockade of T4 to T3 release and ß-adrenergic blockade results in incomplete responses. Direct blockade of the TR would bypass the time requirement and the incomplete effectiveness of the ß-adrenergic blockers.

To our knowledge, no compound with clear TR antagonist activity has been reported. The cardiac antiarrhythmic agent amiodarone (11) and its metabolite desethylamiodarone (12) have been reported to have TR antagonist activity (13, 14, 15). However, amiodarone has been found not to inhibit T3 binding to bacterially expressed TRs in vitro (14, 15), and its inhibition of thyroid hormone response in vivo is noncompetitive in nature (data not shown) and may occur via inhibition of T4 transport across the cell membrane (16). Moreover, amiodarone is toxic to cells at concentrations in the TR antagonist IC50 range. Desethylamiodarone does act as a competitive inhibitor of T3 binding to hTR{alpha} (14) and a noncompetitive inhibitor of T3 binding to hTRß in vitro (15) and blocks the TR interactions with its coactivator protein, glucocorticoid receptor interacting protein-1 [GRIP1 (17)]. However, desethylamiodarone has been reported to be more toxic to cells in culture than amiodarone (18, 19) and has not been reported to have TR antagonist activity in cells in culture or animals. Thus, there remains a clear need for development of new TR antagonists.

We have previously reported x-ray crystallographic structures of TR ligand-binding domains (LBDs) complexed with several different agonist ligands (20, 21, 22). The surprising feature of these structures was that the ligands are completely buried in the receptor’s interior and form part of its hydrophobic core. Comparative analyses of the structures of several other LBDs, both unliganded (23, 24) and liganded ( 25, 26, 27, 28), suggested that the TR overall folding reflects that of most nuclear receptor superfamily members (29). These observations implied that folding of the TR-LBD, and the LBDs of other nuclear receptors around the ligand, is a prerequisite for ligand-mediated activation of receptor regulatory properties. Indeed, one of the dominant effects of agonist ligands is to order the carboxyl-terminal LBD helix (helix 12) so that it packs into the body of the LBD. This results in formation of a surface involving helices 3, 5, 6, and 12 (28, 30, 31) that bind coactivator proteins that mediate receptor stimulation of transcription (1).

The fact that the folded TR-LBD completely encloses the ligand leads to a simple prediction. If a compound could bind to the receptor but not induce the folding that occurs with agonists, the compound might have antagonist activity (29, 32, 33, 34). Inspection of the structures of most antagonists reveals that they resemble agonists, but in addition they have extensions relative to the agonist that might prevent formation of the activator surface. We termed this formulation the "extension hypothesis" (32, 34). The subsequent solution of structures of the ER liganded to the agonists E2 (26) and diethylstilbestrol ( 31) and the mixed antagonists raloxifene (26) and tamoxifen (31) and the structures of PRs liganded to the agonist progesterone and modeled with the antagonist RU-486 (27) provided validation of this hypothesis. In all three cases, folding of helix 12 in the antagonist-bound structures was aberrant relative to agonist-induced folding. These observations suggest that this principle could be generally used for pharmaceutical design (29, 32, 33, 34).

In the current studies, we report the synthesis and examination of a compound that contains an extension relative to an agonist. This compound blocks binding of agonists to the TR, formation of the coactivator-binding surface, and positive and negative TR-mediated agonist responses. The results suggest that the overall approach hypothesis can be applied for antagonist design and that this compound or analogues of it could be useful for medical therapy and research.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Synthesis of DIBRT and MIBRT
DIBRT (3,5-dibromo-4-(3',5'-diisopropyl-4'-hydroxyphenoxy)benzoic acid) [7].
In step A, a mixture of 2,6-diisopropyl phenol [1] (20 g, 0.11 mol), potassium carbonate (62 g, 0.45 mol), acetone (160 ml), and methyl iodide (28 ml, 0.45 mole) was refluxed for 3 d. The reaction mixture was filtered through celite, evaporated, dissolved in ether, washed twice with 1 M sodium hydroxide, dried over magnesium sulfate, and concentrated under vacuum on a rotary film evaporator to afford 15.1 g (0.08 mol, 70%) of 2,6-diisopropyl anisole [2] as a slightly yellow oil.

In step B (35), fuming nitric acid (12.4 ml, 265 mmol) was added dropwise to 31.4 ml acetic anhydride cooled in a dry ice/carbon tetrachloride bath. Iodine (11.3 g, 44.4 mmol) was added in one portion followed by dropwise addition of trifluoroacetic acid (20.5 ml, 266 mmol). The reaction mixture was stirred at room temperature until all the iodine was dissolved. Nitrogen oxides were removed by flushing nitrogen into the vessel. The reaction mixture was concentrated; the residue was dissolved in 126 ml acetic anhydride and cooled in a dry ice/carbon tetrachloride bath. To the stirred solution 2,6-diisopropylanisole [2] (51 g, 266 mmol) in 150 ml acetic anhydride and 22.6 ml trifluoroacetic acid was added dropwise. The reaction mixture was left to stand at room temperature overnight and then concentrated. The residue was taken up in 150 ml methanol and treated with 150 ml 10% aqueous sodium bisulfite solution and 1 liter of 2 M sodium borotetrafluoride solution. After the precipitate had aggregated, petroleum ether was added and the supernatant was decanted. The precipitate was triturated with petroleum ether, filtered, washed with petroleum ether, and dried at room temperature in vacuo. This afforded 34 g (57 mmol, 65%) of bis(3,5-diisopropyl-4-methoxyphenyl)iodonium tetrafluoroborate [3] as a white solid.

For step C, thionyl chloride (3 ml) was added dropwise to a stirred solution of 3,5-dibromo-4-hydroxybenzoic acid [4] (12 g, 40.5 mmol) in 250 ml methanol. The reaction mixture was refluxed for 5 d, water was added, and the precipitated product was filtered off. The residue was dissolved in ethyl acetate. From the aqueous phase, methanol was removed by concentration. The aqueous phase was then saturated with sodium chloride and extracted with ethyl acetate. The combined organic phases were dried over magnesium sulfate, filtered, and concentrated. This gave 12.5 g (40.5 mmol, 100%) 3,5-dibromo-4-hydroxymethyl benzoate [5] as a white crystalline solid.

In step D, the products obtained in steps B and C were reacted with each other according to the following protocol. To bis(3,5-diisopropyl-4-methoxyphenyl)iodonium tetrafluoroborate [3] (2.86 g, 4.8 mmol) and copper bronze (0.42 g, 6.4 mmol) in 7 ml dichloromethane at 0 C was added dropwise a solution of 3,5-dibromo-4-hydroxymethyl benzoate [5] (1.0 g, 3.2 mmol) and triethylamine (0.36 g, 3.5 mmol) in 5 ml dichloromethane. The reaction mixture was stirred in the dark for 8 d and then filtered through celite. The filtrate was concentrated and the residue was purified by column chromatography (silica gel, 97:3 petroleum ether/ethyl acetate) to give 0.62 g (1.2 mmol, 39%) of 3,5-dibromo-4-(3',5'-diisopropyl-4'-methoxyphenoxy)methyl benzoate [6] as a solid.

In step E, the product from step D [6] (0.2 g, 0.4 mmol) was dissolved in 2 ml dichloromethane, put under nitrogen, and cooled to -40 C. To the stirred solution was added 1 M boron tribromide (1.2 ml, 1.2 mmol) dropwise. The reaction mixture was allowed to reach room temperature and then left overnight. It was cooled to 0 C and then hydrolyzed with water. Dichloromethane was removed by concentration and the aqueous phase was extracted with ethyl acetate. The organic phase was washed with 1 M hydrochloric acid and brine. Then it was dried over magnesium sulfate, filtered, and concentrated. The residue was chromatographed (silica, 96:3.6:0.4 dichloromethane/methanol/acetic acid), producing 93 mg (0.2 mmol, 51%) of 3,5-dibromo-4-(3',5'-diisopropyl-4'-hydroxyphenoxy)benzoic acid [7] as a white solid. 1H nmr (CDCl3) {delta} 1.23 (doublet, 12H, methyl), 3.11 (multiplat, 2H, CH), 6.50 (singlet, 2H, 2,6-H), 8.33 (singlet, 2H, 2',6'-H).

MIBRT (3,5-dibromo-4-(3'-isopropyl-4'-hydroxyphenoxy)benzoic acid) [10].
In step F, a solution of 3,5-dibromo-4-hydroxymethyl benzoate [5] (5.3 g, 17.0 mmol) and triethylamine (1.89 g, 18.7 mmol) in 26 ml of dichloromethane was added dropwise to bis(3-isopropyl-4-methoxyphenyl)iodonium tetrafluoroborate [8] (13 g, 25.5 mmol) (35) and copper bronze (2.14 g, 33.7 mmol) in dichloromethane (40 ml) at 0 C. The reaction mixture was stirred in the dark for 8 d and then filtered through celite. The filtrate was concentrated and the residue was purified by column chromatography (silica gel, 98:2 petroleum ether/ethyl acetate) to give 5.96 g (12.9 mmol, 76%) of 3,5-dibromo-4-(3'-isopropyl-4'-methoxyphenoxy)methyl benzoate [9] as a solid.

In step G, the product from step F [9] (1.0 g, 2.2 mmol) was dissolved in 10 ml dichloromethane, put under nitrogen, and cooled -40 C. To the stirred solution was added 1 M BBr3 (6.5 ml, 6.5 mmol) dropwise. The reaction mixture was allowed to reach room temperature and stirred for 2 h. It was cooled to 0 C and then hydrolyzed with water. Dichloromethane was removed by concentration and the aqueous phase was extracted with ethyl acetate. The organic phase was washed with 1 M hydrochloric acid and brine. Then it was dried over magnesium sulfate, filtered, and concentrated. The residue was chromatographed (silica, 96:3.7:0.3 dichloromethane/methanol/acetic acid) producing 610 mg (1.4 mmol, 65%) of 3,5-dibromo-4-(3'-diisopropyl-4'-hydroxyphenoxy)benzoic acid [10] as a white solid. 1H nmr (CDCl3) {delta} 1.17 (doublet, 6H, methyl), 3.15 (multiplat, 1H, CH), 6.36 (doublet of doublets, 1H, 6'-H, coupling constant = 8.7, 3.0 Hz), 6.60 (doublet, 1H, 5'-H, coupling constant = 8.7 Hz), 6.79 (doublet, 1H, 2'-H, coupling constant = 3.0 Hz).

Vector constructs
The cDNAs encoding full-length human TR{alpha}1 and TRß1 were cloned in the mammalian expression vector pMT-hGH (36, 37, 38). The pDR4-ALP reporter vector contains one copy of the direct repeat sequence AGGTCAnnnnAGGTCA, fused upstream of the core promoter sequences of the mouse mammary tumor virus long terminal repeat (MMTV), replacing the glucocorticoid response elements. The DR4-MMTV promoter fragment was then cloned 5' of the cDNA encoding human placental alkaline phosphatase (ALP) (39), followed in the 3'-end by the polyA-signal sequence of the human GH gene (38). The TSHß-ALP reporter vector was generated by replacing the DR4-MMTV promoter in the DR4-ALP reporter vector with a DNA fragment, -1192 to +37, of the human TSHß promoter (40).

DNA transfections
Stable transfections were done by using the OptiMEM/lipofectAMINE procedure according to the supplier’s recommendations (Life Technologies, Inc., Rockville, MD).

Reporter cell lines
Chinese hamster ovary (CHO) K1 cells (ATCC no. CCL 61) were transfected in two steps, first with the receptor expression vectors pMT-TR{alpha}1 and pMT-TRß1, respectively, and the drug-resistance vector pSV2-Neo (41) and, in the second step, with the reporter vector pDR4-ALP and the drug-resistance vector pKSV-Hyg (42). Individual drug-resistant clones were isolated and selected on the basis of responsiveness to T3. One stable reporter cell clone each of CHO/TR{alpha}1 and CHO/TRß1 were chosen for further study in response to various thyroid hormone agonists and antagonists.

The pituitary TSHß-ALP reporter cell line was generated by DNA cotransfection of GH3 cells (ATCC no. CCL 82.1) with the TSHß-ALP reporter vector and pSV2-Neo. Individual, drug-resistant clones were tested for T3-dependent suppression of ALP protein expression from the TSHß-ALP reporter vector. One clone was selected based on its prominent suppression of TSHß-ALP activity in a T3 dose-dependent manner.

Assay procedure for hormonal effects on CHO/TR{alpha}1, CHO/TRß1, and GH3/TSHß-ALP reporter cells
The procedure for characterization of agonism/antagonism of ligands has previously been described in detail (37). Cells were exposed to hormones in serum-free medium for 72 h before harvest and analysis for effect on reporter gene expression. Triplicate determinations of reporter protein levels in the conditioned media for each concentration of compound were performed in all experiments.

Toxicity was assessed by microscopic evaluation of cell morphology and by the MTS/PMS assay (CellTiter 96-cell proliferation assay, Promega Corp., Madison, WI), in which the mitochondrial formation of a colored tetrazolium salt is measured spectrophotometrically at 492 nm (Promega Corp., Madison, WI; Technical Bulletin No. 169). Absorbance is directly proportional to the number of living cells in culture.

Assay for human placental alkaline phosphatase
The level of alkaline phosphatase expressed from the different reporter cell lines was determined by a chemiluminescent assay as follows: a 10-µl aliquot of heat-treated (at 65 C for 40 min) conditioned cell culture medium was mixed with 200 µl of assay buffer [10 mM diethanolamine pH 10.0; 1 mM MgCl2 and 0.5 mM CSPD (Tropix Inc., Boston, MA)] in white microtiter plates (Dynex Corp. Laboratories Inc., Chantilly, VA) and incubated at 37 C for 20 min before being transferred to a microplate format luminometer (Luminoskan, Labsystems, Espoo, Finland). The setting of the Luminoskan was integral measurement with a 1-sec reading of each well. The ALP activity is expressed in light units, which is directly proportional to the level of ALP expressed from the cells.

TR-binding assays
Hormone binding and analog competition assays were carried out as described by Apriletti et al. (43) and the T3-binding equilibrium dissociation constant (Kd) values were calculated from the competition data using the Prism computer program (GraphPad Software, Inc., San Diego, CA).

Coactivator-binding assay
The vector to express wild-type hTRß1 was created by ligating a cDNA that encodes the full-length 461 amino acid hTRß1 sequence into pCMV vector (30) and used to produce radiolabeled full-length receptors in vitro with TnT-coupled Reticulocyte lysate system (Promega Corp.) and [35S]Met (DuPont, Wilmington, DE). The Kd value for in vitro translated WT [35S]Met TR was measured by using [125I] in gel filtration-binding assays as described (43). The glutathione S-transferase (GST)-GRIP1 (amino acids 563-1121, from M. Stallcup, USC) fusion protein was produced in Escherichia coli strain HB101 according to the manufacturer’s protocol (Pharmacia Biotech). Binding experiments were performed by gently mixing glutathione-coated Sepharose 4B beads (Amersham Pharmacia Biotech/Pharmacia, Uppsala, Sweden) containing 10 µg of GST fusion proteins (Coomassie plus protein assay reagent, Pierce Chemical Co., Rockford, IL) with 1–2 µl of the [35S]-labeled hTRß1 (25 fmol, 4000 cpm of receptor) in a final volume of 150 µl of binding buffer (20 mM HEPES, 150 mM KCl, 25 mM MgCl2, 10% glycerol, 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, protease inhibitors, 2 µg/ml BSA) for 1.5 h at 4 C in the presence or absence of 10 nM T3 or 10 µM DIBRT. The binding assay was stopped by washing the beads three times with 1 ml binding buffer, the bound [35S]-labeled hTRß1 proteins were separated with use of 10% SDS-PAGE, and visualized by autoradiography using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA) and quantitated using the manufacturer’s software (ImageQuant).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Design and synthesis of DIBRT
Examination of crystallographic structures of liganded TR-LBD complexes revealed that the H atom in the 5'-position of the thyronine structure that is common to TR agonists is packed tightly against the body of the receptor. This atom is packed against hTRß Phe 439, which is located in a loop between helix 11 and the C-terminal activation helix 12. Thus, an extension at this position might not prevent the analog from binding to the TR but might impair folding of helix 12 and prevent formation of the coactivator-binding surface. Studies of TR ligands with groups larger than H on the 5'-position of the thyronine structure do bind to TRs, albeit with a lower affinity than those containing an H moiety (44, 45).

We synthesized a thyronine with an isopropyl extension in the DIBRT 5'-position (Fig. 1Go; also see Materials and Methods). Because this compound relative to T3 has Br replacing I in the 3 and 5 positions, formate replacing aminopropionic in the 1 position and isopropyl instead of I and H in the 3' and 5' positions, respectively, we synthesized for comparison, an analog expected to be an agonist that differed from DIBRT only in that an H is present in the MIBRT 5'-position (Fig. 1Go; also see Materials and Methods).



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  		Figure 1. Structures of DIMIT, MIBRT, and T3. The isopropyl extension that is believed to distort formation of the coactivator-binding surface is shaded.

 
Binding of DIBRT to the TR
The activities of DIBRT, MIBRT, and T3 to compete with [125I]T3 for binding to human TRs are shown in Fig. 2Go (also see Materials and Methods). Each compound displayed similar affinities for the hTRß relative to hTR{alpha}. Using nonlinear regression analysis, the Kd for T3 was calculated to be 0.12 ± 0.008 nM, and the Ki for MIBRT was 0.94 ± 0.14 nM and for DIBRT was 1380 ± 60 nM (13% and 0.01% of the activity of T3, respectively).



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  		Figure 2. Activities of DIBRT, MIBRT, and T3 for binding to TRs. Standard thyroid hormone-binding assays were prepared containing 1 nM [125I]T3, purified TR (hTR{alpha}, open symbols; hTRß, filled symbols), and various concentrations of unlabeled competitors: DIBRT ({square}, {blacksquare}), MIBRT ({triangleup}, {blacktriangleup}), and T3 ({circ}, {bullet}).

 
DIBRT blockade of coactivator binding
The hypothesis that DIBRT prevents folding of the TR to form the coactivator-binding surface was tested directly by examining binding of the p160 class coactivator GRIP-1 (see Materials and Methods). This coactivator stimulates T3-mediated TR responses and has been extensively characterized (30, 46, 47). As shown in Fig. 3Go, T3 readily stimulated binding of the coactivator GRIP-1 to the hTRß. By contrast, this reaction was not stimulated by DIBRT. Further, DIBRT blocked the ability of T3 to stimulate coactivator binding. Thus, DIBRT disrupts the coactivator-binding surface.



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  		Figure 3. Influence of DIBRT and T3 on TR binding to the coactivator GRIP1. The figure shows a phosphorimage of an SDS-polyacrylamide gel used to separate 35S-methionine labeled TR that was bound to GST-GRIP1 protein complexed with GST beads. Maximum bound TR was obtained with T3 added alone (lane 2) and represented about 4% of the input radioactivity. Lane 1 (no hormone added) contained 41% of the maximum, lane 3 (DIBRT alone) was 37% of maximal, and lane 4 (T3 + DIBRT) was 40% of maximal.

 
DIBRT blockade of a positive TR response
The activity of DIBRT in mediating a thyroid hormone response through the hTR{alpha} or hTRß was tested in cell culture. For these experiments, CHO K1 cells that were stably transfected with vectors that express either the hTR{alpha} or hTRß and a reporter gene that is regulated positively by TRs were employed (Materials and Methods and Fig. 4Go). This reporter contained a thyroid hormone response element (TRE) with a single copy of a direct repeat of the consensus TR DNA-binding site spaced by 4 bp (DR4 elements), a naturally occurring thyroid hormone response element (48). The DR4 elements replaced the native glucocorticoid response elements placed upstream of MMTV core promoter cloned upstream of alkaline phosphatase coding sequences (Materials and Methods).



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  		Figure 4. Transcriptional effects of T3, MIBRT, and DIBRT on a DR4 response element. Cells were exposed for 72 h to the indicated concentrations of ligand in CHO/TR{alpha} (A) or CHO/TRß (B) reporter cells to assess the agonist effect of MIBRT ({triangleup}) and DIBRT ({square}), respectively, in comparison with T3 ({circ}). The T3 antagonist activity of DIBRT was analyzed in the presence of the EC50 concentration for T3 ({triangleup}) in CHO/TR{alpha} (C) and CHO/TRß (D) cells, respectively. The activity of DIBRT alone ({circ}) is shown as the control. The response value for each concentration of ligand is the mean of triplicate determinations with the SD for each value indicated. The error bars are barely visible at some concentrations because they are smaller than the symbol.

 
As shown in Fig. 4Go, DIBRT displayed no activity for activating the hTR{alpha} and only a slight dose-dependent ability to activate the reporter gene through the hTRß. The extent of the full response with the hTRß was about 10% that of T3, suggesting that the compound has very weak partial agonist activity when bound to this receptor isoform in this assay. By contrast, MIBRT displayed agonist activity, and the extent of the full response with both the hTR{alpha} and hTRß was as great as that of T3 (Fig. 4Go). The concentrations of MIBRT for activating the response with the hTRß and hTR{alpha} were similar (~10–20 nM), although T3 shows some selectivity for the hTR{alpha} in this system. It is unlikely that these differences are owing to differences in the levels of TRs in the cells because these have been measured several times and found to be similar, in the 2,000–3,000 receptors/cell range.

Figure 4Go also shows that DIBRT inhibits in a dose- dependent fashion the activity of T3 for mediating responses through both receptors. The compound inhibited the response by 50% at 750 nM for hTR{alpha} and 2500 nM for hTRß. These concentrations are comparable with those that are required for inhibition of T3 binding to TR as described above for Fig. 2Go (1380 nM). The maximal level of inhibition was 100% for the hTR{alpha} and 80% for the hTRß. These data provide evidence that DIBRT has TR antagonist activity.

The effects of DIBRT on cell toxicity were also examined. This involved use of MTS solution (obtained from Promega Corp. and performed according to the manufacturer’s recommendations) that is taken up by healthy cells and reduced into a colored soluble product whose absorbance can be measured at 490 nM. The quantity of formazan is directly proportional to the number of living cells in culture. DIBRT did not display toxicity at concentrations in excess of those required to exhibit antagonist activity (to 10 µM). Toxicity was observed above 10 µM (data not shown).

DIBRT blockade of a negative TR response
The activity of DIBRT in mediating a negative thyroid hormone response through endogenous TRs was also tested in GH3 cells in culture. Here, however, the GH3 cells were stably transfected with a reporter vector that expresses alkaline phosphatase (ALP) under control of the TSHß promoter, which is regulated negatively by TRs (49, 50, 51).

Effects of T3 and DIBRT on this negative T3-regulated response element are shown in Fig. 5Go. As indicated, T3 at a submaximal dose decreased the activity of the TSHß promoter by 71% to 29% of the basal. By contrast, DIBRT had a minor effect at very high doses, decreasing promoter activity by about 23% at 1 µM, suggesting weak partial agonist activity. However, DIBRT was able to block the suppression induced by T3 to the level of basal expression in a dose-dependent fashion, with maximal effects observed at 10 µM. The concentration of DIBRT required for 50% inhibition of T3 activity was about 3 µM. Thus, DIBRT is also an antagonist for negative T3-regulated responses.



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  		Figure 5. The T3 agonist/antagonist effect of DIBRT on the regulation of the TSHß promoter in GH3 rat pituitary cells. The effect on TSHß-ALP gene expression of increasing concentrations of DIBRT in the absence ({triangleup}) or presence ({circ}) of 0.5 nM T3 in GH3 cells was assessed 72 h following the addition of the indicated ligands. Response values are the mean from triplicate samples for each concentration of ligand.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the current studies, we report the synthesis and testing of a compound designed to have thyroid hormone antagonist activity. Design of this TR antagonist was based on an understanding of the atomic structure of the TR-LBD complexed with agonist ligands (20, 21, 22). Because the TR-LBD completely encloses the agonist ligand, we predicted that larger TR ligands with extensions would not be completely accommodated within the ligand-binding pocket and would disrupt both the overall folding of the LBD and its subsequent protein-protein interactions. In this case, we used these principles to design a novel TR ligand (DIBRT) with an isopropyl group that protrudes from the H moiety of the 5'-position of the thyronine ring of TR-binding ligands. We expected that the positioning of this isopropyl group would disrupt the loop between helix 11 and 12, which forms a key part of the coactivator-binding surface but would not prevent the compound from binding to TR. Accordingly, DIBRT binds to the TR with an affinity of about 1000 nM and inhibits binding of the agonists T3 and MIBRT. It blocks the ability of TR agonists to induce TR formation of a coactivator-binding surface and a positive TR response with both the hTR{alpha} and hTRß and a negative TR response with the hTRß. DIBRT showed weak partial agonist activity (10–23% that of T3) with the hTRß and no partial agonist activity with the hTR{alpha}. Thus, the main influence is antagonism, and thus the compound fits a profile of a relatively general TR antagonist designed from first principles.

The idea that nuclear receptor antagonists should resemble agonists, but with extensions that disrupt the LBD surface, is broadly applicable to design of nuclear receptor ligands. To our knowledge, existing nuclear receptor antagonists were not specifically designed with this principle in mind, but many antagonists possess extensions, compared with agonists (29). However, we stress that although placement of the inhibitory extension is a crucial factor for antagonist design, the nature of the extension may also be important. We and other colleagues have produced a series of additional TR-binding compounds that are analogs of the TR agonist GC-1 (52) but with bulky extensions at a position that should disrupt the TR surface (53, 54). Although some of these compounds did have antagonist or partial agonist activity, most of them behaved as agonists. We also found that T4 with a 5'iodine group has agonist activity (55). Thus, there is a lot more to be learned about what properties make ligand antagonists. We note that mutation of an ER{alpha} surface residue that contacts the extensions of both tamoxifen and raloxifene transforms both antagonists into agonists (56, 57). Thus, contacts between residues in the surface of the LBD and the extension could help to stabilize the antagonist conformation.

DIBRT was designed to block formation of the TR-LBD coactivator-binding surface that mediates receptor stimulation of transcription. This was found to be the case because concentrations of DIBRT that blocked T3 and MIBRT binding to the TR blocked TR binding of the p160 coactivator GRIP-1 and T3-dependent transactivation. However, although we predicted that DIBRT should block positive TR responses, we also found that DIBRT blocked a negative effect of T3. Transrepression is a complex phenomenon that probably involves more than one type of mechanism (58), and little is known overall about the effects of various antagonists on negative responses. Some GR and RAR antagonists can dissociate transactivation and transrepression at AP-1 sites (59, 60). However, the behavior of DIBRT in blocking both positive and negative effects is mirrored by the progesterone and glucocorticoid receptor antagonist mifepristone (RU38–486), which blocks glucocorticoid-induced apoptosis and suppression of the hypothalamic-pituitary-adrenal axis (61) and also by ER antagonists, which block negative regulation of genes regulated by nuclear factor-{kappa}B (62, 63, 64). There are two possible explanations for the ability of DIBRT to block positive and negative responses. First, DIBRT could disrupt the coactivator-binding surface but also interfere with other aspects of receptor structure, including the surfaces that mediate negative regulation. In this regard, the region of the ER{alpha} LBD that mediates negative regulation of nuclear factor-{kappa}B sites in the intercellular adhesion molecule promoter is distinct from the coactivator-binding site, yet both processes are inhibited by tamoxifen (64). Alternatively, the behavior of DIBRT could be explained if the coactivator surface mediates both positive and negative responses to nuclear receptor ligands. We recently obtained data that coactivators participate in estrogen-mediated negative control of the TNF promoter; the negative responses could be impaired by mutations in the coactivator-binding surface, and the impairments could be alleviated by coactivator overexpression (63). Moreover, a similar requirement for the ER{alpha} activation surface in negative regulation of erythroid specific genes has also been observed (65). These data taken together suggest that the coactivator surface and coactivators may participate in positive as well as negative responses to ligands that interact with nuclear receptors.

The current studies also indicate that DIBRIT can have activity through either the TR{alpha}1 or TRß1 isoforms. This was demonstrated for the positive TR responses, which were mediated by TRs expressed from transfected genes. However, we cannot know this for the negative TR response because we used GH3 cells in which the endogenous receptors mediate the TR responses (we have been unable to get good responses using cells stably transfected with genes that express TRs). However, other data suggest that the negative response on the TSH promoter is mediated primarily by TRß2 (66, 67, 68). However, others have studied GH3 cells in this regard and found that, although they contain mostly TRß2, they also contain TRß1 and TR{alpha}1 (69, 70). Thus, it is likely that some of the response is because of TRß2, but we cannot know this for certain.

The affinity of DIBRT for the TR is in the micromolar range, and micromolar concentrations of DIBRT were required to block actions of T3 (1 nM) that are 20-fold those of the Kd for T3 for binding to the TR. At these and higher concentrations of DIBRT, the compound also did not show toxicity for the cells. By contrast, higher concentrations of the cardiac antiarrhythmic agent and putative TR antagonist amiodarone are required for an equivalent inhibition of TR-mediated responses (5–10 µM for inhibition of a positive response, data not shown). Thus, DIBRT or similar compounds could represent a significant improvement over existing strategies to block thyroid hormone action. As mentioned above, thyroid hormone antagonists should have clinical utility for treating thyroid hormone excess states and possibly certain types of cardiac arrhythmias. TR antagonists should block thyroid hormone action much more rapidly than is achieved with the current modality of blocking thyroid hormone release from the gland, and their overall effectiveness should be equal to that of blocking the gland and greater than that obtained with current blockade of T4 to T3 conversion or of ß-adrenergic receptor action. The time required for such actions should be limited by the time required for TR-induced responses to subside, which is likely to be hours to a few days in duration in most cases. The availability of TR antagonists also provides an added means to investigate the antiarrhythmic properties of blockade of TR action and whether amiodarone acts analogously to T3 in mediating its potent antiarrhythmic actions.

Whereas the compound that we have reported here has intrinsic TR antagonist activity, we cannot know whether this compound will be active in animals. Many factors such as uptake from the gut or into tissue compartments, metabolic degradation, and different response elements could impair activity. These will need to be addressed before we can know whether given compounds will have utility. Nevertheless, the compound developed demonstrates that once these factors are addressed, intrinsic TR antagonist activity can be obtained.


   Acknowledgments
 
We thank D. Bodenner for his kind gift of the human TSHß promoter. Dr. Baxter has proprietary interests in and serves as a consultant and deputy director to Karo Bio AB, which has commercial interests in this area of research.


   Footnotes
 
This work was supported by NIH Grants DK-41842 and DK-51281.

Abbreviations: ALP, Human placental alkaline phosphatase; CHO, Chinese hamster ovary; DIBRT, 3,5-dibromo-4-(3',5'-diisopropyl-4'-hydroxyphenoxy)benzoic acid; DR-4, direct repeat of the consensus TR DNA-binding site spaced by 4 bp; GRIP1, glucocorticoid receptor interacting protein-1; GST, glutathione S-transferase; helix 12, carboxyl-terminal LBD helix; Kd, equilibrium constant; LBD, ligand-binding domain; MIBRT, 3,5-dibromo-4-(3'-isopropyl-4'-hydroxyphenoxy)benzoic acid; MMTV, mouse mammary tumor virus long terminal repeat; TRE, thyroid hormone response element.

Received July 25, 2001.

Accepted for publication October 11, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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