Selective modulation of thyroid hormone receptor action

Journal of Steroid Biochemistry & Molecular Biology 76 (2001) 31 – 42 www.elsevier.com/locate/jsbmb

Selective modulation of thyroid hormone receptor action John D. Baxter a,*, Wolfgang H. Dillmann b, Brian L. West a, Russ Huber c, J. David Furlow d, Robert J. Fletterick c, Paul Webb a, James W. Apriletti a, Thomas S. Scanlan e a

Metabolic Research Unit, Department of Medicine, Uni6ersity of California, San Francisco, CA 94143, USA b Department of Medicine, Uni6ersity of California, San Diego, CA 92093 -0618, USA c Metabolic Research Unit, Department of Biochemistry and Biophysics, Uni6ersity of California, San Francisco, CA 94143, USA d Section of Neurobiology, Physiology and Beha6ior, Uni6ersity of California, Da6is, Da6is, CA 95616, USA e Metabolic Research Unit, Department of Pharmaceutical Chemistry and Molecular and Cellular Pharmacology, Uni6ersity of California, San Francisco, CA 94143, USA

Abstract Thyroid hormones have some actions that might be useful therapeutically, but others that are deleterious. Potential therapeutically useful actions include those to induce weight loss and lower plasma cholesterol levels. Potential deleterious actions are those on the heart to induce tachycardia and arrhythmia, on bone to decrease mineral density, and on muscle to induce wasting. There have been successes in selectively modulating the actions of other classes of hormones through various means, including the use of pharmaceuticals that have enhanced affinities for certain receptor isoforms. Thus, there is reason to pursue selective modulation of thyroid hormone receptor (TR) function, and several agents have been shown to have some b-selective, hepatic selective and/or cardiac sparring activities, although development of these was largely not based on detailed understanding of mechanisms for the specificity. The possibility of selectively targeting the TRb was suggested by the findings that there are aand b-TR forms and that the TRa-forms may preferentially regulate the heart rate, whereas many other actions of these hormones are mediated by the TRb. We determined X-ray crystal structures of the TRa and TRb ligand-binding domains (LBDs) complexed with the thyroid hormone analog 3,5,3%-triiodithyroacetic acid (Triac). The data suggested that a single amino acid difference in the ligand-binding cavities of the two receptors could affect hydrogen bonding in the receptor region, where the ligand’s 1-position substituent fits and might be exploited to generate b-selective ligands. The compound GC-1, with oxoacetate in the 1-position instead of acetate as in Triac, exhibited TRb-selective binding and actions in cultured cells. An X-ray crystal structure of the GC-1-TRb LBD complex suggests that the oxoacetate does participate in a network of hydrogen bonding in the TR LBD polar pocket. GC-1 displayed actions in tadpoles that were TRb-selective. When administered to mice, GC-1 was as effective in lowering plasma cholesterol levels as T3, and was more effective than T3 in lowering plasma triglyceride levels. At these doses, GC-1 did not increase the heart rate. GC-1 was also less active than T3 in modulating activities of several other cardiac parameters, and especially a cardiac pacemaker channel such as HCN-2, which may participate in regulation of the heart rate. GC-1 showed intermediate activity in suppressing plasma thyroid stimulating hormone (TSH) levels. The tissue/plasma ratio for GC-1 in heart was also less than for the liver. These data suggest that compounds can be generated that are TR-selective and that compounds with this property and/or that exhibit selective uptake, might have clinical utility as selective TR modulators. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Thyroid hormone analogs; Thyroid hormone receptor

1. Introduction 

Proceedings of the 14th International Symposium of the Journal of Steroid Biochemistry and Molecular Biology ‘‘Recent Advances in Steroid Biochemistry and Molecular Biology’’ (Quebec, Canada 24 – 27 June 2000). * Corresponding author. Tel.: + 1-415-4763166; fax: + 1-4154761660. E-mail address: [email protected] (J.D. Baxter).

Thyroid hormones have diverse effects on metabolism and development [1–4]. Some of these, such as to lower plasma cholesterol levels or weight might be medically useful as therapeutic goals when these hormones are administered. Unfortunately, these

0960-0760/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0960-0760(01)00052-8

32

J.D. Baxter et al. / Journal of Steroid Biochemistry & Molecular Biology 76 (2001) 31–42

actions are counterbalanced by deleterious effects of the hormones, such as on the heart rate and to produce arrhythmias, that limit their utility. Thus, it would be desirable to have compounds that elicit beneficial effects while avoiding the deleterious actions. Such compounds would be called selective thyroid hormone receptor modulators (STRMs). Thyroid hormone receptors (TRs) reside in the cell nucleus where they regulate transcription of specific genes [1,5 – 8]. These receptors belong to a class of regulatory proteins, encoded by one of the largest gene families, termed nuclear receptors [1,5– 10]. There is also a need for selective modulation of many of the other classes of nuclear receptors, including those for estrogens, glucocorticoids, retinoids, vitamin D and fatty acids. Although the field is in its infancy, there has been headway in utilizing novel hormone analogues for selective modulation of actions of other nuclear receptors, particularly with estrogens [11] and glucocorticoids [12]. Efforts have also been made to do this with thyroid hormones (discussed below). Current advances in understanding TRs and physiological and pathological responses that they elicit, have provided new and expanded means to address this issue. In this article, we describe our efforts to selectively modulate the b-form of the TR as a possible means to attack hypercholesterolemia and obesity while avoiding deleterious effects on the heart.

Fig. 1. Structures of GC-1, Triac and T3.

2. Physiological actions of thyroid hormones The major form of thyroid hormone secreted from the thyroid gland is T4 (thyroxine, 3,5,3%,5%-tetraiodoL-thyronine), but T3 (3,5,3%-triiodi-L-thyronine, Fig. 1) accounts for most actions of thyroid hormones. T3 has about a 50-fold higher affinity for the TR than T4 [1–3]. About 30% of the T3 is released by the thyroid gland [1]. The other 70% is generated through intracellular deiodination of T4 [1]. This latter step can be regulated by thyroid hormones and provides a mechanism for tissue selective modulation of thyroid hormone action through varying thyroid hormone levels. The diverse effects of thyroid hormones on metabolism in humans and animals can be illustrated by the thyroid hormone excess state, thyrotoxicosis and when thyroid hormones are administered [1–3]. Influences on fat metabolism, most likely to degrade cholesterol through influences on specific lipid lowering liver enzymes [13–18] and to increase levels of the low density lipoprotein (LDL) receptors [13,15,19], lead to a lowering of plasma low-density lipoprotein (LDL) cholesterol levels. The cholesterol lowering actions of thyroid hormones are probably also increased through induction of hepatic cholesterol 7a-hydroxylase, which participates in the conversion of cholesterol to bile acids [15]. Thyroid hormone excess can also reduce plasma levels of triglycerides [14]. Thyroid hormones increase the levels of hepatic HMG CoA reductase [14] and therefore may actually increase cholesterol synthesis. Thus, thyroid hormone analogue treatment may have synergistic actions with inhibitors of HMG CoA reductase. Although there have been variations in the effects on high-density lipoproteins (HDL), thyroid hormones increase the expression of the gene that encodes apolipoprotein AI (Apo AI) [14,20] a main component of HDL. Thus, the analogues should also be considered for their potential to increase HDL, which would decrease the risk of developing atherosclerosis. Actions to increase the basal metabolic rate can lead to weight loss [21]. Effects on the heart increase the heart rate, and the susceptibility to developing atrial arrhythmias and heart failure [22–26]. Actions on muscle lead to muscle wasting and weakness [1–3]. In bone, the hormones increase reabsorption that can lead to osteoporosis; however, the bone resorption induced by these hormones is apparently due to secondary effects through osteoblasts [1–3,27–30]. Effects in the central nervous system (CNS) and pituitary include suppression of the release of thyroid-stimulating hormone (TSH) [1–3], enhancement of the responsiveness to agents used for treatment of depression [31,32], increased attention span and overall hyperactivity [1–3].

J.D. Baxter et al. / Journal of Steroid Biochemistry & Molecular Biology 76 (2001) 31–42

3. Medical indications for selective modulation of thyroid hormone receptor action Several actions of thyroid hormones mentioned above might be beneficial if they were elicited without the deleterious effects of the hormones. Actions to reduce weight would be of great value. Obesity is a major medical problem in Western countries. It is associated with an increased mortality and morbidity, and it increases the overall risk of developing heart attacks, strokes and type 2 diabetes mellitus [33]. Current means to treat this disorder are largely insufficient [34]. LDL cholesterol is a major risk factor for the development of heart attacks and strokes. There has been major progress in treating hypercholesterolemia. In particular, introduction of the 3-hydroxy-3-methyglutaryl coenzyme A reductase (HMG CoA reductase) inhibitors has had a major impact [35,36]. Increased use of nicotinic acid has also been beneficial, although the side effects of this drug limit its use. In addition to the fact that thyroid hormones increase levels of LDL receptors and clearance of cholesterol, these hormones also increase HMG CoA reductase levels [14]. These considerations, combined with recent data suggesting that reducing plasma LDL to very low levels can even reverse atherosclerotic lesions [37,38] indicates a continued need for improved therapies. There is a relatively limited armamentarium of pharmaceuticals that lower plasma triglyceride levels, and these are independent risk factors for atherosclerosis, especially in certain situations such as with diabetes [39]. Thus, actions of thyroid hormones to lower triglyceride levels might be useful [14]. Actions on the CNS might be useful for enhancement of responsiveness to agents used for treatment of depression [31,32]. Currently, T4 is used to suppress TSH release in patients with thyroid cancer [1 –3]. Improved agents that suppress TSH might have medical utility. Of the deleterious effects of thyroid hormones, those on the heart have been of the greatest concern, although effects on muscle and bone must also be considered. In excess thyroid hormones induce an increase in the heart rate (tachycardia) [22– 26]. This can be severe enough to induce heart failure, particularly in individuals with compromised cardiac function due, for example, to atherosclerosis [22– 26]. The hormone excess can also induce atrial arrhythmias, both atrial fibrillation and paroxysmal atrial tachycardia [22– 26]. Both of these can also lead to heart failure, and atrial fibrillation increases the risk for arterial emboli, as clots can form in the dysfunctional atrium that can dislodge and reach the brain. Muscle wasting can lead to significant muscle loss and decreased strength. Thyroid hormoneinduced osteoporosis occurs predominately in postmenopausal women [40,41]. Further, the calcium loss can be blunted or possibly reversed by treatment with

33

bone resorption inhibitors [30,40]. As discussed above, thyroid hormones act on osteoblasts that induce bone deposition with secondary effects on osteoclasts, to induce bone resorption. Thus, it is conceivable that thyroid hormones could actually induce bone deposition if effects on resorption were blocked with antiresorptive therapy [27].

4. Previous attempts at selective modulation of thyroid hormone action A number of attempts at selective modulation of thyroid hormone action have been made. In the late 1960s it was thought that the D-form of T4 (D-T4) might lower plasma cholesterol without having a deleterious effect on the heart. A large trial of D-T4 therapy was conducted [42]. Whereas D-T4 has some thyromimetic activity [43,44], the preparations used in this study were heavily contaminated with L-T4 [45]. Thus, in retrospect, the trial was essentially one with L-T4. This trial was terminated due to sudden deaths in the T4-treated groups [42]. Of note is that the deaths occurred in patients that had significant cardiovascular risk factors at the initiation of the study. These included angina pectoris (indicative of substantial coronary stenosis), congestive heart failure, and tachycardia. If people with these risk factors were excluded, the overall survival in the T4-treated group was greater than with the controls. This study emphasises the cardiovascular risks associated with thyroid hormone therapy. However, it also provides a hint that thyroid hormone analogues that spare the heart might be useful therapeutically. There have been other attempts to develop cardiacsparing thyromimetics. These thyroid hormone analogues were designed without a detailed knowledge of specific cardiac effects of T3 receptor isoforms or of the TR ligand-binding pocket. In the 1980s, a series of 3,5-diodo-3%-aryl-substituted-thyronines were reported that showed promising properties in pre-clinical assays [46]. These compounds showed potent cholesterol-lowering effects in hypercholesterolemic rats without the undesired tachycardia induced by T3. The tissue selectivity of these compounds is likely a result of liver-selective uptake and not TR subtype selectivity. Subsequent results from human or other trials have not been published. A conjugate of cholic acid with T3 was prepared to generate a liver-selective thyromimetic [47]. This compound had about 15 times the cholesterol lowering activity that it had on cardiac functions. Thus, hepatic selectivity in actions could avoid the heart, although such compounds might not have other systemic effects of thyroid hormones that may be important. A series of novel thyronine-like derivatives were reported that contained an oxamic acid group in place of the alanine side

34

J.D. Baxter et al. / Journal of Steroid Biochemistry & Molecular Biology 76 (2001) 31–42

chain at the one position [13]. These compounds carried the dimethyl, isopropyl substituents at the 3,5 and 3% positions instead of halogens. These compounds were also shown to be cardiac-sparing thyromimetics in the hypercholesterolemic rat model [13], and were found to have selectivity for activation of TRb over TRa in transient transfection reporter gene assays [20]. A differential effect on the heart was observed for the compound 3,5-diiodothyropropionic acid (DITPA), which increased cardiac contractility without increasing heart rate and MHC isoform expression [48]. The actual mechanism of action of this compound is also not clear, as DIPTA differs in several aspects from the chemical structure of thyroid hormones and its binding to thyroid hormone receptors was not studied.

5. Selective modulation of actions of other nuclear receptors Selective targeting of receptor isoforms has had a great impact on medicine. This is illustrated by the usefulness of antagonists to the b-adrenergic receptors [49] and the type 1 angiotensin receptors [50]. Pharmaceuticals with this selectivity that largely bypass the a-adrenergic receptors and the type 2 angiotensin receptors have a great advantage over general antagonists. With nuclear receptors, it appears that targeting the g-isoform of the peroxisomal receptor (PPAR) has medical utility [51], and other receptors such as the estrogen receptor b are under investigation [52]. Selective modulation of the action of nuclear receptors in other ways has also been demonstrated. These include the estrogen, glucocorticoid, vitamin D, retinoid and peroxisomal proliferator receptors. The two most advanced are the selective estrogen and glucocorticoid receptor modulators. Selective estrogen receptor modulators (SERMs) were developed with the hope of eliciting favorable estrogen agonist actions in tissues such as brain, bone and cardiovascular, while having antagonist activities in breast or uterus to impair development of breast and uterine cancer [53,54]. Tamoxifen and raloxifene are the two SERMs that have been examined in the greatest detail [11,53]. These compounds have antagonist actions in breast and agonist actions in bone. They do not prevent hot flushes in postmenopausal women and tamoxifen does not have antagonist activity in the uterus. Thus, whereas these compounds have advanced the field in a major way, they still fall short of the ideal. An intensive search is underway in academia and pharmaceutical and biotechnology companies to develop improved SERMs. Information about how SERMs show these differential effects is accumulating [11,53]. These compounds bind to the estrogen receptors (ERs) and induce release

of their associated heat shock proteins. At the same time, the ligands distort the ER helix 12, thereby impairing activation function two (AF-2) of the ER, especially when bound to DNA elements (estrogen response elements, EREs). However, this distortion of helix 12 does not impair the ER’s ability to act through another activation function (AF-1) when ER is bound to DNA and by cross talk with the transcription factor AP-1 (and conceivably others). Thus, a logical explanation of why these compounds are SERMs is that the deleterious ER effects in breast are predominately mediated through AF-2 action at EREs, whereas those in bone may be through AF-1 action at EREs or via cross talk with AP-1 or other transcription factors. When the glucocorticoid cortisone began to be used in the mid 1940s, these compounds were heralded as wonder drugs when dramatic results were reported for rheumatoid arthritis [55,56]. The Nobel Prize was awarded to Kendall, Reichstein and Hench the very next year, one of the shortest periods between the discovery and the Prize [55,56]. However, euphoria over the drugs turned to near despair when the deleterious effects of this class of pharmaceuticals were found. This led to one of the largest searches in pharmaceutical history to find improved compounds. Early efforts resulted in some improvement [56]. Some effects of cortisol, especially that to retain sodium and excrete potassium are mediated through mineralocorticoid receptors, and the improved steroids such as prednisone (which like cortisone needs to be 11b-hydroxlyated to be active) and dexamethasone did not show as much mineralocorticoid activity as cortisone [56]. However, these steroids still elicited most of the undesirable side effects observed with cortisone. Further, when glucocorticoid receptors (GRs) were found in the late 1960s and early 70s, it was thought that selective modulation might be impossible, since all of the effects were though to be mediated through the same receptor [57]. Selective modulation of GR action has now been achieved in a major way with the development of topically applied steroids, especially bronchial sprays [12]. Thus, with the use of these sprays, the glucocorticoids can relieve bronchoconstriction and inflammation in the lung in asthmatics, for example, while eliciting much less of the systemic glucocorticoid effects seen with orally or parenterally administered steroids. These most desirable of these steroid preparations have a high local penetration, but are degraded rapidly in the circulation.

6. Different isoforms of thyroid hormone receptors Products of two different genes are responsible for TR action [5,6,58,59]. The TRa gene encodes two main TR forms due to differential RNA processing. The

J.D. Baxter et al. / Journal of Steroid Biochemistry & Molecular Biology 76 (2001) 31–42

Fig. 2. Pathophysiology in RTH leading to tachycardia.

TRa1 form encodes a ligand-binding receptor, whereas the TRa2 isoform does not bind hormone and acts predominately to suppress expression of genes containing thyroid hormone regulatory elements (TREs) near their promoters. The TRb1 and TRb2 isoforms are both ligand-binding receptors. The TRa1 and TRb1 isoforms are widely distributed in various TR-responsive tissues, but the levels vary. The TRb2 isoform is found mostly in the pituitary and is undetectable or present in very low levels in most tissues. Whereas activities of the TRa1, TRb1 and TRb2 isoforms in many cases are similar, there are also differences. On negative TREs, TRb2 is more potent on ligand-independent activation than TRa1 or TRb1 [60,61]. RXR modulates the ability of the TRa1 and TRb1, but not the b2, to repress expression of the thyroid hormone releasing hormone gene [62]. There are differential effects of corepressor on transcriptional activity of TRa1 and TRb1 versus TRb2 [63]. TRb1 can be more potent than TRb2 [63 – 65].

7. The thyroid hormone receptor-b as a target for selective modulation of thyroid hormone receptor action Our impetus to develop TRb-selective ligands was obtained from an analysis of patients with the syndrome of resistance to thyroid hormone (RTH) (Fig. 2) [66]. These patients have mutations in the TRb gene that result in a defective TRb product. The mutation is recessive in that the product of the other TRb gene is normal as are those from both chromosomes expressing the TRa gene. Resistance is due to the fact that these abnormal TRb gene products inhibit the function of unliganded receptors to either inhibit transcription of genes, whose expression is positively regulated by T3, or to stimulate expression of genes that are negatively regulated by T3. The genes encoding the thyroid stimulating hormone releasing hormone (TRH) and thyroid stimulating hormone (TSH) are included in this latter group. The abnormal receptors also block the

35

ability of thyroid hormone to stimulate or inhibit gene expression. There are variations in the presentation of the syndrome and in the nature of the receptor defects [66,67]. However, the most prevalent pattern is as follows (Fig. 2). The mutated TRb gene products lead to impaired feedback suppression of TSH gene expression. This leads to increased levels of TSH and consequently of T3 and T4. The elevated thyroid hormone levels tend overall to normalize the defect, and result in a mostly euthyroid state. However, this may vary according to the specific thyroid hormone response and also if certain actions of thyroid hormone are mediated predominately by the TRa, then the high thyroid hormone levels could result in hyperstimulation of TRa-mediated responses. Nevertheless patients with RTH tend to appear mostly euthyroid. There are three main exceptions [66]. First, these patients frequently have a tachycardia. Second, they sometimes have an attention deficit. Third, they have a goiter. Administration of thyroid hormone tends to ameliorate the attention deficit, and can suppress goiter development, but exacerbates the tachycardia. These observations could be explained if the attention deficit were TRb-mediated and the endogenous thyroid hormone levels albeit elevated, are insufficient to overcome the defect that is correctable with more hormone. This could not explain the tachycardia that is not ameliorated by more thyroid hormone. In this case, an explanation could be that the tachycardia is due to hyperstimulation of the normal TRa that is present. Although the observations did not come from human studies, a second line of evidence that tachycardia is mediated by the TRa comes from transgenic mice in which the TRa and TRb genes are selectively knocked out. The TRa knockout mice have a slow pulse rate and do not develop a tachycardia with large doses of T3 [68,69]. TRb knockout animals have an increase in T4 levels and heart rate. If the TRa, but not the TRb, stimulates the heart rate, whereas TRb elicits many other actions of thyroid hormones, then a compound with TRb-selective activity might elicit a number of thyroid hormone responses that could be beneficial without causing a tachycardia. For example, TRb is the predominant TR isoform in the liver, the target for cholesterol lowering, accounting for 80% of T3-receptor binding in this organ [48]. Thus, we sought to obtain a TRb-selective agonist. 8. Clues to structures of potential TR b-selective compounds based on X-ray crystal structures of thyroid hormone receptors We previously determined X-ray crystal structures of both the rat (r) TRa ligand-binding domain (LBD) [70] and the human (h) TRb LBD [70] complexed to 3,5,3%-

36

J.D. Baxter et al. / Journal of Steroid Biochemistry & Molecular Biology 76 (2001) 31–42

triiodithyroacetic acid (Triac; Fig. 1). Triac was used as the ligand in the comparative structures, since at that time we were unable to obtain a high resolution X-ray crystal structure for the hTRb complexed to T3 (we have now obtained a high resolution data set of the T3-liganded hTRb, unpublished). From the rTRa structure we could also model the hTRa structure. The data revealed that the residues that lined the ligand-binding pockets of the two receptors were identical except for one residue, Asn331 in hTRb, which is Ser277 in rTRa. Placement of Asn331 appears to reposition a b-hairpin comprised of strands (Ss) three and four (Fig. 3). The b-hairpin in hTRb shifts 0.9A, closer to the carboxylate of the ligand. The repositioning of the hairpin precludes an inward conformation of an arginine residue (Arg228) that is adopted in rTRa (Fig. 3), such that its side chain is located further into the polar pocket where the aminoproprionate side chain of T3 fits. Most residues in the polar pockets of both receptors adjust to adopt the same conformations and interactions with the ligand, with Arg266 (Arg316 in hTRa) forming a charge pair with the negatively-charged acetate moiety of Triac (Fig. 3). However, in rTRb, Arg228 also forms a hydrogen bond with the carboxylate of Triac, whereas in hTRa the homologous residue Arg282 points away from the ligand. These considerations suggest that this region in the TR polar pocket might be exploited for designing TR subtype selective ligands. 9. Development of GC-1 as a b-selective TR agonist We had prepared a compound, GC-1 [3,5-dimethyl4-(4%-hydroy-3%-isopropylbenzyl)-phenoxy acetic acid;

Fig. 1] that had potential properties of a TRb-selective compound [71]. GC-1 has comparable dimensions to T3 but with a greater ring separation (methylene vs. ether), smaller side groups (methyl vs. iodide), lower molecular weight (due to differences in methyl vs. iodine moieties), and a flexible 1-position substituent. Importantly, the oxyacetic acid side chain provides opportunities for hydrogen bonding in the polar part of the ligand-binding pocket where the TRa –TRb differences are found. GC-1 was in fact found to bind to the hTRb with five–seven-fold the affinity of the hTRa [71]. When GC-1 was tested in HeLa cells in culture transfected with either TRa or TRb and a TRE-driven reporter gene, the drug showed an even greater selectivity for the TRb of ten–100-fold versus TRa [71]. Thus, GC-1 has true b-selectivity in its binding and actions. It is of interest that GC-1 has such a high affinity for the TRb, 70 pM (equilibrium dissociation constant), in spite of the fact that it has methyl rather than iodine moieties at the three and five positions. Substitution of methyl for iodine in T3 at these positions results in a reduction in affinity of the compound by 100-fold relative to T3 [72]. 10. Atomic mechanism of b-selectivity of GC-1 Based on the previous discussions, GC-1 might be TRb-selective because the oxoacetate at position one is capable of forming more hydrogen bonds with the arginine residues in the receptor’s polar pocket. To obtain information about the interactions of GC-1 with the hTRb, we determined the X-ray crystal structure of this compound complexed with the hTRb LBD at 2.9 A, [70].

Fig. 3. Triac (A) fitted into the ligand-binding pocket of rTRa and GC-1 (B) fitted into the ligand-binding pocket of hTRb. The a-carbon backbone of the receptor is shown in light purple. The ligands are shown in yellow. Three Arg residues and one Asn residue that protrude into the polar pocket of the ligand-binding cavity are shown in green with oxygens in red and nitrogens in blue. Potential hydrogen bonds are shown by the dotted lines.

J.D. Baxter et al. / Journal of Steroid Biochemistry & Molecular Biology 76 (2001) 31–42

Fig. 4. Affinity (equilibrium association constant, Ka) for GC-1 binding to wild type and mutated hTRa and hTRb. The mutant for the hTRb contains a Ser residue substituted for Asn 331 (designated N331S) to generate the binding cavity of the hTRa. The reciprocal mutant for hTRa contains an Asn substituted for Ser 277, (designated S277N). Hormone binding assays were performed by competing [125I] T3 from each of these receptors and receptor variants with GC-1. Data taken from Wagner et al. [70].

37

forms a charge interaction with the negatively charged carboxylate (3.0A, ) as seen with Triac. Unlike the case with Triac, Arg282 bends inwards, in building a polar network with Asn331, to the carboxylate of GC-1 (2.8A, .). The carbonyl of Leu330 and the ester oxygen of the oxyacetic acid are 3.4 and 2.9 A, , respectively away from Arg282. These interactions are enabled by a 0.9 A, shift in backbone of the b-hairpin containing Asn331. Thus it appears that the larger network of hydrogen bonds present in the hTR-b with GC-1 relative to Triac participates in the mechanism of the TR-b specificity. Formation of this polar network seems to be organized by the side chain of Asn331 and the positions of the oxygen atoms of GC-1. However, the contribution of these interactions to affinity and specificity cannot be quantified. 11. Role of the Ser/Asn difference in b-selectivity of GC-1

Superimposition of the GC-1 and Triac-complexed molecules reveals that atoms of the outer thyronine ring overlap, with the hydrogen bond between the phenolic hydroxyl to His435 maintained and the 3%-isopropyl and 3%-iodo occupying the same pocket. However, the inner ring lies 0.3 A, closer to the polar pocket due to the longer bridge between the thyronine rings. This shift positions the three- and five-position methyl groups slightly forward of the iodines. Thus, the smaller volumes of the methyl (relative to I) moieties facilitated high affinity binding of GC-1, as they prevent a steric clash with pocket residues. This fitting also leads to a small difference of 17° in the torsion angle of the two rings. In the hydrophobic portion of the hormone-binding cavity, receptor residues adopt similar conformations, with the exception of Met310, which rotates about the Cg, creating a small void in the pocket’s interior. The hormone-binding pocket is inside the lower domain of the LBD and protected from water. Side chains, rather than backbone, make most of the contacts with the hormone. All but four of the 20 side chains that are near the hormone are hydrophobic. The walls of the hormone-binding pocket are of two distinct physical classes. One is very hydrophobic and shelters the two phenyl rings with their three iodine atoms. The hydroxyl of the distal phenyl ring is in this hydrophobic environment, but it forms a hydrogen bond with a His345. The second class of interaction is polar and charged moieties from the LBD cluster around the amino acid group of the hormone. The pocket walls here are made from backbone atoms, which contain oxygen and nitrogen atoms and four polar side chains, one Asn and three Args. One of the Arg side chains forms a distant polar interaction with GC-1. Arg320

The data suggest the single Ser to Asn difference between the TRa and TRb participates in differential isoform recognition [70]. To address this issue we mutated Asn331 in hTRb to Ser to produce a ligand-binding pocket with amino acids of the TRa (designated N331S), and Ser277 to Asn in hTRa to produce a binding pocket with amino acids of hTRb [70]. Fig. 4 shows Ka values calculated from competition data. Whereas wild type TRb binds GC-1 more tightly than TRa, N331S binds GC-1 with an affinity comparable to the hTRa. Conversely, the mutation of Ser277 to Asn in S277N-hTRa increases the affinity of the hTRa for GC-1 to that of the hTRb. Thus, this amino acid difference largely accounts for selective binding of GC1 to TRb.

12. Animal studies with GC-1 We have initiated studies in animals to determine whether GC-1 has an activity profile that differs from that of T3. The animals include tadpoles, mice and hypercholesterolemic rats.

12.1. Effects on tadpole metamorphosis Tadpole metamorphosis is a classic developmental model for investigating the effects of thyroid hormones. Metamorphosis is dependent on thyroid hormone and TRa and TRb appear to mediate different events in different tissues. TRa is expressed in the limb buds that undergo proliferation into limbs during the early stages of the program, whereas TRb expression becomes predominant in the tail and gills which undergo resorption at the climax of the program. The TRb selectivity of

38

J.D. Baxter et al. / Journal of Steroid Biochemistry & Molecular Biology 76 (2001) 31–42

GC-1 produces striking effects when administered to premetamorphic tadpoles. The GC-1 treated tadpoles display little or no limb development but show marked resorption of tail and gills. (Furlow, J.D. and Scanlan, T.S., unpublished data). These effects correlate with the TRb selectivity of GC-1 as the resulting thyroid hormone action occurs in the tissues that predominantly express TRb.

12.2. Mice studies Mice are readily adaptable for experimental manipulation, and data obtained from them can be compared with those from gene knockout animals. We made animals hypothyroid using propylthiouracil in the drinking water, and then initially administered equimolar doses of either 3.5 ng/g body weight (BW) T3 or 1.8 ng/g BW GC-1 (1 ×T3, 1 × GC-1) [73]; whereas we did not have information about the metabolic clearance of

Fig. 5. Effect of 1 ×GC-1 and 1×T3 on cholesterol (Chol), triglyceride (TG), TSH and heart rate (HR) in hypothyroid mice. Data are presented as % of hypothyroid values. Data obtained from Trost et al. [73].

Fig. 6. Effect of 1 × GC-1 and 1 × T3 on cardiac parameters, heart rate (HR), maximum speed of contraction (dP/dt), mRNA for myosin heavy chain-a (MHalpha), mRNA for sarcoplasmic calcium pump protein (SERCA2) and mRNA for the cardiac pacemaker channel HCN2 (HCN2). Data are taken from Trost et al. [73] and the results expressed as a percentage of the change from the hypothyroid to the euthyroid level.

GC-1, it has an affinity for the TR that is nearly equal to that of T3 [71].

12.2.1. Plasma lipids Fig. 5 shows the effects of 1× T3 and 1 × GC-1 on plasma lipid levels [73]. Plasma cholesterol levels were elevated in hypothyroid mice to 165 mg/dl compared to 749 3 mg/dl in euthyroid mice (PB 0.0001). Both T3 and GC-1 decreased plasma cholesterol levels to normal (65 and 75 mg/dl, respectively). Plasma triglyceride levels were elevated in hypothyroid mice to 126915 mg/dl compared to 709 15 mg/dl in euthyroid animals (P B0.05). GC-1 treatment resulted in a lowering of plasma triglyceride levels to those of euthyroid animals (ca. 70 mg/dl at 1 × GC-1 and 35 mg/dl at 4.5×GC-1 vs. 65 mg/dl in normals) whereas 1× T3 failed to decrease triglyceride levels. Other doses of T3 and GC-1 were also used at comparative molar ratios [73]. T3 and GC-1 induced lowering of plasma cholesterol levels were similar at doses of GC-1 of 1.8 ng/g BW (1 ×GC-1), 8.1 ng/g BW (4.5× GC-1) and 16.2 ng/g BW (9 × GC-1). However, the GC-1 induced lowering of triglyceride levels was more efficient at all doses in comparison to T3. 12.2.2. Heart rate Heart rate in hypothyroid mice (Fig. 5 and 6) was markedly decreased to 335921 beats/min from a rate in control mice of 4729 26 beats/min (P= 0.001 control vs. hypothyroid) [73]. Treatment with 1×T3 increased the rate to 502 beats/min (PB0.001 hypothyroid versus 1×T3 treated). By contrast, treatment with 1 × GC-1 retained a low heart rate of 3589 28 beats/min (PB 0.01 control vs. 1× GC-1 treated). Higher equimolar doses of the compounds let to similar increases in heart rate: 4.5× T3 and 4.5×GC-1 increased heart rates to 5369 24 and 5279 35 beats/min, respectively, and treatment with 9× T3 and 9× GC-1 resulted in heart rates of 5849 28 and 5709 31 beats/ min respectively. 12.2.3. Other hemodynamic parameters Other hemodynamic parameters measured were systolic and diastolic arterial pressure (Pa sys and Pa dias), endsystolic left ventricular pressure (LVESP), and maximum speed of contraction (dP/dtmax) and relaxation (dP/dtmin) [73]. These were all decreased markedly in hypothyroid mice (Fig. 6). 1× GC-1 showed a positive inotropic effect and increased dP/dtmax from 29459 273 mmHg/s in hypothyroid mice to 44249510 mmHg/s (PB 0.05 hypothyroid vs. 1× GC-1 treated), but this was less than the effect of 1× T3 of a dP/dtmax of 62249 888 mmHg/s (PB 0.05 1× T3 treated vs. 1 × GC-1 treated). 1×GC-1 failed to increase Pa sys, Pa dias, LVESP and dP/dtmin whereas treatment with 1×

J.D. Baxter et al. / Journal of Steroid Biochemistry & Molecular Biology 76 (2001) 31–42

Fig. 7. Effect of 1 × GC-1 and 1×T3 on cholesterol, TSH and heart rate in hypothyroid (Hypo), T3-treated and GC1-treated hypercholesterolemic rats. Data taken from Trost et al. [73].

T3 increased all of these parameters (P B 0.05 1× T3 treated vs. hypothyroid).

12.2.4. Inotropic response to isoproterenol Administration of increasing doses of isoproterenol iv resulted in positive inotropic effects in all groups [73]. With 500 ng isoproterenol the percentage increases from the basal level in arterial pressure for control, hypothyroid, 1×T3 treated and 1×GC-1 treated mice were 259 5, 239 2, 25 9 5 and 3699% respectively, dP/dtmax increased by 1696, 25 911, 149 5 and 179 9% respectively. These data show that the sympathetic response of the hypothyroid, 1×GC1 and 1 × T3 treated groups was not changed from the control group. 12.2.5. Le6els of mRNAs coding for cardiac proteins Hearts of hypothyroid mice showed the expected high level of myosin heavy chain (MHC) b mRNA and almost no MHCa mRNA [73]. Treatment of these mice with 1×T3 restored MHCa mRNA levels, and reduced MHCb mRNA levels to undetectable. In contrast, 1× GC-1 did not increase MHCa expression and decreased MHCb mRNA to a lesser extent than with 1× T3 treated mice [73]. Treatment with 9× GC-1 increased MHCa mRNA to euthyroid levels, but MHCb mRNA was still detectable [73]. Hypothyroidism decreased by 70% mRNA for the sarcoplasmic calcium pump protein (Serca2) and almost completely abolished mRNA for the cardiac pacemaker channel HCN2 [73]. Changes in Serca2 were reversed in mice treated with T3, whereas this was not observed with GC-1, but was observed with 9×GC-1. mRNA levels for HCN2 were restored by 1× T3, but not by 1× GC-1 or 4.5×GC-1, but were restored by treatment with 9×GC-1 [73]. The cardiac pace maker channel If, appears to play an essential role in setting the heart rate [74.75]. This hyperpolarization activated inward rectifier channel

39

conducts the monovalent cations potassium and sodium. This channel has been termed hyperpolarization activated cyclic nucleotide regulated (HCN) channel. Four isoforms of it occur in the brain (HCN1-HCN4) [74,75]. HCN2 and to a lesser extent HCN4, are expressed in the heart. Thus, expression of this gene might mediate effects of thyroid hormone on heart rate. HCN2 was decreased in hypothyroid animals [73]. 1× T3 increased mRNA levels encoding HCN2 to euthyroid levels [73]. By contrast, this was not observed with 1×GC-1 [73]. Thus, the discrepant heart rate and HCN2 responses to T3 and GC-1 may be due to differences in effects on expression of the HCN2 channel [73].

12.3. Hypercholesterolemic rats Dose–response studies in hypercholesterolemic rats confirmed the preferential GC-1 effect of influencing cholesterol and thyroid stimulating hormone (TSH) more than heart rate (Fig. 7) [73]. Thus GC-1 represents a prototype for new drugs to treat hypercholesterolemia, hypertriglyceridemia, obesity and other indications.

13. Selective uptake as an additional mechanism for generating cardiac-sparing actions of thyroid hormone analogues Whereas GC-1 clearly exhibits TRb-selectivity in its binding and actions, the results to date do not indicate that this is the exclusive mechanism for the cardiac sparing actions of this compound in animals. To obtain an initial indication of whether selective cardiac uptake could also contribute to the cardiac-sparing actions of GC-1, we measured the GC-1 and T3 content of heart and liver following acute administration of the compounds [73]. The tissue to plasma ratios in the liver and plasma differed only slightly for T3 and GC-1 (5.869 1.93 and 2.099 0.53, respectively). By contrast, the tissue/plasma ratio for heart was 30-fold higher for T3 than GC-1 (3.619 0.69 and 0.129 0.002). Whereas these data suggest that the cardiac sparing activities of GC-1 may be due in part to the b-selectivity of the compound, tissue uptake differences may also contribute heavily.

Acknowledgements This work was supported by grants from the NIH (RJF and JDB). 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.

40

J.D. Baxter et al. / Journal of Steroid Biochemistry & Molecular Biology 76 (2001) 31–42

References [1] R.D. Utiger, The thyroid: physiology, thyrotoxicosis, hypothyroidism, and the painful thyroid, in: P.F. Felig, J.D. Baxter, L.A. Frohman (Eds.), Endocrinology and Metabolism, McGraw-Hill, New York, 1995, pp. 435 – 519. [2] L.E. Braverman, R.D. Utiger (Eds.), Werner’s and Ingbar’s The Thyroid: A Fundamental and Clinical Text, Lippincott Williams & Wilkins, Philadelphia, 2000. [3] F.S. Greenspan, The thyroid gland, in: F.S. Greenspan, D.G. Gardner (Eds.), Basic & Clinical Endocrinology, McGraw-Hill, New York, 2001, pp. 201 –272. [4] L.J. DeGroot, P.R. Larsen, G. Hennemann, The Thyroid and its Diseases, Churchill Livingstone, New York, 1996. [5] R.C.J. Ribeiro, P.J. Kushner, J.D. Baxter, The nuclear hormone receptor gene superfamily, Annu. Rev. Med. 46 (1995) 443– 453. [6] R.C.J. Ribeiro, J.W. Apriletti, B.L. West, R.L. Wagner, R.J. Fletterick, F. Schaufele, J.D. Baxter, The molecular biology of thyroid hormone action, Ann. NY Acad. Sci. 758 (1995) 366 – 389. [7] J.W. Apriletti, R.C.J. Ribeiro, R.L. Wagner, W. Feng, P. Webb, P.J. Kushner, et al., Molecular and Structural Biology of Thyroid Hormone Receptors, Clin. Exp. Pharmacol. Physiol. Suppl. 25 (1998) S2 –S11. [8] R.C.J. Ribeiro, J.W. Apriletti, R.L. Wagner, B.L. West, W. Feng, R. Huber, P.J. Kushner, S. Nilsson, T.S. Scanlan, R.J. Fletterick, F. Schaufele, J.D. Baxter, Mechanisms of thyroid hormone action: insights from X-ray crystallographic and functional studies, Recent Prog. Horm. Res. 53 (1998) 351 – 394. [9] D.J. Mangelsdorf, R.M. Evans, The RXR heterodimers and orphan receptors, Cell 83 (1995) 841 – 850. [10] B. O’Malley, Thirty years of steroid hormone action: personal recollections of an investigator, Steroids 60 (1995) 490 –498. [11] J.A. Gustafsson, Therapeutic potential of selective estrogen receptor modulators, Curr. Opin. Chem. Biol. 2 (1998) 508 – 511. [12] J.D. Baxter, Advances in glucocorticoid therapy, Adv. Intern. Med. 45 (2000) 317 –349. [13] N. Yokoyama, G.N. Walker, A.J. Main, J.L. Stanton, M.M. Morrissey, C. Boehm, et al., Synthesis and structure –activity relationships of oxamic acid and acetic acid derivatives related to L-thyronine, J. Med. Chem. 38 (1995) 695 – 707. [14] G.C. Ness, D. Lopez, C.M. Chambers, W.P. Newsome, P. Cornelius, C.A. Long, H.J. Jr. Harwood, Effects of L-triiodothyronine and the thyromimetic L-94901 on serum lipoprotein levels and hepatic low-density lipoprotein receptor, 3hydroxy-3-methylglutaryl coenzyme A reductase, and apo A-I gene expression, Biochem. Pharmacol. 56 (1998) 121 –129. [15] G.C. Ness, D. Lopez, Transcriptional regulation of rat hepatic low-density lipoprotein receptor and cholesterol 7 alpha hydroxylase by thyroid hormone, Arch. Biochem. Biophys. 323 (1995) 404 – 408. [16] N.D. Ridgway, P.J. Dolphin, Serum activity and hepatic secretion of lecithin:cholesterol acyltransferase in experimental hypothyroidism and hypercholesterolemia, J. Lipid Res. 26 (1985) 1300 – 1313. [17] W.C. Hulsmann, M.C. Oerlemans, M.M. Geelhoed-Mieras, Effect of hypothyroidism, diabetes and polyunsaturated fatty acids on heparin-releasable rat liver lipase, Biochem. Biophys. Res. Commun. 79 (1977) 784 –788. [18] P. Hansson, S. Valdemarsson, P. Nilsson-Ehle, Experimental hyperthyroidism in man: effects on plasma lipoproteins, lipoprotein lipase and hepatic lipase, Horm. Metab. Res. 15 (1983) 449 –452.

[19] L. Scarabottolo, E. Trezzi, P. Roma, A.L. Catapano, Experimental hypothyroidism modulates the expression of the low density lipoprotein receptor by the liver, Atherosclerosis 59 (1986) 329 – 333. [20] A.H. Taylor, Z.F. Stephan, R.E. Steele, N.C. Wong, Beneficial effects of a novel thyromimetic on lipoprotein metabolism, Mol. Pharmacol. 52 (1997) 542 – 547. [21] C.N. Mariash, The Thyroid and Obesity Revisited, Thyroid Today 21 (1998) 1 – 9. [22] A.M. Galloe, M. Rolff, H. Nordin, S.D. Ladefoged, N.B. Mogensen, Cardiac performance and thyroid function. The correlation between systolic time intervals, heart rate and thyroid hormone levels, Dan. Med. Bull. 40 (1993) 492 – 495. [23] K.A. Woeber, Thyrotoxicosis and the heart, New Engl. J. Med. 327 (1992) 94 – 98. [24] W.H. Dillmann, Biochemical basis of thyroid hormone action in the heart, Am. J. Med. 88 (1990) 626 – 630. [25] K. von Olshausen, S. Bischoff, G. Kahaly, S. Mohr-Kahaly, R. Erbel, J. Beyer, J. Meyer, Cardiac arrhythmias and heart rate in hyperthyroidism, Am. J. Cardiol. 63 (1989) 930 –933. [26] W.H. Dillmann, Cardiac function in thyroid disease: clinical features and management considerations, Ann. Thorac. Surg. 56 (1993) S9 – 14. [27] J.M. Britto, A.J. Fenton, W.R. Holloway, G.C. Nicholson, Osteoblasts mediate thyroid hormone stimulation of osteoclastic bone resorption, Endocrinology 134 (1994) 169 – 176. [28] A. Schoutens, E. Laurent, E. Markowicz, J. Lisart, V. Maertelaer de, Serum triiodothyronine, bone turnover, and bone mass changes in euthyroid pre- and postmenopausal women, Calcif. Tissue Int. 49 (1991) 95 – 100. [29] J.J. Stepan, Z. Limanova, Biochemical assessment of bone loss in patients on long-term thyroid hormone treatment, Bone Miner. 17 (1992) 377 – 388. [30] B. Ongphiphadhanakul, L.G. Jenis, L.E. Braverman, S. Alex, G.S. Stein, J.B. Lian, D.T. Baran, Etidronate inhibits the thyroid hormone-induced bone loss in rats assessed by bone mineral density and messenger ribonucleic acid markers of osteoblast and osteoclast function, Endocrinology 133 (1993) 2502 – 2507. [31] I.M.D. Jackson, E.O. Asamoach, Thyroid function in clinical depression: insights and uncertainties, Thyroid Today 22 (1999) 1 – 11. [32] R. Joffe, S.T. Sokolov, W. Singer, Thyroid hormone treatment of depression, Thyroid 5 (1995) 235 – 239. [33] S.M. Grundy, Metabolic Complications of Obesity, Endocrine 13 (2000) 155 – 165. [34] L.A. Campfield, F.J. Smith, P. Burn, Strategies and potential molecular targets for obesity treatment, Science 280 (1998) 1383 – 1387. [35] Randomised trial of cholesterol lowering in 4444 patients with coronary heart disease: the Scandinavian Simvastatin Survival Study (4S), Lancet 344 (1994) 1383 – 1389. [36] G.N. Levine, J.F. Jr. Keaney, J.A. Vita, Cholesterol reduction in cardiovascular disease. Clinical benefits and possible mechanisms, New Engl. J. Med. 332 (1995) 512 – 521. [37] J.P. Kane, M.J. Malloy, T.A. Ports, N.R. Phillips, J.C. Diehl, R.J. Havel, Regression of coronary atherosclerosis during treatment of familial hypercholesterolemia with combined drug regimens, J. Am. Med. Assoc. 264 (1990) 3007 – 3012. [38] G. Brown, J.J. Albers, L.D. Fisher, S.M. Schaefer, J.T. Lin, C. Kaplan, X.Q. Zhao, et al., Regression of coronary artery disease as a result of intensive lipid- lowering therapy in men with high levels of apolipoprotein B, New Engl. J. Med. 323 (1990) 1289 – 1298. [39] M.A. Austin, J.E. Hokanson, K.L. Edwards, Hypertriglyceridemia as a cardiovascular risk factor, Am. J. Cardiol. 81 (1998) 7B – 12B.

J.D. Baxter et al. / Journal of Steroid Biochemistry & Molecular Biology 76 (2001) 31–42 [40] D.L. Schneider, E.L. Barrett-Connor, D.J. Morton, Thyroid hormone use and bone mineral density in elderly women, Effects of estrogen, J. Am. Med. Assoc. 271 (1994) 1245 –1249. [41] D.L. Schneider, E.L. Barrett-Connor, D.J. Morton, Thyroid hormone use and bone mineral density in elderly men, Arch. Intern. Med. 155 (1995) 2005 –2007. [42] The coronary drug project. Findings leading to further modifications of its protocol with respect to dextrothyroxine. The coronary drug project research group, J. Am. Med. Assoc. 220, 1972 996 – 1008. [43] K. Ichikawa, L.J. DeGroot, Purification and characterization of rat liver nuclear thyroid hormone receptors, Proc. Natl. Acad. Sci. USA 84 (1987) 3420 – 3424. [44] B. Busnardo, N. Simioni, M.E. Girelli, T. Rampazzo, A. Pagnan, G. Zanetti, S. Dotto, N. Ciuccio, Acute and chronic effects of dextro-thyroxine on pituitary thyroid axis and on reverse triiodothyronine production in euthyroid subjects, J. Endocrinol. Invest. 10 (1987) 73 –77. [45] W.F. Young Jr., C.A. Gorman, N.S. Jiang, D. Machacek, I.D. Hay, L-thyroxine contamination of pharmaceutical D-thyroxine: probable cause of therapeutic effect, Clin. Pharmacol. Ther. 36 (1984) 781 –787. [46] P.D. Leeson, J.C. Emmett, V.P. Shah, G.A. Showell, R. Novelli, H.D. Prain, M.G. Benson, D. Ellis, N.J. Pearce, A.H. Underwood, Selective thyromimetics. Cardiac-sparing thyroid hormone analogues containing 3%-arylmethyl substituents, J. Med. Chem. 32 (1989) 320 –336. [47] Z.F. Stephan, E.C. Yurachek, R. Sharif, J.M. Wasvary, R.E. Steele, C. Howes, Reduction of cardiovascular and thyroxinesuppressing activities of L-T3 by liver targeting with cholic acid, Biochem.Pharmacol. 43 (1992) 1969 –1974. [48] H.L. Schwartz, K.A. Strait, N.C. Ling, J.H. Oppenheimer, Quantitation of rat tissue thyroid hormone binding receptor isoforms by immunoprecipitation of nuclear triiodothyronine binding capacity, J. Biol. Chem. 267 (1992) 11794 –11799. [49] N.C. Prichard, J.M. Cruickshank, Beta blockade in hypertension: past present and future, in: J.H. Laragh, B.M. Brenner (Eds.), Hypertension, Raven Press, New York, 1995, pp. 2827 – 2859. [50] P.B. Timmermans, Angiotensin II receptor antagonists: an emerging new class of cardiovascular therapeutics, Hypertens. Res. 22 (1999) 147 –153. [51] B.M. Spiegelman, PPAR-gamma: adipogenic regulator and thiazolidinedione receptor, Diabetes 47 (1998) 507 –514. [52] G.G. Kuiper, J.G. Lemmen, B. Carlsson, J.C. Corton, S.H. Safe, P.T. van der Saag, B. van der Burg, J.A. Gustafsson, Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta, Endocrinology 139 (1998) 4252 –4263. [53] H.U. Bryant, W.H. Dere, Selective estrogen receptor modulators: an alternative to hormone replacement therapy, Proc. Soc. Exp. Biol. Med. 217 (1998) 45 –52. [54] A.E. Wakeling, Similarities and distinctions in the mode of action of different classes of antioestrogens, Endocr. Relat. Cancer 7 (2000) 17 –28. [55] P.S. Hench, E.C. Kendall, C.H. Slocumb, H.F. Polley, The effect of a hormone of the adrenal cortex (17-hydroxy-11-dehydrocorticosterone compound E) and of pituitary adrenocorticotrophic hormone on rheumatoid arthritis, Proc. Staff. Meet. Mayo. Clin. 24, 1949. [56] J.D. Baxter, Cortisone and the adrenal cortex, Trans. Assoc. Am. Physicians 99 (1986) clxvii –clxxxviii. [57] J.D. Baxter, Glucocorticoid hormone action, Pharmacol. Ther. [B] 2 (1976) 605 – 669. [58] M.A. Lazar, Thyroid hormone receptors: multiple forms, multiple possibilities, Endocr. Rev. 14 (1993) 184 –193. [59] E.D. Abel, M.E. Boers, C. Pazos-Moura, E. Moura, H. Kaulbach, M. Zakaria, B. Lowell, S. Radovick, M.C. Liber-

[60]

[61]

[62]

[63]

[64]

[65]

[66] [67]

[68]

[69]

[70]

[71]

[72]

[73]

41

man, F. Wondisford, Divergent roles for thyroid hormone receptor beta isoforms in the endocrine axis and auditory system, J. Clin. Invest. 104 (1999) 291 – 300. L. Ng, D. Forrest, B.R. Haugen, W.M. Wood, T. Curran, N-terminal variants of thyroid hormone receptor beta: differential function and potential contribution to syndrome of resistance to thyroid hormone, Mol. Endocrinol. 9 (1995) 1202 – 1213. J.D. Safer, M.F. Langlois, R. Cohen, T. Monden, D. JohnHope, J. Madura, A.N. Hollenberg, F.E. Wondisford, Isoform variable action among thyroid hormone receptor mutants provides insight into pituitary resistance to thyroid hormone, Mol. Endocrinol. 11 (1997) 16 – 26. O. Cohen, T.R. Flynn, F.E. Wondisford, Ligand-dependent antagonism by retinoid X receptors of inhibitory thyroid hormone response elements, J. Biol. Chem. 270 (1995) 13899 – 13905. A.N. Hollenberg, T. Monden, J.P. Madura, K. Lee, F.E. Wondisford, Function of nuclear co-repressor protein on thyroid hormone response elements is regulated by the receptor A/B domain, J. Biol. Chem. 271 (1996) 28516 – 28520. M.F. Langlois, K. Zanger, T. Monden, J.D. Safer, A.N. Hollenberg, F.E. Wondisford, A unique role of the beta-2 thyroid hormone receptor isoform in negative regulation by thyroid hormone. Mapping of a novel amino-terminal domain important for ligand-independent activation, J. Biol. Chem. 272 (1997) 24927 – 24933. H. Tomura, J. Lazar, M. Phyillaier, V.M. Nikodem, The Nterminal region (A/B) of rat thyroid hormone receptors alpha 1, beta 1, but not beta 2 contains a strong thyroid hormonedependent transactivation function, Proc. Natl. Acad. Sci. USA 92 (1995) 5600 – 5604. S. Refetoff, R.E. Weiss, S.J. Usala, The syndromes of resistance to thyroid hormone, Endocr. Rev. 14 (1993) 348 –399. Y. Hayashi, R.E. Weiss, D.H. Sarne, P.M. Yen, T. Sunthornthepvarakul, C. Marcocci, W.W. Chin, S. Refetoff, Do clinical manifestations of resistance to thyroid hormone correlate with the functional alteration of the corresponding mutant thyroid hormone-beta receptors?, J. Clin. Endocrinol. Metab. 80 (1995) 3246 – 3256. A. Fraichard, O. Chassande, M. Plateroti, J.P. Roux, J. Trouillas, C. Dehay, et al., The T3R alpha gene encoding a thyroid hormone receptor is essential for post-natal development and thyroid hormone production, EMBO J. 16 (1997) 4412 – 4420. L. Wikstrom, C. Johansson, C. Salto, C. Barlow, A. Campos Barros, F. Baas, et al., Abnormal heart rate and body temperature in mice lacking thyroid hormone receptor alpha 1, EMBO J. 17 (1998) 455 – 461. R.L. Wagner, B.R. Huber, A.K. Shiau, A.E. Kelly, T.S. Scanlan, J.W. Apriletti, J.D. Baxter, B.L. West, R.J. Fletterick, Hormone selectivity in thyroid hormone receptors, Mol. Endocrinol. 15, (2001) 398 – 410. G. Chiellini, J.W. Apriletti, H.A.I. Yoshihara, J.D. Baxter, R.C. Ribeiro, T.S. Scanlan, A high-affinity subtype-selective agonist ligand for the thyroid hormone receptor, Chem. Biol. 5 (1998) 299 – 306. S.W. Dietrich, M.B. Bolger, P.A. Kollman, E.C. Jorgensen, Thyroxine analogues. 23. Quantitative structure-activity correlation studies of in vivo and in vitro thyromimetic activities, J. Med. Chem. 20 (1977) 863 – 880. S.U. Trost, E. Swanson, B. Gloss, D.B. Wang-Iverson, H. Zhang, T. Volodarsky, G.J. Grover, J.D. Baxter, G. Chiellini, T.S. Scanlan, W.H. Dillmann, The thyroid hormone receptorbeta-selective agonist GC-1 differentially affects plasma lipids and cardiac activity, Endocrinology 141 (2000) 3057 – 3064.

42

J.D. Baxter et al. / Journal of Steroid Biochemistry & Molecular Biology 76 (2001) 31–42

[74] A. Ludwig, X. Zong, M. Jeglitsch, F. Hofmann, M. Bie, A family of hyperpolarization-activated mammalian cation channels, Nature 393 (1998) 587 –591. [75] B. Santoro, S.G. Grant, D. Bartsch, E.R. Kandel, Interactive

.

cloning with the SH3 domain of N-src identifies a new brain specific ion channel protein, with homology to eag and cyclic nucleotide-gated channels, Proc. Natl. Acad. Sci. USA 94 (1997) 14815 – 14820.