Biomedical subjects
Publications and source records attributed to Thomas S Scanlan.
A novel class of phenylpyrazole fused Wieland-Miescher ketone derivatives are high affinity, receptor specific, selective modulators of glucocorticoid receptor (GR) mediated transcription in vitro, dissociating transactivation, AP-1 repression, and NF-kappaB repression from each other.
Thyroxine (T(4)) is the predominant form of thyroid hormone (TH). Hyperthyroidism, a condition associated with excess TH, is characterized by increases in metabolic rate, core body temperature and cardiac performance. In target tissues, T(4) is enzymatically deiodinated to 3,5,3'-triiodothyronine (T(3)), a high-affinity ligand for the nuclear TH receptors TR alpha and TR beta, whose activation controls normal vertebrate development and physiology. T(3)-modulated transcription of target genes via activation of TR alpha and TR beta is a slow process, the effects of which manifest over hours and days. Although rapidly occurring effects of TH have been documented, the molecules that mediate these non-genomic effects remain obscure. Here we report the discovery of 3-iodothyronamine (T(1)AM), a naturally occurring derivative of TH that in vitro is a potent agonist of the G protein-coupled trace amine receptor TAR1. Administering T(1)AM in vivo induces profound hypothermia and bradycardia within minutes. T(1)AM treatment also rapidly reduces cardiac output in an ex vivo working heart preparation. These results suggest the existence of a new signaling pathway, stimulation of which leads to rapid physiological and behavioral consequences that are opposite those associated with excess TH.
The thyroid hormone receptor regulates a diverse set of genes that control processes from embryonic development to adult homeostasis. Upon binding of thyroid hormone, the thyroid receptor releases corepressor proteins and undergoes a conformational change that allows for the interaction of coactivating proteins necessary for gene transcription. This interaction is mediated by a conserved motif, termed the NR box, found in many coregulators. Recent work has demonstrated that differentially assembled coregulator complexes can elicit specific biological responses. However, the mechanism for the selective assembly of these coregulator complexes has yet to be elucidated. To further understand the principles underlying thyroid receptor-coregulator selectivity, we designed a high-throughput in vitro binding assay to measure the equilibrium affinity of thyroid receptor to a library of potential coregulators in the presence of different ligands including the endogenous thyroid hormone T3, synthetic thyroid receptor beta-selective agonist GC-1, and antagonist NH-3. Using this homogenous method several coregulator NR boxes capable of associating with thyroid receptor at physiologically relevant concentrations were identified including ones found in traditional coactivating proteins such as SRC1, SRC2, TRAP220, TRBP, p300, and ARA70; and those in coregulators known to repress gene activation including RIP140 and DAX-1. In addition, it was discovered that the thyroid receptor-coregulator binding patterns vary with ligand and that this differential binding can be used to predict biological responses. Finally, it is demonstrated that this is a general method that can be applied to other nuclear receptors and can be used to establish rules for nuclear receptor-coregulator selectivity.
A major challenge in understanding nuclear hormone receptor function is to determine how the same ligand can cause very different tissue-specific responses. Tissue specificity may result from the presence of more than one receptor subtype arising from multiple receptor genes or alternative splicing. Recently, high affinity analogs of nuclear receptor ligands have been synthesized that show subtype selectivity. These analogs can greatly facilitate the study of receptor subtype-specific functions in organisms where mutational analysis is problematic or where it is desirable for receptors to be expressed in their normal physiological contexts. We describe here the effects of the synthetic thyroid hormone analog GC-1 on the metamorphosis of the frog Xenopus laevis. The most potent natural thyroid hormone, 3,5,3'-triidothyronine or T3, shows similar binding affinity and transactivation dose-response curves for both thyroid hormone receptor isotypes, designated TRalpha and TRbeta. GC-1, however, binds to and activates TRbeta at least an order of magnitude better than it does TRalpha. GC-1 efficiently induces death and resorption of premetamorphic tadpole tissues such as the gills and the tail, two tissues that strongly induce thyroid hormone receptor beta during metamorphosis. GC-1 has less effect on the growth of adult tissues such as the hindlimbs, which express high TRalpha levels. The effectiveness of GC-1 in inducing tail resorption and tail gene expression correlates with increasing TRbeta levels. These results illustrate the utility of subtype selective ligands as probes of nuclear receptor function in vivo.
Thyroid hormones (TH) are essential regulators of vertebrate development and metabolism. Central mechanisms governing their production have evolved, with the beta-TH receptor (TRbeta) playing a key regulatory role in the negative feedback effects of circulating TH levels on production of hypothalamic TRH and hypophyseal TSH. Both TRbeta-isoforms (TRbeta1 and TRbeta2) are expressed in the hypothalamus and pituitary. However, their respective roles in TH-dependent transcriptional regulation of TRH are undefined. We confirmed the preferential role of TRbeta vs. TRalpha isoforms in TRH regulation in wild-type mice in vivo by using the TRbeta preferential agonist GC-1. We next determined the effects of tissue-specific rescue of TRbeta1 and TRbeta2 isoforms by somatic gene transfer in hypothalami of TRbeta null (TRbeta(-/-)) mice. TH-dependent TRH transcriptional repression was impaired in TRbeta(-/-) mice, but was restored by cotransfection of either TRbeta1 or TRbeta2 into the hypothalamus. TRbeta1, but not TRbeta2, displayed a role in ligand-independent activation. In situ hybridization was used to examine endogenous TRH expression in the paraventricular nucleus of the hypothalamus of TRbeta(-/-) or TRalpha null (TRalpha(o/o)) mice under different thyroid states. In contrast to published data on TRbeta2(-/-) mice, we found that both ligand-independent TRH activation and ligand-dependent TRH repression were severely impaired in TRbeta(-/-) mice. This study thus provides functional in vivo data showing that both TRbeta1 and TRbeta2 isoforms have specific roles in regulating TRH transcription.
Current drug therapies for obesity are ineffective, and existing treatments for lipid disorders can be further improved. Thyroid hormones affect both conditions, although currently available nonselective thyromimetics are not clinically useful for such treatment due to cardiac side effects. Recent studies suggest that thyroid hormone receptor subtype beta (TRbeta) selective agonists have a profile in which cholesterol can be reduced with minimal tachycardia. The purpose of this study was to determine whether modest (5-10%) increases in metabolic rate could also be observed with minimal tachycardia after TRbeta stimulation. For these studies, the TRbeta selective agonist, GC-1, was used to assess selectivity for lipid-lowering and metabolic rate changes relative to tachycardia. Studies in cholesterol-fed rats (7 d treatment) showed that GC-1 reduced cholesterol (ED(50) = 190 nmol/kg x d) approximately 30 times more potently than it induced tachycardia (ED(15) = 5451 nmol/kg x d). T(3) showed no potency difference between cholesterol lowering and tachycardia. GC-1 showed approximately 10-fold selectivity for increasing metabolic rate (ED(5) = 477 nmol/kg x d) relative to tachycardia compared with T(3), which showed no selectivity. In cynomolgus monkeys treated for 7 d, significant cholesterol-lowering and lipoprotein (a) reduction was noted for both T(3) and GC-1, whereas no tachycardia was observed for GC-1, unlike T(3). T(3) and GC-1 caused a significant (approximately 4%) reduction in body weight in these animals. Therefore, selective TRbeta activation may be a potentially usefully treatment for obesity and reduction of low density lipoprotein cholesterol and reduction of the atherogenic risk factor lipoprotein (a).
Selective therapeutics for nuclear receptors would revolutionize treatment for endocrine disease. Specific control of nuclear receptor activity is challenging because the internal cavities that bind hormones can be virtually identical. Only one highly selective hormone analog is known for the thyroid receptor, GC-24, an agonist for human thyroid hormone receptor beta. The compound differs from natural hormone in benzyl, substituting for an iodine atom in the 3' position. The benzyl is too large to fit into the enclosed pocket of the receptor. The crystal structure of human thyroid hormone receptor beta at 2.8-A resolution with GC-24 bound explains its agonist activity and unique isoform specificity. The benzyl of GC-24 is accommodated through shifts of 3-4 A in two helices. These helices are required for binding hormone and positioning the critical helix 12 at the C terminus. Despite these changes, the complex associates with coactivator as tightly as human thyroid hormone receptor bound to thyroid hormone and is fully active. Our data suggest that increased specificity of ligand recognition derives from creating a new hydrophobic cluster with ligand and protein components.
Although the effects of thyroid hormones on the development of neurons and oligodendrocytes are well documented, less is known about the hormonal effects on astrocytes. Our analyses of cerebellar slices from 2-month-old T(3) receptor protein (TR)alpha1-deficient mice show that mature astrocytes, Golgi epithelial cells, and their Bergmann processes had strongly reduced glial fibrillary acidic protein (GFAP) and nestin immunoreactivity, in contrast to wild-type mice. Furthermore, the Bergmann processes exhibited an irregular GFAP staining. A similar expression of nestin and GFAP was observed in 11-d-old (P11) mutant pups. Surprisingly, however, hypothyroidism normalized the appearance of these markers in the P11 mutants, suggesting that liganded TR beta is detrimental to astroglial cell differentiation in the absence of TR alpha 1. To test this hypothesis, hypothyroid mice were treated from birth until P11 with the TR beta-selective ligand GC-1. This treatment was devastating in the TR alpha 1(-/-) mice, causing little if any nestin or GFAP immunoreactivity, whereas the wild-type mice were normal. The results thus indicate an important interplay between thyroid hormone receptor isoforms in astroglial cell maturation.
Classically, 17beta-estradiol (E2) is thought to control homeostatic functions such as reproduction, stress responses, feeding, sleep cycles, temperature regulation, and motivated behaviors through transcriptional events. Although it is increasingly evident that E2 can also rapidly activate kinase pathways to have multiple downstream actions in CNS neurons, the receptor(s) and the signal transduction pathways involved have not been identified. We discovered that E2 can alter mu-opioid and GABA neurotransmission rapidly through nontranscriptional events in hypothalamic GABA, proopiomelanocortin (POMC), and dopamine neurons. Therefore, we examined the effects of E2 in these neurons using whole-cell recording techniques in ovariectomized female guinea pigs. E2 reduced rapidly the potency of the GABAB receptor agonist baclofen to activate G-protein-coupled, inwardly rectifying K+ channels in hypothalamic neurons. These effects were mimicked by the membrane impermeant E2-BSA and selective estrogen receptor modulators, including a new diphenylacrylamide compound, STX, that does not bind to intracellular estrogen receptors alpha or beta, suggesting that E2 acts through a unique membrane receptor. We characterized the coupling of this estrogen receptor to a Galpha(q)-mediated activation of phospholipase C, leading to the upregulation of protein kinase Cdelta and protein kinase A activity in these neurons. Moreover, using single-cell reverse transcription-PCR, we identified the critical transcripts, PKCdelta and its downstream target adenylyl cyclase VII, for rapid, novel signaling of E2 in GABA, POMC, and dopamine neurons. Therefore, this unique Gq-coupled estrogen receptor may be involved in rapid signaling in hypothalamic neurons that are critical for normal homeostatic functions.
Thyrotoxicosis is frequently associated with increased bone turnover and decreased bone mass. To investigate the role of thyroid hormone receptor-beta (TR beta) in mediating the osteopenic effects of triiodothyronine (T3), female adult rats were treated daily (64 days) with GC-1 (1.5 microg/100 g body wt), a TR beta-selective thyromimetic compound. Bone mass was studied by dual-energy X-ray absorptiometry of several skeletal sites and histomorphometry of distal femur, and the results were compared with T3-treated (3 microg/100 g body wt) or control animals. As expected, treatment with T3 significantly reduced bone mineral density (BMD) in the lumbar vertebrae (L2-L5), femur, and tibia by 10-15%. In contrast, GC-1 treatment did not affect the BMD in any of the skeletal sites studied. The efficacy of GC-1 treatment was verified by a reduction in serum TSH (-52% vs. control, P < 0.05) and cholesterol (-21% vs. control, P < 0.05). The histomorphometric analysis of the distal femur indicated that T3 but not GC-1 treatment reduced the trabecular volume, thickness, and number. We conclude that chronic, selective activation of the TR beta isoform does not result in bone loss typical of T3-induced thyrotoxicosis, suggesting that the TR beta isoform is not critical in this process. In addition, our findings suggest that the development of TR-selective T3 analogs that spare bone mass represents a significant improvement toward long-term TSH-suppressive therapy.
The availability of synthetic thyroid hormone receptor agonists provides a valuable tool to analyze whether specific receptor isoforms mediate specific physiological responses to thyroid hormone. GC-1 is a thyroid hormone analog displaying selectivity for thyroid hormone receptor beta. We have analyzed the effect of GC-1 on expression of thyroid hormone target genes in the cerebrum and cerebellum. Congenitally hypothyroid rats were treated with single daily doses of either T3 or GC-1. Both compounds similarly induced Purkinje cell protein-2 (PCP-2) in the cerebellum. Expression of RC3 and Rhes in the caudate, and hairless, neurotrophin-3, Reelin, and Rev-ErbAalpha in the cerebellum, was analyzed by in situ hybridization on postnatal d 16. Hypothyroidism strongly decreased expression of RC3 and Rhes in the caudate, and hairless, Rev-ErbAalpha, and neurotrophin-3 in the cerebellum, and increased Reelin. T3 treatment normalized the expression of all genes. However, GC-1 effectively normalized expression of Rhes and Reelin only. The lack of a GC-1 effect on most cerebellar genes can be explained by the known distribution of thyroid hormone receptor alpha and beta isoforms. However, in the caudate, RC3 and Rhes are expressed in the same cells, and therefore, they may represent specific gene responses linked to specific thyroid hormone receptor isoforms.
The thyromimetic GC-1 shows a preference for binding the beta form of the thyroid hormone receptor (TR). GC-1 was designed as an analogue of the thyromimetic DIMIT, which has a lower affinity for TR and is not selective. GC-1 has a methylene group linking its two aromatic rings and an oxyacetic acid polar side chain, while DIMIT has an ether oxygen linking its aromatic rings and an l-alanine polar side chain. The structural features of GC-1 that confer its greater affinity and selectivity compared to DIMIT were analyzed with the preparation of analogues that bear only one of their two different structural features. The analogue of GC-1 with a biaryl ether has selectivity comparable to that of GC-1, while the analogue of DIMIT with a methylene group linking its aromatic rings is only slightly selective. These results demonstrate that the oxyacetic acid side chain of GC-1 is critical in conferring TR-beta selectivity.
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Direct hydroxide attack on the scissile carbonyl of the substrate has been suggested as a likely mechanism for esterase antibodies elicited by phosphonate haptens, which mimic the transition states for the alkaline hydrolysis of esters.1 The unique amidase activity of esterase antibody 43C9 has been attributed to nucleophilic attack by an active-site histidine residue.2 Yet, the active site of 43C9 is strikingly similar to those of other esterase antibodies, particularly 17E8. We have carried out quantum mechanical calculations, molecular dynamics simulations, and free energy calculations to assess the mechanism involving direct hydroxide attack for 43C9. Results support this mechanism and suggest that the mechanism is plausible for other antiphosphonate antibodies that catalyze the hydrolysis of (p-nitro)phenyl esters.
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Thyroid hormone regulates many important processes in vertebrates. Analysis of the symptoms that accompany hypo- and hyperthyroidism, the most common disorders of the thyroid, suggests that there are certain desirable biological effects brought about by an excess or deficiency of thyroid hormone, and that selective thyroid hormone receptor modulators (STRMs) would be potentially useful therapeutic agents. This review will provide an introduction to thyroid hormone biology, and will discuss the chemistry and pharmacology of the first generation STRMs.
Recent efforts have focused on the design and synthesis of thyroid hormone (T(3)) antagonists as potential therapeutic agents and chemical probes to understand hormone-signaling pathways. We previously reported the development of novel first-generation T(3) antagonists DIBRT, HY-4, and GC-14 using the "extension hypothesis" as a general guideline in hormone antagonist design.(1-3) These compounds contain extensions at the 5'-position (DIBRT, GC-14) of the outer thyronine ring or from the bridging carbon (HY-4). All of these compounds have only a modest affinity and potency for the thyroid hormone receptor (TR) that limits studies of their antagonistic actions. Here, we report the design and synthesis of a novel series of 5'-phenylethynyl derivatives sharing the GC-1 halogen-free thyronine scaffold.(4) One compound (NH-3) is a T(3) antagonist with negligible TR agonist activity and improved TR binding affinity and potency that allow for further characterization of its observed activity. One mechanism for antagonism appears to be the ability of NH-3 to block TR-coactivator interactions. NH-3 will be a useful pharmacological tool for further study of T(3) signaling and TR function.
We have characterized the newly developed thyroid hormone antagonist NH-3 in both cell culture and in vivo model systems. NH-3 binds Xenopus laevis thyroid hormone receptors directly in vitro and induces a conformation distinct from agonist-bound receptors. Transcriptional activation of a thyroid hormone response element-containing reporter gene is strongly inhibited by NH-3 in a dose-dependent manner. In addition, NH-3 prevents X. laevis thyroid hormone receptors from binding to the p160 family of co-activators GRIP-1 and SRC-1 in a two-hybrid assay. To assess the potency of the compound in vivo, we used induced and spontaneous X. laevis tadpole metamorphosis, a thyroid hormone-dependent developmental process. NH-3 inhibits thyroid hormone-induced morphological changes in a dose-dependent manner and inhibits the up-regulation of endogenous thyroid hormone-responsive genes. Spontaneous metamorphosis is efficiently and reversibly arrested by NH-3 with at least the same effectiveness as the thyroid hormone synthesis inhibitor methimazole. Therefore, NH-3 is the first thyroid hormone antagonist to demonstrate potent inhibition of thyroid hormone action in both cell culture- and whole animal-based assays.