Ngoc Ha Nguyen

Rapid nongenomic actions of thyroid hormone

The binding of thyroid hormone to the thyroid hormone receptor (TR) mediates important physiological effects. However, the transcriptional effects of TR mediated by the thyroid response element (TRE) cannot explain many actions of thyroid hormone. We postulate that TR can initiate rapid, non-TRE-mediated effects in the cardiovascular system through cross-coupling to the phosphatidylinositol 3-kinase (PI3-kinase)/protein kinase Akt pathway. In vascular endothelial cells, the predominant TR isoform is TRα1. Treatment of endothelial cells with l-3,5,3′-triiodothyronine (T3) increased the association of TRα1 with the p85α subunit of PI3-kinase, leading to the phosphorylation and activation of Akt and endothelial nitric oxide synthase (eNOS). The activation of Akt and eNOS by T3 was abolished by the PI3-kinase inhibitors, LY294002 and wortmannin, but not by the transcriptional inhibitor, actinomycin D. To determine the physiological relevance of this PI3-kinase/Akt pathway, we administered T3 to mice undergoing transient focal cerebral ischemia. Compared with vehicle, a single bolus infusion of T3 rapidly increased Akt activity in the brain, decreased mean blood pressure, reduced cerebral infarct volume, and improved neurological deficit score. These neuroprotective effects of T3 were greatly attenuated or absent in eNOS−/− and TRα1−/−β−/− mice and were completely abolished in WT mice pretreated with LY294002 or a T3 antagonist, NH-3. These findings indicate that the activation of PI3-kinase/Akt pathways can mediate some of the rapid, non-TRE effects of TR and suggest that the activation of Akt and eNOS contributes to some of the acute vasodilatory and neuroprotective effects of thyroid hormone.

Polybrominated diphenyl ethers induce developmental neurotoxicity in a human in vitro model: evidence for endocrine disruption.

Background Polybrominated diphenyl ethers (PBDEs) are persistent and bioaccumulative flame retardants, which are found in rising concentrations in human tissues. They are of concern for human health because animal studies have shown that they possess the potential to be developmentally neurotoxic. Objective Because there is little knowledge of the effects of PBDEs on human brain cells, we investigated their toxic potential for human neural development in vitro. Moreover, we studied the involvement of thyroid hormone (TH) disruption in the effects caused by PBDEs. Methods We used the two PBDE congeners BDE-47 and BDE-99 (0.1–10 μM), which are most prominent in human tissues. As a model of neural development, we employed primary fetal human neural progenitor cells (hNPCs), which are cultured as neurospheres and mimic basic processes of brain development in vitro: proliferation, migration, and differentiation. Results PBDEs do not disturb hNPC proliferation but decrease migration distance of hNPCs. Moreover, they cause a reduction of differentiation into neurons and oligodendrocytes. Simultaneous exposure with the TH receptor (THR) agonist triiodothyronine rescues these effects on migration and differentiation, whereas the THR antagonist NH-3 does not exert an additive effect. Conclusion PBDEs disturb development of hNPCs in vitro via endocrine disruption of cellular TH signaling at concentrations that might be of relevance for human exposure.

Polychlorinated biphenyls disturb differentiation of normal human neural progenitor cells: clue for involvement of thyroid hormone receptors.

Polychlorinated biphenyls (PCBs) are ubiquitous environmental chemicals that accumulate in adipose tissues over the food chain. Epidemiologic studies have indicated that PCBs influence brain development. Children who are exposed to PCBs during development suffer from neuropsychologic deficits such as a lower full-scale IQ (intelligence quotient), reduced visual recognition memory, and attention and motor deficits. The mechanisms leading to these effects are not fully understood. It has been speculated that PCBs may affect brain development by interfering with thyroid hormone (TH) signaling. Because most of the data are from animal studies, we established a model using primary normal human neural progenitor (NHNP) cells to determine if PCBs interfere with TH-dependent neural differentiation. NHNP cells differentiate into neurons, astrocytes, and oligodendrocytes in culture, and they express a variety of drug metabolism enzymes and nuclear receptors. Like triiodothyronine (T3), treatment with the mono-ortho-substituted PCB-118 (2,3′,4,4′,5-pentachlorobiphenyl; 0.01–1 μM) leads to a dose-dependent increase of oligodendrocyte formation. This effect was congener specific, because the coplanar PCB-126 (3,3′,4,4′,5-pentachlorobiphenyl) had no effect. Similar to the T3 response, the PCB-mediated effect on oligodendrocyte formation was blocked by retinoic acid and the thyroid hormone receptor antagonist NH-3. These results suggest that PCB-118 mimics T3 action via the TH pathway.

A thyroid hormone antagonist that inhibits thyroid hormone action in vivo.

We have characterized the newly developed thyroid hormone antagonist NH-3 in both cell culture and in vivomodel systems. NH-3 binds Xenopus laevisthyroid 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.

Improved synthesis of the iodine-free thyromimetic GC-1.

Synthesis of the TRbeta-selective thyromimetic GC-1 has been improved using methoxymethyl (MOM) and triisopropylsilyl (TiPS) substituents as phenolic protecting groups. The new synthetic route is adaptable to analogue design.

Pharmacological Profile of the Thyroid Hormone Receptor Antagonist NH3 in Rats

NH3 is a thyroid hormone receptor (TR) antagonist that inhibits binding of thyroid hormones to their receptor and that inhibits cofactor recruitment. It was active in a tadpole tail resorption assay, with partial agonist activity at high concentrations. We determined the effect of NH3 on the cholesterol-lowering, thyroid stimulating hormone (TSH)-lowering, and tachycardic action of thyroid hormone (T3) in rats. Cholesterol-fed, euthyroid rats were treated for 7 days with NH3, and a dose response (46.2-27,700 nmol/kg/day) was determined. We also determined the effect of two doses of T3 on the NH3 dose-response curve. NH3 decreased heart rate modestly starting at 46.2 nmol/kg/day, but the effect was lost at >2920 nmol/kg/day. At 27,700 nmol/kg/day, tachycardia was seen, suggesting partial agonist activity. NH3 increased plasma cholesterol to a maximum of 27% at 462 nmol/kg/day. At higher doses, cholesterol was reduced, suggesting partial agonist activity. Plasma TSH was increased from 46.2 to 462 nmol/kg/day NH3, but at higher doses, this effect was lost, and partial agonist effects were apparent. T3 at 15.4 and 46.2 nmol/kg/day increased heart rate, reduced cholesterol, and reduced plasma TSH. NH3 inhibited the cholesterol-lowering, TSH-lowering and tachycardic effects of 15.4 nmol/kg/day T3, but much of the effect was lost at >924 nmol/kg/day doses. NH3 had no effect on the cholesterol-lowering action of 46.2 nmol/kg/day T3, but it inhibited the tachycardic and TSH suppressant effects up to the 924 nmol/kg/day dose. Single doses of 462 and 27,700 nmol/kg caused no TR inhibitory effects. In conclusion, NH3 has TR antagonist properties on T3-responsive parameters, but it has partial agonist properties at higher doses.

Complex actions of thyroid hormone receptor antagonist NH-3 on gene promoters in different cell lines.

It is desirable to obtain new antagonists for thyroid hormone receptors (TRs) and other nuclear receptors (NRs). We previously used X-ray structural models of TR ligand binding domains (LBDs) to design compounds, such as NH-3, that impair coactivator binding to activation function 2 (AF-2) and block thyroid hormone (triiodothyronine, T(3)) actions. However, TRs bind DNA and are transcriptionally active without ligand. Thus, NH-3 could modulate TR activity via effects on other coregulator interaction surfaces, such as activation function (AF-1) and corepressor binding sites. Here, we find that NH-3 blocks TR-LBD interactions with coactivators and corepressors and also inhibits activities of AF-1 and AF-2 in transfections. While NH-3 lacks detectable agonist activity at T(3)-activated genes in GC pituitary cells it nevertheless activates spot 14 (S14) in HTC liver cells with the latter effect accompanied by enhanced histone H4 acetylation and coactivator recruitment at the S14 promoter. Surprisingly, T(3) promotes corepressor recruitment to target promoters. NH-3 effects vary; we observe transient recruitment of N-CoR to S14 in GC cells and dismissal and rebinding of N-CoR to the same promoter in HTC cells. We propose that NH-3 will generally behave as an antagonist by blocking AF-1 and AF-2 but that complex effects on coregulator recruitment may result in partial/mixed agonist effects that are independent of blockade of T(3) binding in some contexts. These properties could ultimately be utilized in drug design and development of new selective TR modulators.

Design and Synthesis of Receptor Ligands

Publisher Summary This chapter focuses on the design and synthesis of receptor ligands. The synthetic ligands of nuclear receptors have a number of properties that make them useful as pharmacological probes in the study of nuclear receptor action. These properties include selective action, either in specifically binding to a receptor orthologue, isoform, or subtype; the selective activation of specific hormone response elements; or antagonism, where a ligand competes with the natural hormone for binding to the receptor but does not activate it. Nuclear hormone receptors play important roles in development and homeostasis and are implicated in a variety of diseases such as cancer, diabetes, and various endocrine disorders. The roles of the RXR, TR, LXR, FXR, and PPAR receptors in regulating lipid homeostasis also make them potentially attractive targets to treat or prevent cardiovascular diseases. The selective activators (or inhibitors) of nuclear receptors have the promise to reduce these side effects, thereby acting as safer and more useful drugs. The classic steroid hormones are derived from cholesterol by enzymatic modification. Other cholesterol metabolites, including oxysterols and bile acids, serve as ligands for LXR and FXR, respectively. Retinoids are produced from the oxidative cleavage of β-carotene, an essential nutrient. While natural ligands of NRs are produced by a variety of different pathways, the ligands themselves share common features. They are largely hydrophobic in character, consisting mostly of aliphatic, aromatic, or olefinic hydrocarbons.

Differential effects of TR ligands on hormone dissociation rates: evidence for multiple ligand entry/exit pathways.

Some nuclear receptor (NR) ligands promote dissociation of radiolabeled bound hormone from the buried ligand binding cavity (LBC) more rapidly than excess unlabeled hormone itself. This result was interpreted to mean that challenger ligands bind allosteric sites on the LBD to induce hormone dissociation, and recent findings indicate that ligands bind weakly to multiple sites on the LBD surface. Here, we show that a large fraction of thyroid hormone receptor (TR) ligands promote rapid dissociation (T(1/2)<2h) of radiolabeled T(3) vs. T(3) (T(1/2) approximately 5-7h). We cannot discern relationships between this effect and ligand size, activity or affinity for TRbeta. One ligand, GC-24, binds the TR LBC and (weakly) to the TRbeta-LBD surface that mediates dimer/heterodimer interaction, but we cannot link this interaction to rapid T(3) dissociation. Instead, several lines of evidence suggest that the challenger ligand must interact with the buried LBC to promote rapid T(3) release. Since previous molecular dynamics simulations suggest that TR ligands leave the LBC by several routes, we propose that a subset of challenger ligands binds and stabilizes a partially unfolded intermediate state of TR that arises during T(3) release and that this effect enhances hormone dissociation.