Alternative ligands for thyroid hormone receptors.

Thyroid hormone receptors (TRs) are ligand-dependent transcription factors that activate or repress gene transcription, resulting in the regulation of numerous physiological programs. While 3,3',5-L-triiodothyronine is the TR cognate ligand, these receptors can also be activated by various alternative ligands, including endogenous and synthetic molecules capable of inducing diverse active receptor conformations that influence thyroid hormone-dependent signaling pathways. This review mainly discusses current knowledge on 3,5-diiodo-L-thyronine and 3,5,3'-triiodothyroacetic acid, two endogenous molecules that bind to TRs and regulate gene expression; and the molecular interactions between TRs and ligands, like synthetic thyromimetics developed to target specific TR isoforms for tissue-specific regulation of thyroid-related disorders, or endocrine disruptors that have allowed the design of new analogues and revealed essential amino acids for thyroid hormone binding.

Environmental photochemical fate and UVC degradation of sodium levothyroxine in aqueous medium.

The synthetic hormone sodium levothyroxine (LTX) is one of the most prescribed drugs in the world and the most effective in hypothyroidism treatment. The presence of LTX in the environment has become a matter of major concern due to the widespread use of this hormone and by the fact that it is only partially removed in conventional water and sewage treatment plants. However, information regarding the photochemical fate of this hormone in environmental or engineered systems is scarce in the literature. In this work, the sunlight-driven direct and indirect LTX degradation was investigated by determining the photolysis quantum yield, ΦLTX = 3.80 (± 0.02) × 10-5, as well as the second-order kinetic constants of the reactions with hydroxyl radicals, kLTX,•OH = 1.50 (± 0.01) × 1010 L mol-1 s-1 and singlet oxygen, kLTX,1O2 = 1.47 (± 0.66) × 108 L mol-1 s-1. Mathematical simulations indicate that LTX photodegradation is favored in shallow, nitrite-rich, and dissolved organic matter (DOM)-poor environments, with LTX half-life times varying from less than 10 days to about 80 days. LTX removals of 85 and 95% were achieved by UVC photolysis and UVC/H2O2 after 120 min, respectively. Three transformation products, triiodothyronine, diiodothyronine, and diiodotyrosine, were identified during LTX degradation by the UVC-based processes studied. The results herein regarding photo-induced kinetics coupled with environmental fate simulations may help evaluate LTX persistence and also the design of water and wastewater treatment processes.

[Thyroid hormones and their precursors I. Biochemical properties]. / Pajzsmirigyhormonok és eloanyagaik I. Biokémiai tulajdonságok.

This paper and the following one (see the next issue of Acta Pharmaceutica Hungarica) survey the biological roles and the related site-specific physico-chemical parameters (basicity and lipophilicity) of the presently known thyroid hormones (thyroxine, liothyronine and reverse liothyronine) and their biological precursors (monoiodotyrosine and diiodotyrosine). Here the literature of the thyroid hormone biochemistry, biosynthesis, plasma- and membrane transport is summarized, focusing on the pH-dependent processes. Biosyntheses of the thyroid hormones take place by oxidative coupling of two iodotyrosine residues catalyzed by thyreoperoxidase in thyreoglobulin. The protonation state of the precursors, especially that of the phenolic OH is crucial for the biosynthesis, since anionic iodotyrosine residues can only be coupled in the thyroid hormone biosyntheses. In the blood more than 99% of the circulating thyroid hormone is bound to plasma proteins among which the thyroxine-binding globulin and transthyretin are crucial. The amphiphilic character of the hormones is assumed to be the reason why their membrane transport is an energy-dependent, transport-mediated process, in which the organic anion transporter family, mainly OATP1C1, and the amino acid transporters, such as MCT8 play important roles. Liothyronine is the biologically active hormone; it binds the thyroid hormone receptor, a type of nuclear receptor. There are two major thyroid hormone receptor (TR) isoforms, alfa (TRalpha) and beta (TRbeta). The activation of the TRalpha is associated with modifications in cardiac behavior, while activation of the TRbeta is associated with increasing metabolic rates, resulting in weight loss and reduction of blood plasma lipid levels. The affinity of the thyroid hormones for different proteins depends on the ionization state of the ligands. The site-specific physico-chemical characterization of the thyroid hormones is of fundamental importance to understand their (patho)physiological behavior and also, to influence their therapeutic properties at the molecular level.

Photochemical transformation of the thyroid hormone levothyroxine in aqueous solution.

PURPOSE: The direct aqueous photolysis of the thyroid hormone levothyroxine (T(4)) has been studied. METHODS AND RESULT: One of the major photoproducts, i.e., 4-[4-(2-amino-2-carboxy-ethyl)-2,6-diiodo-phenoxy]-penta-2,4-dienoic acid (P1), was isolated by liquid chromatography and structurally assigned by mass spectrometric (MS) and nuclear magnetic resonance spectroscopic methods. The identity of a second major product, i.e., 3,5-diiodo-L: -thyrosine (P3), was confirmed through access to a commercially available standard. Furthermore, the structures of three additional transformation products are proposed on the basis of data obtained by high-resolution MS analyses. UV absorption spectra were determined for T(4) and the two photoproducts P1 and P3. Disappearance quantum yields were calculated for T(4) (ϕ = 0.014 at pH 12) and P3 (ϕ = 0.024 at pH 12 and ϕ = 0.010 at pH 8.5), whereas the compound P1 was found to be stable under the studied conditions (T(1/2) = 600 min). CONCLUSION: The results indicate that solar UV light may have a significant impact on the fate of T(4) in the aquatic environment.

Thyroid hormone interactions with DMPC bilayers. A molecular dynamics study.

The structure and dynamics of thyroxine (T4), distal and proximal conformers of 3',3,5-triiodo-l-thyronine (T3d and T3p), and 3,5-diiodo-l-thyronine (T2) upon interaction with DMPC membranes were analyzed by means of molecular dynamics simulations. The locations, the more stable orientations, and the structural changes adopted by the hormones in the lipid medium evidence that the progressive iodine substitution on the beta ring lowers both the possibility of penetration and the transversal mobility in the membrane. However, the results obtained for T3d show that the number of iodine atoms in the molecule is not the only relevant factor in the hormone behavior but also the orientation of the single iodine substitution. The electrostatic interactions between the zwitterion group of the hormones with specific groups in the hydrophilic region of the membrane as well as the organization of the alkyl chains around the aromatic beta ring of the hormone were evaluated in terms of several radial distribution functions.

Differentiation of diiodothyronines using electrospray ionization tandem mass spectrometry.

Diiodothyronines 3,5-diiodothyronine (3,5-T2), 3',5'-diiodothyronine (3',5'-T2), and 3,3'-diiodothyronine (3,3'-T2) are important metabolites of 3,5,3'-triiodothyronine (T3) and 3,3',5'-triiodothyronine (rT3; reverse T3). In this paper, a novel and rapid method for identifying and quantifying 3,5-T2, 3',5'-T2 and 3,3'-T2 has been introduced using electrospray ionization tandem mass spectrometry (ESI-MS/MS). Fragmentation patterns were proposed on the basis of our data obtained by ESI-MS/MS. MS2 spectra in either negative ionization mode or positive ionization mode can be used to differentiate 3,5-T2, 3',5'-T2 and 3,3'-T2. On the basis of the relative abundance of fragment ions in MS2 spectra under the positive ionization mode, quantification of the 3,5-T2, 3',5'-T2 and 3,3'-T2 isomers in mixtures is also achieved without prior separation.

Short-term effects of thyroid hormone in prenatal development and cell differentiation.

Extranuclear or nongenomic effects of thyroid hormones do not require interaction with the nuclear receptor, but are probably mediated by specific membrane receptors. This review will focus on the extranuclear effects of thyroid hormones on plasma membrane transport systems in non mammalian cells: chick embryo hepatocytes at two different stages of development, 14 and 19 days. At variance with mammals, the chick embryo develops in a closed compartment, beyond the influence of maternal endocrine factors. Thyroid hormones inhibit the Na+/K+-ATPase but stimulate the Na+/H+ exchanger and amino acid transport System A with different dose-responses: a bell-shaped curve in the case of the exchanger and a classic saturation curve in the case of System A. These effects are mimicked by the analog 3,5-diiodothyronine. Signal transduction is mediated by interplay among kinases, mainly protein kinase C and the MAPK pathway, initially primed by second messengers such as Ca2+, IP3, and DAG as in mammalian cells. Thyroid hormones and 3,5-diiodothyronine stimulate thymidine incorporation and DNA synthesis, associated with the increased levels and activity of cyclins and cyclin-dependent kinases involved in the G1/S transition, and also these effects have their starting point at the plasma membrane. Increasing evidence now demonstrates that thyroid hormones act as growth factors for chick embryo hepatocytes and their extranuclear effects are important for prenatal development and differentiation.

Effect of iodine and selenium upon thyroid function.

Iodine is an essential element with unique role in organism: it is indispensable component of thyroid hormones. After binding with specific nuclear receptor, T3/T4 induce transcription of genetic code via mRNA and regulate proteosynthesis in most tissues. Thyroid hormones regulate rate of metabolic processes and consequently development of organism. Czech Republic was in past typical region with moderate to severe iodine deficiency. Therefore epidemiological survey was started in randomly selected samples of peoples. Thereafter a complex program of improving iodine supply was realized. Prompt effects of this changes were recorded, namely increase of ioduria and decrease of thyroid volume. Essential trace element selenium has a fundamental importance to the cell and body metabolism regulation by thyroid hormones. Activities of selenoenzymes deiodinases lead to the activation of prohormone T4 to active hormone T3 and the inactivation of T3 and of T4, as well. There is unfortunately moderate to mild Se deficit in the CR. Average serum Se concentrations for the populations from 6 to 65 years are in the regions of the CR between 42 and 62 micrograms/l and urine Se values are between 8 and 15 micrograms/l. We have found statistically significant correlations among indexes of selenium status and indexes of thyroid hormone metabolism and function. Especially dangerous are concomitant deficiencies of both key elements for thyroid hormone metabolism--I and Se--from the point of thyroid hormone regulative functions.

Comparative evaluation of 11 scoring functions for molecular docking.

Eleven popular scoring functions have been tested on 100 protein-ligand complexes to evaluate their abilities to reproduce experimentally determined structures and binding affinities. They include four scoring functions implemented in the LigFit module in Cerius2 (LigScore, PLP, PMF, and LUDI), four scoring functions implemented in the CScore module in SYBYL (F-Score, G-Score, D-Score, and ChemScore), the scoring function implemented in the AutoDock program, and two stand-alone scoring functions (DrugScore and X-Score). These scoring functions are not tested in the context of a particular docking program. Instead, conformational sampling and scoring are separated into two consecutive steps. First, an exhaustive conformational sampling is performed by using the AutoDock program to generate an ensemble of docked conformations for each ligand molecule. This conformational ensemble is required to cover the entire conformational space as much as possible rather than to focus on a few energy minima. Then, each scoring function is applied to score this conformational ensemble to see if it can identify the experimentally observed conformation from all of the other decoys. Among all of the scoring functions under test, six of them, i.e., PLP, F-Score, LigScore, DrugScore, LUDI, and X-Score, yield success rates higher than the AutoDock scoring function. The success rates of these six scoring functions range from 66% to 76% if using root-mean-square deviation < or =2.0 A as the criterion. Combining any two or three of these six scoring functions into a consensus scoring scheme further improves the success rate to nearly 80% or even higher. However, when applied to reproduce the experimentally determined binding affinities of the 100 protein-ligand complexes, only X-Score, PLP, DrugScore, and G-Score are able to give correlation coefficients over 0.50. All of the 11 scoring functions are further inspected by their abilities to construct a descriptive, funnel-shaped energy surface for protein-ligand complexation. The results indicate that X-Score and DrugScore perform better than the other ones at this aspect.

Thyroid hormones and their effects: a new perspective.

The thyroid hormones are very hydrophobic and those that exhibit biological activity are 3',5',3,5-L-tetraiodothyronine (T4), 3',5,3-L-triiodothyronine (T3), 3',5',3-L-triiodothyronine (rT3) and 3,5',-L-diiothyronine (3,5-T2). At physiological pH, dissociation of the phenolic -OH group of these iodothyronines is an important determinant of their physical chemistry that impacts on their biological effects. When non-ionized these iodothyronines are strongly amphipathic. It is proposed that iodothyronines are normal constituents of biological membranes in vertebrates. In plasma of adult vertebrates, unbound T4 and T3 are regulated in the picomolar range whilst protein-bound T4 and T3 are maintained in the nanomolar range. The function of thyroid-hormone-binding plasma proteins is to ensure an even distrubtion throughout the body. Various iodothyronines are produced by three types of membrane-bound cellular deiodinase enzyme systems in vertebrates. The distribution of deiodinases varies between tissues and each has a distinct developmental profile. Thyroid hormones. (1) the nuclear receptor mode is especially important in the thyroid hormone axis that controls plasma and cellular levels of these hormones. (2) These hormones are strongly associated with membranes in tissues and normally rigidify these membranes. (3) They also affect the acyl composition of membrane bilayers and it is suggested that this is due to the cells responding to thyroid-hormone-induced membrane rigidificataion. Both their immediate effects on the physical state of membranes and the consequent changes in membrane composition result in several other thyroid hormone effects. Effects on metabolism may be due primarily to membrane acyl changes. There are other actions of thyroid hormones involving membrane receptors and influences on cellular interactions with the extracellulara matrix. The effects of thyroid hormones are reviewed and appear to b combinations of these various modes of action. During development, vertebrates show a surge in T4 and other thyroid hormones, as well as distinctive profiles in the appearance of the deiodinase enzymes and nuclear receptors. Evidence from the use of analogues supports multiple modes of action. Re-examination of data from th early 1960s supports a membrane action. Findings from receptor 'knockout' mice supports an important role for receptors in the development of the thyroid axis. These iodothyronines may be better thought of as 'vitamone'-like molecules than traditional hormonal messengers.

Conformational dynamics of thyroid hormones by variable temperature nuclear magnetic resonance: the role of side chain rotations and cisoid/transoid interconversions.

1H NMR spectra of the thyroid hormone thyroxine recorded at low temperature and high field show splitting into two peaks of the resonance due to the H2,6 protons of the inner (tyrosyl) ring. A single resonance is observed in 600 MHz spectra at temperatures above 185 K. An analysis of the line shape as a function of temperature shows that the coalescence phenomenon is due to an exchange process with a barrier of 37 kJ mol-1. This is identical to the barrier for coalescence of the H2',6' protons of the outer (phenolic) ring reported previously for the thyroid hormones and their analogues. It is proposed that the separate peaks at low temperature are due to resonances for H2,6 in cisoid and transoid conformers which are populated in approximately equal populations. These two peaks are averaged resonances for the individual H2 and H6 protons. Conversion of cisoid to transoid forms can occur via rotation of either the alanyl side chain or the outer ring, from one face of the inner ring to the other. It is proposed that the latter process is the one responsible for the observed coalescence phenomenon. The barrier to rotation of the alanyl side chain is > or = 37 kJ mol-1, which is significantly larger than has previously been reported for Csp2-Csp3 bonds in other Ph-CH2-X systems. The recent crystal structure of a hormone agonist bound to the ligand-binding domain of the rat thyroid hormone receptor (Wagner et al. Nature 1995, 378, 690-697) shows the transoid form to be the bound conformation. The significant energy barrier to cisoid/transoid interconversion determined in the current study combined with the tight fit of the hormone to its receptor suggests that interconversion between the forms cannot occur at the receptor site but that selection for the preferred bound form occurs from the 50% population of the transoid form in solution.

Development of a thyroid hormone analogue for the treatment of congestive heart failure.

The possibility that thyroid hormone or a thyroid hormone analogue that improves cardiac performance might be useful in the treatment of heart failure has-been examined. In the rat postinfarction model of heart failure, treatment with low doses (1.5 micrograms/100 g) of thyroxine (T4) for 3 days produced a positive inotropic response, including an increase in left ventricular (LV) dP/dt and a decrease in LV end-diastolic pressure (LVEDP). When treatment with T4 was continued at the same or higher doses (3 to 15 micrograms/100 g) for 10-12 days, heart rate was increased and improvement in LVEDP was not sustained. To identify an analogue with a more favorable hemodynamic profile, single- and double-ring compounds related to T4 were screened for thyromimetic activity in heart cell cultures and for their ability to bind thyroid hormone receptors. One of the analogues selected, 3,5-diiodothyropropionic acid (DITPA), was found to have inotropic selectivity in hypothyroid rats. When administered (375 micrograms/100 g) to rats with ventricular dysfunction after myocardial infarction in combination with captopril, there was improvement of the resting and stressed cardiac index and LV filling pressure. Similar improvement in cardiac performance was obtained when DITPA was administered to rabbits after infarction. Thus a thyroid hormone analogue with inotropic selectivity may be a useful adjunct to other measures in the treatment of heart failure.

1H and 13C NMR relaxation studies of molecular dynamics of the thyroid hormones thyroxine, 3,5,3'-triiodothyronine, and 3,5-diiodothyronine.

1H and 13C NMR spin-lattice relaxation times and 13C{1H} nuclear Overhauser enhancement factors have been measured for the thyroid hormones thyroxine, 3,5,3'-triiodothyronine, and 3,5-diiodothyronine, with the aim of determining the internal molecular dynamics in these molecules. Spin-lattice relaxation times of protons on the two aromatic rings of these hormones show remarkable differences, with values for the hydroxyl-bearing ring being a factor of 4-12 times larger than those for the alanyl-bearing ring. This difference is not mirrored in the 13C relaxation times, which are identical within experimental error for the two rings. The 13C data show that the mobility of the two rings is similar, and therefore the difference in proton spin-lattice relaxation times arises because the protons of the alanyl-bearing ring are efficiently relaxed by interactions with neighboring protons on the side chain. Quantitative analysis of the 13C relaxation data shows that there must be a significant degree of internal flexibility in the thyroid hormone molecules. The NMR data suggest that in methanol the molecules tumble with an overall correlation time of approximately 0.35 ns, but that rapid internal motion (in the form of jumps between two stable conformations) occurs on a 30-fold faster time scale. When combined with previous variable temperature NMR studies that show interconversion between proximal and distal forms of the outer ring on the microsecond time scale, the results provide a complete description of the conformations and both fast and slow internal motions in the thyroid hormones. The findings suggest that modeling studies of thyroid hormone interactions with receptor proteins should take into account the possibility that these internal motions are present. In effect, the thyroid hormones may likely populate a larger range of conformations in the bound state than might be inferred from just the lowest energy forms seen in the crystal and solution states.

Mechanism of molecular recognition. Structural aspects of 3,3'-diiodo-L-thyronine binding to human serum transthyretin.

The three-dimensional structure of the thyroid hormone metabolite, 3,3'-diiodo-L-thyronine (3,3'-T2), complex with human serum transthyretin (TTR) has been refined to R = 18.5% for 8-2 A resolution data. This is the first detailed description of a thyroid hormone metabolite binding to a thyroid transport protein. The four TTR monomeric subunits form a tetramer in the same manner as the native transthyretin reported earlier (Blake, C. C. F., Geisow, M. J., Oatley, S. J., Rerat, B., and Rerat, C. (1978) J. Mol. Biol. 121, 339-356). The two hormone binding sites of the TTR tetramer are occupied by 3,3'-T2. A statistical disorder model for the ligand was applied with a 50% occupancy to account for the discrepancy between the crystallographic 2-fold symmetry of the binding sites and the lack of such symmetry for 3,3'-T2. The bound metabolite has an overall transoid conformation with the either bridge intermediate between skewed and perpendicular. The hormone metabolite is bound 3.5 A deeper and with a different orientation in the channel than observed for thyroxine (T4), thereby revealing the presence of another set of halogen binding sites close to the center of the tetramer. When compared with the binding of T4, these data show that the 3-iodine of 3,3'-T2 occupies the same site as the 3'-iodine of T4, and the metabolite 3'-iodine occupies the water site observed in the T4 complex. The binding affinity of 3,3'-T2, which is 100-fold lower than that of T4, reflects the lack of the second pair of iodine atoms interacting in the channel. In order to understand the tighter binding of T4 observed in the Ala-109----Thr mutant, modeling studies were carried out that indicate that this modification could shorten the contacts between thyroid hormone iodines and residues 108-110 of the binding site.