Genetically encoding thyronine for fluorescent detection of peroxynitrite.

Peroxynitrite is a highly reactive oxidant effecting cell signaling and cell death. Here we report a fluorescent protein probe to selectively detect peroxynitrite. A novel unnatural amino acid, thyronine (Thy), was genetically encoded in E. coli and mammalian cells by evolving an orthogonal tRNAPyl/ThyRS pair. Incorporation of Thy into the chromophore of sfGFP or cpsGFP afforded a virtually non-fluorescent reporter. Upon treatment with peroxynitrite, Thy was converted into tyrosine via O-dearylation, regenerating GFP fluorescence in a time- and concentration-dependent manner. Genetically encoded thyronine may also be valuable for other redox applications.

Endogenous 3-Iodothyronamine (T1AM) and Synthetic Thyronamine-like Analog SG-2 Act as Novel Pleiotropic Neuroprotective Agents Through the Modulation of SIRT6.

3-iodothyronamine (T1AM) and the recently developed analog SG-2 are rapidly emerging as promising multi-target neuroprotective ligands able to reprogram lipid metabolism and to produce memory enhancement in mice. To elucidate the molecular mechanisms underlying the multi-target effects of these novel drug candidates, here we investigated whether the modulation of SIRT6, known to play a key role in reprogramming energy metabolism, might also drive the activation of clearing pathways, such as autophagy and ubiquitine-proteasome (UP), as further mechanisms against neurodegeneration. We show that both T1AM and SG-2 increase autophagy in U87MG cells by inducing the expression of SIRT6, which suppresses Akt activity thus leading to mTOR inhibition. This effect was concomitant with down-regulation of autophagy-related genes, including Hif1α, p53 and mTOR. Remarkably, when mTOR was inhibited a concomitant activation of autophagy and UP took place in U87MG cells. Since both compounds activate autophagy, which is known to sustain long term potentiation (LTP) in the entorhinal cortex (EC) and counteracting AD pathology, further electrophysiological studies were carried out in a transgenic mouse model of AD. We found that SG-2 was able to rescue LTP with an efficacy comparable to T1AM, further underlying its potential as a novel pleiotropic agent for neurodegenerative disorders treatment.

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.

Electrochemical sensing of the thyroid hormone thyronamine (T0AM) via molecular imprinted polymers (MIPs).

Recent studies have shown that besides the well-known T3 (triiodothyronine) and T4 (thyroxine) there might be other important thyroid hormones, in particular T0AM (thyronamine) and T1AM (3-iodothyronamine). The absence of a large number of studies showing their precise importance might be explained by the limited number of analytical methodologies available. This work aims to show an electroanalytical alternative making use of electropolymerized molecularly imprinted polymer (MIPs). The MIPs' polymerization is performed on the surface of screen-printed carbon electrodes (SPCEs), using 4-aminobenzoic acid (4-ABA) as the building and functional monomer and the analyte T0AM as the template. The step-by-step construction of the SPCE-MIP sensor was studied by cyclic voltammetry (CV) and by electrochemical impedance spectroscopy (EIS). After optimization, by means of square-wave voltammetry, the SPCE-MIP showed suitable selectivity (in comparison with other thyroid hormones and catechol amines), repeatability (intra-day of 3.9%), a linear range up to 10 µmol L-1 (0.23 × 103 µg dL-1) with an r2 of 0.998 and a limit of detection (LOD) and quantification (LOQ) of 0.081 and 0.27 µmol L-1 (1.9 and 6.2 µg dL-1), respectively.

3-Iodothyronamine reduces insulin secretion in vitro via a mitochondrial mechanism.

PURPOSE: 3-iodothyronamine (3-T1AM), a decarboxylated and deiodinated thyroid hormone metabolite, leads at pharmacological doses to hypoinsulinemia, hyperglucagonemia and hyperglycemia in vivo. As the pancreatic Langerhans islets express thyroid hormone transmembrane transporters (THTT), we tested the hypothesis that not only plasma membrane-mediated 3-T1AM binding to and activation of G-protein coupled receptors, but also 3-T1AM metabolite(s) generated by 3-T1AM uptake and metabolism might decrease glucose-stimulated insulin secretion (GSIS). METHODS: Murine pancreatic ß-cells MIN6 were characterized for gene expression of THTT, deiodinases and monoamine oxidases. 3-T1AM uptake and intracellular metabolism to the corresponding 3-iodothyroacetic acid were analysed by liquid-chromatography tandem mass spectrometry (LC-MS/MS) at different time points in cells as well as the conditioned medium. Mitochondrial activity, especially ATP-production, was monitored real-time after 3-T1AM application using Seahorse Bioanalyzer technique. Effect of 3-T1AM on GSIS into the culture medium was assayed by ELISA. RESULTS: MIN6 cells express classical THTT, proposed to transport 3-T1AM, as well as 3-T1AM metabolizing enzymes comparable to murine primary pancreatic islets. 3-T1AM accumulates in MIN6 cells and is metabolized by intracellular MaoB to 3-iodothyroacetic, which in turn is rapidly exported. 3-T1AM decreases mitochondrial ATP-production concentration dependently. GSIS is diminished by 3-T1AM treatment. Using LC-MS/MS, no further 3-T1AM metabolites except 3-iodothyroacetic were detectable. CONCLUSIONS: This data provides a first link between cellular 3-T1AM uptake and regulation of mitochondrial energy metabolism in ß-cells, resulting in reduced insulin secretion. We conclude that MIN6 is an appropriate cell model to study 3-T1AM-dependent (intra-)cellular biochemical mechanisms affecting insulin production in vitro.

Targeting species-specific trace amine-associated receptor 1 ligands: to date perspective of the rational drug design process.

G-protein-coupled receptors represent main targets of several clinically relevant drugs, playing nowadays a leading part for further drug discovery process. Trace amine-associated receptor's family (TAARs) assumed an intriguing role as druggable target in medicinal chemistry, being TAAR1 the most investigated. Indeed, related ligands proved to be intertwined in several circuits involved in pathological pathways or therapeutic routes. Herein, we highlight relevant efforts in the search of novel agonists, focusing on responsiveness featured by different chemotypes toward rodent and human TAAR1, in order to explore species-specificity preferences. We also discuss the main strategies guiding so far the design of new TAAR1 agonists, giving a perspective of the structure-based methodologies aimed at deriving new insights for more potent and selective derivatives.

Thyronamines and Analogues - The Route from Rediscovery to Translational Research on Thyronergic Amines.

Thyronamines are a novel class of endogenous signaling compounds, structurally related to thyroid hormones (THs). Specific thyronamines, particularly 3-iodothyronamine (T1AM), stimulate with nanomolar affinity trace amine-associated receptor 1 (TAAR1), a G protein-coupled membrane receptor, and may also interact with other TAAR subtypes (particularly TAAR5), adrenergic receptors (particularly α2 receptors), amine transporters, and mitochondrial proteins. In addition to its structural similarities with THs, T1AM also contains the arylethylamine scaffold as in monoamine neurotransmitters, implicating an intriguing role for T1AM as both a neuromodulator and a hormone-like molecule constituting a part of thyroid hormone signaling. A large number of T1AM derivatives have already been synthesized. We discuss the different chemical strategies followed to obtain thyronamine analogues, their potency at TAAR1, and their structure-activity relationship. Preliminary characterization of the functional effects of these synthetic compounds is also provided.

New insights into the structure of the trace amine-associated receptor 2: Homology modelling studies exploring the binding mode of 3-iodothyronamine.

Recent studies have further investigated the trace amine-associated receptor type 2 (TAAR2) pharmacology, revealing its role not only at the olfactory sensory neurons but also at the immune system, being expressed in human leucocytes. In particular, the ability of this receptor to bind the unselective TAAR ligand 3-iodo-thyronamine (T1 AM) was elucidated, making in the meanwhile the discovery of selective compounds a urgent need to derive much more suitable tools for studying TAARs. In this context, we developed our work on TAAR2 applying a structure-based computational protocol, including TAAR2 homology modelling and T1 AM docking studies. The results were compared with those we previously obtained about TAAR1, in order to point out new insights guiding for selectivity between TAAR1 and TAAR2. The in silico strategy applied allowed us to provide for the first time thorough TAAR2 homology models, which are expected to be useful tools for a further design process of more selective TAAR ligands.

Biomimetic deiodination of thyroid hormones and iodothyronamines - a structure-activity relationship study.

Mammalian selenoenzymes, iodothyronine deiodinases (DIOs), catalyze the tyrosyl and phenolic ring deiodination of thyroid hormones (THs) and play an important role in maintaining the TH concentration throughout the body. These enzymes also accept the decarboxylated thyroid hormone metabolites, iodothyronamines (TAMs), as substrates for deiodination. Naphthalene-based selenium and/or sulphur-containing small molecules have been shown to mediate the regioselective tyrosyl ring deiodination of thyroid hormones and their metabolites. Herein, we report on the structure-activity relationship studies of a series of peri-substituted selenium-containing naphthalene derivatives for the deiodination of thyroid hormones and iodothyronamines. Single crystal X-ray crystallographic and 77Se NMR spectroscopic studies indicated that the intramolecular SeX (X = N, O and S) interactions play an important role in the deiodinase activity of the synthetic mimics. Furthermore, the decarboxylated metabolites, TAMs, have been observed to undergo slower tyrosyl ring deiodination than THs by naphthyl-based selenium and/or sulphur-containing synthetic deiodinase mimics and this has been explained on the basis of the strength of SeI halogen bonding formed by THs and TAMs.

Design, Synthesis, and Evaluation of Thyronamine Analogues as Novel Potent Mouse Trace Amine Associated Receptor 1 (mTAAR1) Agonists.

Trace amine associated receptor 1 (TAAR1) is a G protein coupled receptor (GPCR) expressed in brain and periphery activated by a wide spectrum of agonists that include, but are not limited to, trace amines (TAs), amphetamine-like psychostimulants, and endogenous thyronamines such as thyronamine (T0AM) and 3-iodothyronamine (T1AM). Such polypharmacology has made it challenging to understand the role and the biology of TAAR1. In an effort to understand the molecular basis of TAAR1 activation, we rationally designed and synthesized a small family of thyronamine derivatives. Among them, compounds 2 and 3 appeared to be a good mimic of the parent endogenous thyronamine, T0AM and T1AM, respectively, both in vitro and in vivo. Thus, these compounds offer suitable tools for studying the physiological roles of mouse TAAR1 and could represent the starting point for the development of more potent and selective TAAR1 ligands.

Regioselective deiodination of iodothyronamines, endogenous thyroid hormone derivatives, by deiodinase mimics.

Iodothyronine deiodinases (IDs) are mammalian selenoenzymes that play an important role in the activation and inactivation£ of thyroid hormones. It is known that iodothyronamines (TnAMs), produced by the decarboxylation of thyroid hormones, act as substrates for deiodinases. To understand whether decarboxylation alters the rate and/or regioselectivity of deiodination by using synthetic deiodinase mimics, we studied the deiodination of different iodothyronamines. The triiodo derivative 3,3',5-triiodothyronamine (T3 AM) is deiodinated at the inner ring by naphthyl-based deiodinase mimics, which is similar to the deiodination of 3,3',5-triiodothyronine (T3). However, T3 AM undergoes much slower deiodination than T3. Detailed experimental and theoretical investigations suggest that T3 AM forms a weaker halogen bond with selenium donors than T3. Kinetic studies and single-crystal X-ray structures of T3 and T3 AM reveal that intermolecular I⋅⋅⋅I interactions may play an important role in deiodination. The formation of hydrogen- and halogen-bonding assemblies, which leads to the formation of a dimeric species of T3 in solution, facilitates the interactions between the selenium and iodine atoms. In contrast, T3 AM, which does not have I⋅⋅⋅I interactions, undergoes much slower deiodination.

The influence of halogen derivatives of thyronine and fluorescein on the dipole potential of phospholipid membranes.

The effects of halogen derivatives of thyronine (tetraiodotironine and triiodothyronine) and fluorescein (Rose Bengal, phloxine B, erythrosin, eosin Y, and fluorescein) on the dipole potential of membranes composed of diphytanoylphosphocholine, diphytanoylphosphoserine, and diphytanoylphosphoethanolamine were investigated. A quantitative description of the modifying action of the agents was presented as characteristic parameters of the Langmuir adsorption isotherm: the maximum changes in the dipole potential of the membrane at an infinitely high concentration of modifiers and the desorption constant, characterizing their inverse affinities to the lipid phase. It was shown that the iodine-containing hormones led to a less significant reduction in the dipole potential of phospholipid membranes compared to the xanthene dyes, Rose Bengal, phloxine B, and erythrosin. The latter were characterized by the highest affinity for the lipid membranes compared to tetraiodotironine and triiodothyronine. It was found that the effect of iodine-containing hormones and xanthene dyes on the membrane dipole potential was caused by their uncharged and charged forms, respectively.

Histamine mediates behavioural and metabolic effects of 3-iodothyroacetic acid, an endogenous end product of thyroid hormone metabolism.

BACKGROUND AND PURPOSE: 3-Iodothyroacetic acid (TA1) is an end product of thyroid hormone metabolism. So far, it is not known if TA1 is present in mouse brain and if it has any pharmacological effects. EXPERIMENTAL APPROACH: TA1 levels in mouse brain were measured by HPLC coupled to mass spectrometry. After i.c.v. administration of exogenous TA1 (0.4, 1.32 and 4 µg·kg(-1) ) to mice, memory acquisition-retention (passive avoidance paradigm with a light-dark box), pain threshold to thermal stimulus (51.5°C; hot plate test) and plasma glucose (glucorefractometer) were evaluated. Similar assays were performed in mice pretreated with s.c. injections of the histamine H1 receptor antagonist pyrilamine (10 mg·kg(-1) ) or the H2 receptor antagonist zolantidine (5 mg·kg(-1) ). TA1 (1.32 and 4 µg·kg(-1) ) was also given i.c.v. to mice lacking histidine decarboxylase (HDC(-/-) ) and the corresponding WT strain. KEY RESULTS: TA1 was found in the brain of CD1 but not of HDC mice. Exogenous TA1 induced amnesia (at 0.4 µg·kg(-1) ), stimulation of learning (1.32 and 4 µg·kg(-1) ), hyperalgesia (0.4, 1.32 and 4 µg·kg(-1) ) and hyperglycaemia (1.32 and 4 µg·kg(-1) ). All these effects were modulated by pyrilamine and zolantidine. In HDC(-/-) mice, TA1 (1.32 and 4 µg·kg(-1) ) did not increase plasma glucose or induce hyperalgesia. CONCLUSIONS AND IMPLICATIONS: Behavioural and metabolic effects of TA1 disclosed interactions between the thyroid and histaminergic systems.

Characterization of 3-iodothyronamine in vitro dynamics by mathematical modeling.

3-Iodothyronamine (T1AM) is regarded as a hormone-like substance thanks to its endogenous nature, its interaction with specific receptors trace amine-associated receptor 1 and its biological effects. We characterized T1AM transport and conversion in an in vitro culture of H9c2 murine cells, after a T1AM bolus injection. Samples of cell medium culture and cell lysate were assayed by high-performance liquid chromatography coupled to tandem mass spectrometry. We performed comparative experiments by adding to T1AM bolus amino oxidase inhibitors as iproniazid, pargyline (monoamine oxidase, MAO inhibitors), aminoguanidine, and semicarbazide (semicarbazide-sensitive amino oxidase, SSAO inhibitors). A mathematical model was developed, based on the assumption that T1AM is transported with a mechanism that is typical of hormone transport (i.e., EGF or insulin). We noticed that surface receptors downregulation could play a major role in T1AM dynamics. We also estimated that T1AM catabolism is mainly affected by MAO inhibitors, which produce a dramatic decrease in the kinetic constants related to T1AM degradation, while no significant changes were observed in experiments with SSAO inhibitors.

Influence of iodothyronine conjugates of bovine serum albumin and horseradish peroxidase on enzyme immunosorbent assay of thyroid hormones.

Enzyme-linked immunosorbent assays (ELISA's) reported for thyroxine (T4) and 3,5,3'-triiodothyronine (T3), involved coupling of the haptens through (i) carboxylic group to carrier protein for producing antibodies and (ii) amino group to detection labels. To improve the titer and specificity of antibodies, immunogens were prepared by coupling of carboxyl group to bovine serum albumin (BSA) either directly or through adipic acid dihydrazide (ADH), after protecting amino group through acetylation of T4 and T3. Direct coupling resulted in the incorporation of 40-50 moles of T4 and T3 per BSA molecule and helped in improving immunogenic response and specificity, especially of T4. High epitope density of immunogens evoked better antibody response, since attachement of ADH as spacer, introduced 18-27 moles of haptens into carrier protein and had less effect on antibody development, with T3 being exception. Detection labels were prepared by coupling horseradish peroxidase (HRP) to amino group of thyroid hormones directly and after preparing their methyl esters, which provided sensitive displacement curves in combination with the antibodies developed against N-acetylated-T4 and T3. Unlike methyl esters, T4-HRP and T3-HRP showed higher sensitivity and seemed to be related to the affinity of the labels for binding the antibody.

In vivo molecular imaging of [125I]-labeled 3-iodothyronamine: a hibernation-inducing agent.

The present investigation was carried out with the objective of studying in vivo imaging of 3-iodothyronamine (T(1)AM) compound in mice. A simple and efficient synthesis of [(125)I]-T(1)AM was established, and a molecular imaging study was performed using micro-SPECT/CT at 1h post-injection of [(125)I]-T(1)AM. Imaging studies revealed the activity in the gastrointestinal tract and liver, indicating that [(125)I]-T(1)AM was distributed primarily in the liver, and excreted into the gastrointestinal tract through a bile duct.

Insights into the structure and pharmacology of the human trace amine-associated receptor 1 (hTAAR1): homology modelling and docking studies.

Trace amine-associated receptor 1 (TAAR1) is a G protein-coupled receptor that belongs to the family of TAAR receptors and responds to a class of compounds called trace amines, such as ß-phenylethylamine (ß-PEA) and 3-iodothyronamine (T(1)AM). The receptor is known to have a very rich pharmacology and could be also activated by other classes of compounds, including adrenergic and serotonergic ligands. It is expected that targeting TAAR1 could provide a novel pharmacological approach to correct monoaminergic dysfunctions found in several brain disorders, such as schizophrenia, depression, attention deficit hyperactivity disorder and Parkinson's disease. Only recently, the first selective TAAR1 agonist RO5166017 has been identified. To explore the molecular mechanisms of protein-agonist interaction and speed up the identification of new chemical entities acting on this biomolecular target, we derived a homology model for the hTAAR1. The putative protein-binding site has been explored by comparing the hTAAR1 model with the ß(2)-adrenoreceptor binding site, available by X-ray crystallization studies, and with the homology modelled 5HT(1A) receptor. The obtained results, in tandem with docking studies performed with RO5166017, ß-PEA and T(1)AM, provided an opportunity to reasonably identify the hTAAR1 key residues involved in ligand recognition and thus define important starting points to design new agonists.

ApoB-100-containing lipoproteins are major carriers of 3-iodothyronamine in circulation.

3-Iodothyronamine (T(1)AM) is a biogenic amine derivative of thyroid hormone present in tissue and blood of vertebrates. Approximately 99% of the circulating thyroid hormones are bound to plasma proteins, including three major thyroid hormone-binding proteins, and the question arises as to whether circulating T(1)AM is also bound to serum factors. We report here that T(1)AM is largely bound to a single protein component of human serum. Using T(1)AM-affinity chromatography, we isolated this protein, and sequence analysis identified it as apolipoprotein B-100 (apoB-100), the protein component of several low density lipoprotein particles. Consistent with this finding, we demonstrate that >90% of specifically bound T(1)AM in human serum resides in the apoB-100-containing low density lipoprotein fraction. T(1)AM reversibly binds to apoB-100-containing lipoprotein particles with an equilibrium dissociation constant (K(D)) of 17 nm and a T(1)AM/apoB-100 stoichiometry of 1:1. Competition binding assays demonstrate that this binding site is highly selective for T(1)AM. Intracellular T(1)AM uptake is significantly enhanced by apoB-100-containing lipoprotein particles. Modest enhancements to apoB-100 cellular uptake and secretion by T(1)AM were observed; however, multidose T(1)AM treatment did not affect lipid or lipoprotein inventory in vivo. Thus, it appears that apoB-100 serves as a carrier of circulating T(1)AM and affords a novel mechanism by which T(1)AM gains entry to cells.

TRC150094, a novel functional analog of iodothyronines, reduces adiposity by increasing energy expenditure and fatty acid oxidation in rats receiving a high-fat diet.

Chronic overnutrition and modern lifestyles are causing a worldwide epidemic of obesity and associated comorbidities, which is creating a demand to identify underlying biological mechanisms and to devise effective treatments. In rats receiving a high-fat diet (HFD), we analyzed the effects of a 4-wk administration of a novel functional analog of iodothyronines, TRC150094 (TRC). HFD-TRC rats exhibited increased energy expenditure (+24% vs. HFD rats; P<0.05) and body weight (BW) gain comparable to that of standard chow-fed (N) rats [N, HFD, and HFD-TRC rats, +97 g, +140 g (P<0.05 vs. N), and +98 g (P<0.05 vs. HFD)]. HFD-TRC rats had significantly less visceral adipose tissue (vs. HFD rats) and exhibited altered metabolism in two major tissues that are very active metabolically. In liver, mitochondrial fatty acid import and oxidation were increased (+56 and +32%, respectively; P<0.05 vs. HFD rats), and consequently the hepatic triglyceride content was lower (-35%; P<0.05 vs. HFD rats). These effects were independent of the AMP-activated protein kinase-acetyl CoA-carboxylase-malonyl CoA pathway but involved sirtuin 1 activation. In skeletal muscle, TRC induced a fiber shift toward the oxidative type in tibialis anterior muscle, increasing its capacity to oxidize fatty acids. HFD-TRC rats had lower (vs. HFD rats) plasma cholesterol and triglyceride concentrations. If reproduced in humans, these results will open interesting possibilities regarding the counteraction of metabolic dysfunction associated with ectopic/visceral fat accumulation.