Thyroid hormone is known to increase metabolism, core body temperature, and cardiac performance by regulating gene expression through the thyroid hormone nuclear receptors. These transcriptional effects have a slow onset and occur within hours to days. 3-Iodothyronamine (T1AM) is an endogenous, decarboxylated, and deiodinated metabolite of the thyroid hormone thyroxine that is found in the brain, heart, liver, and blood. When administered to mice intraperitoneally, T1AM rapidly induces hypothermia, anergia, and bradycardia; effects of which are opposite those observed with hyperthyroidism. In vitro, T1AM potently activates the orphan G-protein coupled receptor (GPCR) known as the trace amine-associated receptor 1 (TAAR1), inhibits neurotransmitter reuptake by the dopamine (DAT) and norepinephrine transporter (NET), and inhibits vesicular packaging by the vesicular monoamine transporter 2 (VMAT2). To understand the role of TAAR1 in mediating the effects of T1AM, we sought to develop small molecules that regulate the activity of TAAR1.

Structure-activity relationship studies on the ethylamine portion of T1AM showed that TAAR1 can tolerate prominent structural features found in existing GPCR agonists and antagonists. A closer investigation of the ligand-receptor interactions of existing catecholamine receptor drugs revealed a relationship between the functional properties of the ligand and how it interacts with the rotamer toggle switch residues of the receptor. Allowing the rotamer switch residues to toggle to their active conformation was associated with agonism while interfering with this conformational transition resulted in antagonism.

The rotamer toggle switch model of aminergic GPCR activation was a useful guideline in the design and synthesis of rat TAAR1 agonists and antagonists. It provided an insightful approach to understanding the molecular basis of rat TAAR1 activation by T1AM and related analogs, and guided the successful development of rat TAAR1 superagonists ET-36, ET-64, and ET-69 and lead antagonists ET-78, ET-92, and ET-93.

This work is premised on the hypothesis that thyroid hormone may be a substrate for the aromatic L-amino acid decarboxylase (AADC) and that the resulting iodothyronamines would have significant structural and perhaps functional similarity with several biogenic amines including the classical monoamine neurotransmitters. Previous reports have shown that several thyronamines are rapid and potent agonists of the trace amine-associated receptor 1 (TAAR1), a G protein-coupled receptor (GPCR). We hypothesized that thyronamines might function physiologically as classical neurotransmitters or neuromodulators. As such, we sought to screen thyronamines for activity with a variety of known monoamine transporters. Additionally we attempted to clone, express and screen the rat TAAR family of GPCRs for activity with thyronamines and a variety of phenethylamine (PEA), thyroid hormone, and amino acid derivatives.

Using our previously reported synthesis of these presumed thyroid hormone metabolites and a radioactive derivative of T1AM, we found that T1AM inhibits plasma membrane transport of norepinephrine and dopamine in synaptosomes, and also inhibits biogenic amine vesicular packaging in synaptic vesicles. We further demonstrate that T1AM is an inhibitor of the plasma membrane neurotransmitter transporters DAT and NET, and an inhibitor of the vesicular transporter VMAT2. This is a novel and exciting result, because T1AM is the first example of an endogenous substance that inhibits these transporters and suggests that T1AM may play a physiological role in regulating synaptic transmission with catecholamine neurons. In addition, we report that several PEA derivatives act as agonists of rTAAR4, both confirming previously identified compounds and discovering several additional agonists. Lastly, we show that a class of clinically used thyroid hormone derivatives act as agonists of TAAR1 with potential therapeutic relevance. Significantly, we expand on our understanding of trace amine-associated receptor ligand recognition and the physiological relevance of thyronamine/GPCR interactions.

Thyroid hormone orchestrates a multifarious set of physiological activities ranging from fetal, brain, and skeletal maturation to improving vascular tone and controlling cardiac output. Thyroid hormone elicits its effects by binding to the ligand binding domain of the thyroid hormone receptor (TR) for which there are two subtypes for two isoforms: TRα₁, TRα₂, TRβ₁, and TRβ₂. Although TR isoforms, with the exception of TRβ₂ which is mainly expressed in the pituitary, are ubiquitously expressed, these isoforms are expressed in different ratios in different tissues giving rise to tissue-specific isoform action. Selective thyroid hormone analogues, or selective thyromimetics, that discriminate between these two subtypes would serve as useful chemical tools to gain or refine our understanding of the physiological effects of thyroid hormone. These compounds may also prove useful in the clinic as known thyromimetics (i.e. 3,5-diiodothyroproprionic acid and GC-1) show promise in treating cardiac related diseases and metabolic disorders. One way to generate selective thyromimetics with unique pharmacological profiles is to synthesize a small panel of compounds by employing a bioisosteric replacement strategy. This method entails derivatizing one region of the thyronine backbone, the C₁ region, by incorporation of various heterocycles or carbocycles. This strategy led to the production of a small panel of ligands which were screened in ¹²⁵I-T₃ competitive binding and transactivation assays. After this screen, the TRα-specific ligand CO22 was identified; however, it demonstrated poor binding and potency. Replacing the inner-ring methyl groups of CO22 with iodides led to CO23, the first potent thyromimetic to show TRα-specificity in vitro and in vivo. CO23 showed specificity in TRα-mediated transcription in U2OS and HeLa cells and caused Xenopus laevis tadpoles to experience more extensive and rapid hind leg growth, less larval tissue resorption, greater induction of TRα early genes, and less induction of TRβ early and late genes. It was surmised that the imidazolidinedione moiety of the inner-ring confers TRα-specificity; therefore, to engender ligands with improved specificity the 3' outer-ring position was derivatized. This led to several analogues with less potency than CO23, but with a gain in specificity of up to 2-fold.

3-iodothyronamine (T₁AM) is an endogenous thyroid hormone metabolite with distinct, acute biological effects that are largely opposite those of thyroid hormone. Administration of T₁AM to rodents results in rapid and profound reduction in body temperature, heart rate, and metabolism. Since its discovery only five years ago, T₁AM is emerging as a potentially key signaling molecule involved in thyroid hormone endocrinology. The structural similarities between T₁AM and monoamine neurotransmitters as well as its parent compound, thyroid hormone, suggest an intriguing role for T₁AM as both a neuromodulator and a hormone-like molecule that complements or regulates thyroid hormone action.

The known molecular targets of T₁AM include both plasma membrane and intracellular proteins, suggesting that intracellular transport of T₁AM may be an important component of its action. Using various cell lines, we show that, indeed, specific T₁AM uptake occurs in multiple cell types. An RNAi screen targeting the SLC superfamily of transporter genes identifies eight transporters that may contribute to T₁AM uptake in HeLa cells. Moreover, we demonstrate that T₁AM is taken up into the nucleus of HepG2 cells, suggesting that T₁AM might play a role in transcriptional regulation.

We also investigate the effect of T₁AM on cellular entry of thyroid hormones. Transport inhibition studies reveal that T₁AM displays differential inhibition of T₃ and T₄ cellular uptake by the specific thyroid hormone transporter MCT8 and by the multispecific organic anion transporting polypeptides (OATPs) 1A2 and 1C1, but does not affect thyroid hormone transport by OATP1B3. Given that OATP1A2, OATP1C1, and MCT8 are present in the brain, T₁AM may modulate thyroid hormone delivery and activity in specific target regions in the central nervous system.

Finally, we identify α₂-Macroglobulin (α₂M) as a serum binding protein for T₁AM. α₂M is known for various functions in the body, including its unique role as a pan-protease inhibitor, as well as its potential significance in immune defense and modulation of neurotransmitter metabolism. In addition to the possible role of α₂M as a carrier protein for T₁AM, the discovery of α₂M interaction with T₁AM opens another interesting area of investigation into this thyroid hormone derivative and its mechanisms of action.