Annunziatina Laurino

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.

In the brain of mice, 3-iodothyronamine (T1AM) is converted into 3-iodothyroacetic acid (TA1) and it is included within the signaling network connecting thyroid hormone metabolites with histamine.

3-iodothyronamine (T1AM) and its oxidative product, 3-iodotyhyroacetic acid (TTA1A), are known to stimulate learning and induce hyperalgesia in mice. We investigated whether i)TA1 may be generated in vivo from T1AM, ii) T1AM shares with TA1 the ability to activate the histaminergic system. Tandem mass spectrometry was used to measure TA1 and T1AM levels in i) the brain of mice following intracerebroventricular (i.c.v.) injection of T1AM (11μgkg(-1)), with or without pretreatment with clorgyline, (2.5mgkg(-1) i.p.), a monoamine oxidase inhibitor; ii) the medium of organotypic hippocampal slices exposed to T1AM (50nM). In addition, learning and pain threshold were evaluated by the light-dark box task and the hot plate test, respectively, in mice pre-treated subcutaneously with pyrilamine (10mgkg(-1)) or zolantidine (5mgkg(-1)), 20min before i.c.v. injection of T1AM (1.32 and 11μgkg(-1)). T1AM-induced hyperalgesia (1.32 and 11μgkg(-1)) was also evaluated in histidine decarboxylase (HDC(-/-)) mice. T1AM and TA1 brain levels increased in parallel in mice injected with T1AM with the TA1/T1AM averaging 1.7%. Clorgyline pre-treatment reduced the increase in both T1AM and TA1. TA1 was the main T1AM metabolite detected in the hippocampal preparations. Pretreatment with pyrilamine or zolantidine prevented the pro-learning effect of 1.32 and 4μgkg(-1) T1AM while hyperalgesia was conserved at the dose of 11μgkg(-1) T1AM. T1AM failed to induce hyperalgesia in HDC(-/-) mice at all the doses. In conclusion, TA1 generated from T1AM, but also T1AM, appears to act by modulating the histaminergic system.

3‐iodothyroacetic acid, a metabolite of thyroid hormone, induces itch and reduces threshold to noxious and to painful heat stimuli in mice

Itch is associated with increased sensitization to nociceptive stimuli. We investigated whether 3‐iodothyroacetic acid (TA1), by releasing histamine, induces itch and increases sensitization to noxious and painful heat stimuli.

3-iodothyronamine (T1AM), a novel antagonist of muscarinic receptors.

3-iodothyronamine (T1AM) is a trace amine suspected to derive from thyroid hormone metabolism. T1AM was described as a ligand of G-protein coupled monoaminergic receptors, including trace amine associated receptors, suggesting the amine may exert a modulatory role on the monoaminergic transmission. Nothing is known on the possibility that T1AM could also modulate the cholinergic transmission interacting with muscarinic receptors. We evaluated whether T1AM (10nM-100μM) was able to i) displace [3H]-NMS (0.20nM) binding to membrane preparations from CHO cells stably transfected with human muscarinic receptor subtypes (M1-M5); ii) modify basal or acetylcholine induced pERK1/2 levels in CHO expressing the human muscarinic type 3 receptor subtype by Western blot iii) modify basal and carbachol-induced contraction of isolated rat urinary bladder. T1AM fitting within rat muscarinic type 3 receptor was simulated by Docking studies. T1AM recognized all muscarinic receptor subtypes (pKi values in the micromolar range). Interacting at type 3, T1AM reduced acetylcholine-increased pERK1/2 levels. T1AM reduced carbachol-induced contraction of the rat urinary bladder. The fenoxyl residue and the iodide ion were found essential for establishing contacts with the active site of the rat muscarinic type 3 receptor subtype. Our results indicate that T1AM binds at muscarinic receptors behaving as a weak, not selective, antagonist. This finding adds knowledge on the pharmacodynamics features of T1AM and it may prompt investigation on novel pharmacological effects of T1AM at conditions of hyper-activation of the muscarinic tone including the overactive urinary bladder.

Pharmacological perspectives in sarcopenia: A potential role for renin-angiotensin system blockers?

Sarcopenia represents a major health problem highly prevalent in elderly and age-related chronic diseases. Current pharmacological strategies available to prevent and reverse sarcopenia are largely unsatisfactory thus raising the need to identify novel targets for pharmacological intervention and possibly more effective and safe drugs. This review highlights the current knowledge of the potential benefits of renin-angiotensin system blockade in sarcopenia and discuss the main mechanisms underlying the effects.

Pathogenesis of Hypertrophic Cardiomyopathy is Mutation Rather Than Disease Specific: A Comparison of the Cardiac Troponin T E163R and R92Q Mouse Models

Background In cardiomyocytes from patients with hypertrophic cardiomyopathy, mechanical dysfunction and arrhythmogenicity are caused by mutation‐driven changes in myofilament function combined with excitation‐contraction (E‐C) coupling abnormalities related to adverse remodeling. Whether myofilament or E‐C coupling alterations are more relevant in disease development is unknown. Here, we aim to investigate whether the relative roles of myofilament dysfunction and E‐C coupling remodeling in determining the hypertrophic cardiomyopathy phenotype are mutation specific. Methods and Results Two hypertrophic cardiomyopathy mouse models carrying the R92Q and the E163R TNNT2 mutations were investigated. Echocardiography showed left ventricular hypertrophy, enhanced contractility, and diastolic dysfunction in both models; however, these phenotypes were more pronounced in the R92Q mice. Both E163R and R92Q trabeculae showed prolonged twitch relaxation and increased occurrence of premature beats. In E163R ventricular myofibrils or skinned trabeculae, relaxation following Ca2+ removal was prolonged; resting tension and resting ATPase were higher; and isometric ATPase at maximal Ca2+ activation, the energy cost of tension generation, and myofilament Ca2+ sensitivity were increased compared with that in wild‐type mice. No sarcomeric changes were observed in R92Q versus wild‐type mice, except for a large increase in myofilament Ca2+ sensitivity. In R92Q myocardium, we found a blunted response to inotropic interventions, slower decay of Ca2+ transients, reduced SERCA function, and increased Ca2+/calmodulin kinase II activity. Contrarily, secondary alterations of E‐C coupling and signaling were minimal in E163R myocardium. Conclusions In E163R models, mutation‐driven myofilament abnormalities directly cause myocardial dysfunction. In R92Q, diastolic dysfunction and arrhythmogenicity are mediated by profound cardiomyocyte signaling and E‐C coupling changes. Similar hypertrophic cardiomyopathy phenotypes can be generated through different pathways, implying different strategies for a precision medicine approach to treatment.

New Insights into the Potential Roles of 3-Iodothyronamine (T1AM) and Newly Developed Thyronamine-Like TAAR1 Agonists in Neuroprotection

3-Iodothyronamine (T1AM) is an endogenous high-affinity ligand of the trace amine-associated receptor 1 (TAAR1), detected in mammals in many organs, including the brain. Recent evidence indicates that pharmacological TAAR1 activation may offer a novel therapeutic option for the treatment of a wide range of neuropsychiatric and metabolic disorders. To assess potential neuroprotection by TAAR1 agonists, in the present work, we initially investigated whether T1AM and its corresponding 3-methylbiaryl-methane analog SG-2 can improve learning and memory when systemically administered to mice at submicromolar doses, and whether these effects are modified under conditions of MAO inhibition by clorgyline. Our results revealed that when i.p. injected to mice, both T1AM and SG-2 produced memory-enhancing and hyperalgesic effects, while increasing ERK1/2 phosphorylation and expression of transcription factor c-fos. Notably, both compounds appeared to rely on the action of ubiquitous enzymes MAO to produce the corresponding oxidative metabolites that were then able to activate the histaminergic system. Since autophagy is key for neuronal plasticity, in a second line of experiments we explored whether T1AM and synthetic TAAR1 agonists SG1 and SG2 were able to induce autophagy in human glioblastoma cell lines (U-87MG). After treatment of U-87MG cells with 1 μM T1AM, SG-1, SG-2 transmission electron microscopy (TEM) and immunofluorescence (IF) showed a significant time-dependent increase of autophagy vacuoles and microtubule-associated protein 1 light chain 3 (LC3). Consistently, Western blot analysis revealed a significant increase of the LC3II/LC3I ratio, with T1AM and SG-1 being the most effective agents. A decreased level of the p62 protein was also observed after treatment with T1AM and SG-1, which confirms the efficacy of these compounds as autophagy inducers in U-87MG cells. In the process to dissect which pathway induces ATG, the effects of these compounds were evaluated on the PI3K-AKT-mTOR pathway. We found that 1 μM T1AM, SG-1 and SG-2 decreased pAKT/AKT ratio at 0.5 and 4 h after treatment, suggesting that autophagy is induced by inhibiting mTOR phosphorylation by PI3K-AKT-mTOR pathway. In conclusion, our study shows that T1AM and thyronamine-like derivatives SG-1 and SG-2 might represent valuable tools to therapeutically intervene with neurological disorders.

Selective HCN1 block as a strategy to control oxaliplatin-induced neuropathy

ABSTRACT Chemotherapy‐Induced Peripheral Neuropathy (CIPN) is the most frequent adverse effect of pharmacological cancer treatments. The occurrence of neuropathy prevents the administration of fully‐effective drug regimen, affects negatively the quality of life of patients, and may lead to therapy discontinuation. CIPN is currently treated with anticonvulsants, antidepressants, opioids and non‐opioid analgesics, all of which are flawed by insufficient anti‐hyperalgesic efficacy or addictive potential. Understandably, developing new drugs targeting CIPN‐specific pathogenic mechanisms would dramatically improve efficacy and tolerability of anti‐neuropathic therapies. Neuropathies are associated to aberrant excitability of DRG neurons due to the alteration in the expression or function of a variety of ion channels. In this regard, Hyperpolarization‐activated Cyclic Nucleotide‐gated (HCN) channels are overexpressed in inflammatory and neuropathic pain states, and HCN blockers have been shown to reduce neuronal excitability and to ameliorate painful states in animal models. However, HCN channels are critical in cardiac action potential, and HCN blockers used so far in pre‐clinical models do not discriminate between cardiac and non‐cardiac HCN isoforms. In this work, we show an HCN current gain of function in DRG neurons from oxaliplatin‐treated rats. Biochemically, we observed a downregulation of HCN2 expression and an upregulation of the HCN regulatory beta‐subunit MirP1. Finally, we report the efficacy of the selective HCN1 inhibitor MEL57A in reducing hyperalgesia and allodynia in oxaliplatin‐treated rats without cardiac effects. In conclusion, this study strengthens the evidence for a disease‐specific role of HCN1 in CIPN, and proposes HCN1‐selective inhibitors as new‐generation pain medications with the desired efficacy and safety profile. HighlightsNociceptive neurons from oxaliplatin‐treated rats show Ih gain of function.Increased current density is mediated by HCN1‐specific kinetic enhancement.Acceleration of HCN1 kinetics is associated to increased MiRP1 expression.Pan HCN blockers effectively revert neuropathy in vivo but cause bradycardia.HCN1‐selective blocker MEL57A shows intact analgesic efficacy without cardiac effects.

The impact of scopolamine pretreatment on 3-iodothyronamine (T1AM) effects on memory and pain in mice☆

ABSTRACT We previously demonstrated that 3‐iodothyronamine (T1AM), a by‐product of thyroid hormone metabolism, pharmacologically administered to mice acutely stimulated learning and memory acquisition and provided hyperalgesia with a mechanism which remains to be defined. We now aimed to investigate whether the T1AM effect on memory and pain was maintained in mice pre‐treated with scopolamine, a non‐selective muscarinic antagonist expected to induce amnesia and, possibly, hyperalgesia. Mice were pre‐treated with scopolamine and, after 20 min, injected intracerebroventricularly (i.c.v.) with T1AM (0.13, 0.4, 1.32 &mgr;g/kg). 15 min after T1AM injection, the mice learning capacity or their pain threshold were evaluated by the light/dark box and by the hot plate test (51.5 °C) respectively. Experiments in the light/dark box were repeated in mice receiving clorgyline (2.5 mg/kg, i.p.), a monoamine oxidase (MAO) inhibitor administered 10 min before scopolamine (0.3 mg/kg). Our results demonstrated that 0.3 mg/kg scopolamine induced amnesia without modifying the murine pain threshold. T1AM fully reversed scopolamine‐induced amnesia and produced hyperalgesia at a dose as low as 0.13 &mgr;g/kg. The T1AM anti‐amnestic effect was lost in mice pre‐treated with clorgyline. We report that the removal of muscarinic signalling increases T1AM pro learning and hyperalgesic effectiveness suggesting T1AM as a potential treatment as a “pro‐drug” for memory dysfunction in neurodegenerative diseases. HIGHLIGHTST1AM fully reverts scopolamine‐induced amnesia.T1AM effectiveness on memory and pain increases in the presence of scopolamine.T1AM as a pro‐drug for treating memory disorders associated with altered pain perception.

Commentary: 3-Iodothyronamine Reduces Insulin Secretion In Vitro via a Mitochondrial Mechanism

Lehmphul et al. report the effect of 3-iodothyronamine in reducing insulin release in a model of immortalized pancreatic β-cells. Notwithstanding the simplified β-cell model used, this article offers an opportunity to reconsider, possibly under a new light, an old issue of research, which excited people working on amine oxidases (AOs) in the last 20 years. Toward this aim, we would like to propose some points of reflection to the scientific community working on 3-iodothyronamine and thyroid hormone metabolites: 1. the paper indicates that 3-iodothyronamine reduces insulin release with a mechanism mediated, at least in part, by its oxidative metabolite, the 3-iodothyroacetic acid, produced by mitochondrial monoamine oxidase (MAOs), type B (MAO-B) activity. This finding, confirming our observations and hypothesis on the role of 3-iodothyronamine as a source of active metabolites (1, 2), demonstrates for the first time that 3-iodothyronamine is a substrate for MAO-B, the MAO isoform in search of substrates and of functions; 2. the degradation of 3-iodothyronamine by MAO-B, with production of the corresponding aldehyde and hydrogen peroxide (H2O2), potentially represents a self-standing mechanism independently of 3-iodothyronamine receptor activation on pancreatic cells. Amine oxidases are a heterogeneous class of enzymes, including MAOs (type A and B) and semicarbazide-sensitive amine oxidases (SSAOs). While MAOs are ubiquitous enzymes, being linked to the outer mitochondrial membrane (active site facing the cytoplasm), plasma membrane SSAOs can have selective and species-specific tissue/cell expression. In addition, MAOs and SSAOs are distinguishable by inhibitor sensitivity, substrate selectivity and affinity, and subcellular localization. Noradrenaline and serotonin are among MAO-A substrates, dopamine and other trace amines, including tyramine and β-phenylethylamine, are MAO-A, B, and SSAO substrates. Up to now, direct evidence that 3-iodothyronamine is a substrate for MAO-A is lacking. However, now we know that 3-iodothyroanime is a substrate for MAO-B.