Diphenyl-Methane Based Thyromimetic Inhibitors for Transthyretin Amyloidosis.

Thyromimetics, whose physicochemical characteristics are analog to thyroid hormones (THs) and their derivatives, are promising candidates as novel therapeutics for neurodegenerative and metabolic pathologies. In particular, sobetirome (GC-1), one of the initial halogen-free thyromimetics, and newly synthesized IS25 and TG68, with optimized ADME-Tox profile, have recently attracted attention owing to their superior therapeutic benefits, selectivity, and enhanced permeability. Here, we further explored the functional capabilities of these thyromimetics to inhibit transthyretin (TTR) amyloidosis. TTR is a homotetrameric transporter protein for THs, yet it is also responsible for severe amyloid fibril formation, which is facilitated by tetramer dissociation into non-native monomers. By combining nuclear magnetic resonance (NMR) spectroscopy, computational simulation, and biochemical assays, we found that GC-1 and newly designed diphenyl-methane-based thyromimetics, namely IS25 and TG68, are TTR stabilizers and efficient suppressors of TTR aggregation. Based on these observations, we propose the novel potential of thyromimetics as a multi-functional therapeutic molecule for TTR-related pathologies, including neurodegenerative diseases.

TG68, a Novel Thyroid Hormone Receptor-ß Agonist for the Treatment of NAFLD.

Activation of thyroid hormone receptor ß (THRß) has shown beneficial effects on metabolic alterations, including non-alcoholic fatty liver disease (NAFLD). Here, we investigated the effect of TG68, a novel THRß agonist, on fatty liver accumulation and liver injury in mice fed a high-fat diet (HFD). C57BL/6 mice fed HFD for 17 or 18 weeks, a time when all mice developed massive steatohepatitis, were then given TG68 at a dose of 9.35 or 2.8 mg/kg for 2 or 3 weeks, respectively. As a reference compound, the same treatment was adopted using equimolar doses of MGL-3196, a selective THRß agonist currently in clinical phase III. The results showed that treatment with TG68 led to a reduction in liver weight, hepatic steatosis, serum transaminases, and circulating triglycerides. qRT-PCR analyses demonstrated activation of THRß, as confirmed by increased mRNA levels of Deiodinase-1 and Malic enzyme-1, and changes in lipid metabolism, as revealed by increased expression of Acyl-CoA Oxidase-1 and Carnitine palmitoyltransferase-1. The present results showed that this novel THRß agonist exerts an anti-steatogenic effect coupled with amelioration of liver injury in the absence of extra-hepatic side effects, suggesting that TG68 may represent a useful tool for the treatment of NAFLD.

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.

Optimization of a dispersive liquid-liquid microextraction method followed by UHPLC analysis for fluoxetine quantification in environmental water resources.

Fluoxetine is the most prescribed drug for treatment of depression. Recently, its presence in aquatic environment has been receiving a growing interest as several studies assessed its effects on aquatic fauna. Therefore, it's important to have an analytical method capable of monitoring these compounds at low concentrations. In this study, a new method was developed based on dispersive liquid-liquid microextraction to preconcentrate fluoxetine in a small volume of water sample (6 mL) before chromatographic analysis using ultra high performance liquid chromatography with fluorescence detection. Effect of composition and volume of extracting mixture, sample pH, vortexing time and salt addition were evaluated. Optimization of extraction conditions lead to an enrichment factor of 61 ± 18. After extraction optimization, recovery percentages of fluoxetine spiked into different water matrices between 83-110% were obtained. For the optimized method, the calibration curve was obtained in the range of 160-2500 ng/L with a limit of detection of 98.9 ng/L and a limit of quantification of 329.8 ng/L.

Synthetic Thyroid Hormone Receptor-ß Agonists Promote Oligodendrocyte Precursor Cell Differentiation in the Presence of Inflammatory Challenges.

Oligodendrocytes and their precursors are the cells responsible for developmental myelination and myelin repair during adulthood. Their differentiation and maturation processes are regulated by a complex molecular machinery driven mainly by triiodothyronine (T3), the genomic active form of thyroid hormone, which binds to thyroid hormone receptors (TRs), regulating the expression of target genes. Different molecular tools have been developed to mimic T3 action in an attempt to overcome the myelin repair deficit that underlies various central nervous system pathologies. In this study, we used a well-established in vitro model of neural stem cell-derived oligodendrocyte precursor cells (OPCs) to test the effects of two compounds: the TRß1 ligand IS25 and its pro-drug TG68. We showed that treatment with TG68 induces OPC differentiation/maturation as well as both the natural ligand and the best-known TRß1 synthetic ligand, GC-1. We then described that, unlike T3, TG68 can fully overcome the cytokine-mediated oligodendrocyte differentiation block. In conclusion, we showed the ability of a new synthetic compound to stimulate OPC differentiation and overcome inflammation-mediated pathological conditions. Further studies will clarify whether the compound acts as a pro-drug to produce the TRß1 ligand IS25 or if its action is mediated by secondary mechanisms such as AMPK activation.

Design, Synthesis, and In Vitro Evaluation of Novel 8-Amino-Quinoline Combined with Natural Antioxidant Acids.

Overproduction of reactive oxygen species (ROS) and alterations in metallostasis are common and related hallmarks in several neurodegenerative diseases (NDDs). Nature-based derivatives always represent an attractive tool in MTDL drug design, especially against ROS in NDDs. On this notion, we designed a new series of 8-quinoline-N-substituted derivatives with a natural antioxidant portion (i.e., lipoic, caffeic, and ferulic acids). These compounds were shown to chelate copper, a metal involved in ROS-induced degeneration, and scavenger oxygen radicals in DPPH assay. Then, selected compounds 4 and 5 were evaluated in an in vitro model of oxidative stress and shown to possess cytoprotective effects in 661W photoreceptor-like cells. The obtained results may represent a starting point for the application of the proposed class of compounds in retinal neurodegenerative diseases such as retinitis pigmentosa (RP), comprising a group of hereditary rod-cone dystrophies that represent a major cause of blindness in patients of working age, where the progression of the disease is a multifactorial event, with oxidative stress contributing predominantly.

Beyond Antioxidant Effects: Nature-Based Templates Unveil New Strategies for Neurodegenerative Diseases.

The complex network of malfunctioning pathways occurring in the pathogenesis of neurodegenerative diseases (NDDs) represents a huge hurdle in the development of new effective drugs to be used in therapy. In this context, redox reactions act as crucial regulators in the maintenance of neuronal microenvironment homeostasis. Particularly, their imbalance results in the severe compromising of organism's natural defense systems and subsequently, in the instauration of deleterious OS, that plays a fundamental role in the insurgence and progress of NDDs. Despite the huge efforts in drug discovery programs, the identification process of new therapeutic agents able to counteract the relentless progress of neurodegenerative processes has produced low or no effective therapies. Consequently, a paradigm-shift in the drug discovery approach for these diseases is gradually occurring, paving the way for innovative therapeutical approaches, such as polypharmacology. The aim of this review is to provide an overview of the main pharmacological features of most promising nature-based scaffolds for a possible application in drug discovery, especially for NDDs, highlighting their multifaceted effects against OS and neuronal disorders.

Development of potent dual PDK1/AurA kinase inhibitors for cancer therapy: Lead-optimization, structural insights, and ADME-Tox profile.

We report the synthesis of novel first-in-class 2-oxindole-based derivatives as dual PDK1-AurA kinase inhibitors as a novel strategy to treat Ewing sarcoma. The most potent compound 12 is suitable for progression to in vivo studies. The specific attributes of 12 included nanomolar inhibitory potency against both phosphoinositide-dependent kinase-1 (PDK1) and Aurora A (AurA) kinase, with acceptable in vitro ADME-Tox properties (cytotoxicity in 2 healthy and 14 hematological and solid cancer cell-lines; inhibition of PDE4C1, SIRT7, HDAC4, HDAC6, HDAC8, HDAC9, AurB, CYP1A2, CYP2C9, CYP2C19, CYP2D6, and hERG). X-ray crystallography and docking studies led to the identification of the key AurA and PDK1/12 interactions. Finally, in vitro drug-intake kinetics and in vivo PK appear to indicate that these compounds are attractive lead-structures for the design and synthesis of PDK1/AurA dual-target molecules to further investigate the in vivo efficacy against Ewing Sarcoma.

Identification of a Thyroid Hormone Derivative as a Pleiotropic Agent for the Treatment of Alzheimer's Disease.

The identification of effective pharmacological tools for Alzheimer's disease (AD) represents one of the main challenges for therapeutic discovery. Due to the variety of pathological processes associated with AD, a promising route for pharmacological intervention involves the development of new chemical entities that can restore cellular homeostasis. To investigate this strategy, we designed and synthetized SG2, a compound related to the thyroid hormone thyroxine, that shares a pleiotropic activity with its endogenous parent compound, including autophagic flux promotion, neuroprotection, and metabolic reprogramming. We demonstrate herein that SG2 acts in a pleiotropic manner to induce recovery in a C. elegans model of AD based on the overexpression of Aß42 and improves learning abilities in the 5XFAD mouse model of AD. Further, in vitro ADME-Tox profiling and toxicological studies in zebrafish confirmed the low toxicity of this compound, which represents a chemical starting point for AD drug development.

Selective Thyroid Hormone Receptor-Beta (TRß) Agonists: New Perspectives for the Treatment of Metabolic and Neurodegenerative Disorders.

Thyroid hormones (THs) elicit significant effects on numerous physiological processes, such as growth, development, and metabolism. A lack of thyroid hormones is not compatible with normal health. Most THs effects are mediated by two different thyroid hormone receptor (TR) isoforms, namely TRα and TRß, with the TRß isoform known to be responsible for the main beneficial effects of TH on liver. In brain, despite the crucial role of TRα isoform in neuronal development, TRß has been proposed to play a role in the remyelination processes. Consequently, over the past two decades, much effort has been applied in developing thyroid hormone analogs capable of uncoupling beneficial actions on liver (triglyceride and cholesterol lowering) and central nervous system (CNS) (oligodendrocyte proliferation) from deleterious effects on the heart, muscle and bone. Sobetirome (GC-1) and subsequently Eprotirome (KB2115) were the first examples of TRß selective thyromimetics, with Sobetirome differing from the structure of thyronines because of the absence of halogens, biaryl ether oxygen, and amino-acidic side chain. Even though both thyromimetics showed encouraging actions against hypercholesterolemia, non-alcoholic steatohepatitis (NASH) and in the stimulation of hepatocytes proliferation, they were stopped after Phase 1 and Phase 2-3 clinical trials, respectively. In recent years, advances in molecular and structural biology have facilitated the design of new selective thyroid hormone mimetics that exhibit TR isoform-selective binding, and/or liver- and tissue-selective uptake, with Resmetirom (MGL-3196) and Hep-Direct prodrug VK2809 (MB07811) probably representing two of the most promising lipid lowering agents, currently under phase 2-3 clinical trials. More recently the application of a comprehensive panel of ADME-Toxicity assays enabled the selection of novel thyromimetic IS25 and its prodrug TG68, as very powerful lipid lowering agents both in vitro and in vivo. In addition to dyslipidemia and other liver pathologies, THs analogs could also be of value for the treatment of neurodegenerative diseases, such as multiple sclerosis (MS). Sob-AM2, a CNS- selective prodrug of Sobetirome has been shown to promote significant myelin repair in the brain and spinal cord of mouse demyelinating models and it is rapidly moving into clinical trials in humans. Taken together all these findings support the great potential of selective thyromimetics in targeting a large variety of human pathologies characterized by altered metabolism and/or cellular differentiation.

Collecting data through high throughput in vitro early toxicity and off-target liability assays to rapidly identify limitations of novel thyromimetics.

In order to rapidly identify the phenotypic profile and possible off-target liability effects of novel synthesized thyromimetics for selection of lead compounds for further optimization studies, we performed in vitro screening on a new small library of synthetic thyromimetics. A comprehensive panel of early toxicity assays comprising cytotoxicity on 4 different cell lines (osteosarcoma, U2OS; lung fibroblast, hTERT; human breast adenocarcinoma, MCF7; human embryonic kidney, HEK293), hERG liability, cytochrome P450 inhibition (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 isoforms), and off-target liability against selected proteins (Aurora B kinase and phosphodiesterase PDE4C1) and epigenetic enzymes (HDAC4, HDAC6, HDAC8, HDAC9 & SIRT7). All the compounds were screened at 10 µM in at least triplicate using well-established in vitro assays with readouts in luminescence or fluorescence polarization mode. The raw data were processed using Microsoft Excel and the Z' for each assay was calculated (acceptable Z' >0.40). The processed and normalized data were organized in tables and visualized using spider plots. The results which are reported in the present manuscript can be used in prediction studies of early toxicity and off-target liabilities of other thyromimetics using in silico methods. The data reported herein support our research article entitled "Design, synthesis and biological evaluation of novel TRß selective agonists sustained by ADME-Toxicity analysis" by Runfola M., Sestito S., et al. [1].

Potential role of two novel agonists of thyroid hormone receptor-ß on liver regeneration.

OBJECTIVES: Although the hepatomitogenic activity of triiodothyronine (T3) is well established, the wide range of harmful effects exerted by this hormone precludes its use in liver regenerative therapy. Selective agonists of the beta isoform of thyroid hormone receptor (TRß) do not exhibit T3-induced cardiotoxicity and show a good safety profile in patients with NASH. The aim of this study was to investigate whether two novel TRß agonists, the prodrug TG68 and the active compound IS25 could stimulate hepatocyte proliferation without T3/TRα-dependent side effects. METHODS: Rats were treated with three different doses (12.5, 25 and 50 µg/100 g body weight) for one week. Hepatocyte proliferation, liver injury and serum biochemical parameters were measured by immunohistochemistry, qRT-PCR and Western blot. RESULTS: Both drugs increased hepatocyte proliferation as assessed by bromodeoxyuridine incorporation (from 14% to 28% vs 5% of controls) and mitotic activity. Enhanced proliferation occurred in the absence of significant signs of liver injury as shown by lack of increased serum transaminase levels or of apoptosis. No cardiac or renal hypertrophy typically associated with treatment with T3 was observed. Importantly, no proliferation of pancreatic acinar cells, such as that seen after administration of T3 or the TRß agonist GC1 was detected following either TG68 or IS25, demonstrating the hepato-specificity of these novel TRß agonists. CONCLUSIONS: The present study shows that TG68 and IS25 induce massive hepatocyte proliferation without overt toxicity. Hence, these agents may have a significant clinical application for regenerative therapies in liver transplantation or other surgical settings.

Design, synthesis and biological evaluation of novel TRß selective agonists sustained by ADME-toxicity analysis.

Although triiodothyronine (T3) induces several beneficial effects on lipid metabolism, its use is hampered by toxic side-effects, such as tachycardia, arrhythmia, heart failure, bone and muscle catabolism and mood disturbances. Since the α isoform of thyroid hormone receptors (TRs) is the main cause of T3-related harmful effects, several efforts have been made to develop selective agonists of the ß isoform that could induce some beneficial effects (i.e. lowering triglyceride and cholesterol levels reducing obesity and improving metabolic syndrome), while overcoming most of the adverse T3-dependent side effects. Herein, we describe the drug discovery process sustained by ADME-Toxicity analysis that led us to identify novel agonists with selectivity for the isoform TRß and an acceptable off-target and absorption, distribution metabolism, excretion and toxicity (ADME-Tox) profile. Within the small series of compounds synthesized, derivatives 1 and 3, emerge from this analysis as "potentially safe" to be engaged in preclinical studies. In in vitro investigation proved that both compounds were able to reduce lipid accumulation in HepG2 and promote lipolysis with comparable effects to those elicited by T3, used as reference drug. Moreover, a preliminary in vivo study confirmed the apparent lack of toxicity, thus suggesting compounds 1 and 3 as new potential TRß-selective thyromimetics.

Design and synthesis of H2S-donor hybrids: A new treatment for Alzheimer's disease?

Hydrogen sulphide (H2S) is an endogenous gasotransmitter, largely known as a pleiotropic mediator endowed with antioxidant, anti-inflammatory, pro-autophagic, and neuroprotective properties. Moreover, a strong relationship between H2S and aging has been recently identified and consistently, a significant decline of H2S levels has been observed in patients affected by Alzheimer's disease (AD). On this basis, the use of H2S-donors could represent an exciting and intriguing strategy to be pursued for the treatment of neurodegenerative diseases (NDDs). In this work, we designed a small series of multitarget molecules combining the rivastigmine-scaffold, a well-established drug already approved for AD, with sulforaphane (SFN) and erucin (ERN), two natural products deriving from the enzymatic hydrolysis of glucosinolates contained in broccoli and rocket, respectively, endowed both with antioxidant and neuroprotective effects. Notably, all new synthetized hybrids exhibit a H2S-donor profile in vitro and elicit protective effects in a model of LPS-induced microglia inflammation. Moreover, a decrease in NO production has been observed in LPS-stimulated cells pre-treated with the compounds. Finally, the compounds showed neuroprotective and antioxidant activities in human neuronal cells. The most interesting compounds have been further investigated to elucidate the possible mechanism of action.

New Multitarget Approaches in the War Against Glioblastoma: A Mini-Perspective.

Glioblastoma multiforme (GBM) is the most common tumor of the CNS, and the deadliest form of brain cancer. The rapid progression, the anatomic location in the brain and a deficient knowledge of the pathophysiology, often limit the effectiveness of therapeutic interventions. Current pillars of GBM therapies include surgical resection, radiotherapy and chemotherapy, but the low survival rate and the short life expectation following these treatments strongly underline the urgency to identify innovative and more effective therapeutic tools. Frequently, patients subjected to a mono-target therapy, such as Temozolomide (TMZ), develop drug resistance and undergo relapse, indicating that targeting a single cellular node is not sufficient for eradication of this disease. In this context, a multi-targeted therapeutic approach aimed at using compounds, alone or in combination, capable of inhibiting more than one specific molecular target, offers a promising alternative. Such strategies have already been well integrated into drug discovery campaigns, including in the field of anticancer drugs. In this miniperspective, we will discuss the recent progress in the treatment of GBM focusing on innovative and effective preclinical strategies, which are based on a multi-targeted approach.

Hit-to-Lead Optimization of Mouse Trace Amine Associated Receptor 1 (mTAAR1) Agonists with a Diphenylmethane-Scaffold: Design, Synthesis, and Biological Study.

The trace amine-associated receptor 1 (TAAR1) is a G-protein-coupled receptors (GPCR) potently activated by a variety of molecules besides trace amines (TAs), including thyroid hormone-derivatives like 3-iodothyronamine (T1AM), catechol-O-methyltransferase products like 3-methoxytyramine, and amphetamine-related compounds. Accordingly, TAAR1 is considered a promising target for medicinal development. To gain more insights into TAAR1 physiological functions and validation of its therapeutic potential, we recently developed a new class of thyronamine-like derivatives. Among them compound SG2 showed high affinity and potent agonist activity at mouse TAAR1. In the present work, we describe design, synthesis, and SAR study of a new series of compounds (1-16) obtained by introducing specific structural changes at key points of our lead compound SG2 skeleton. Five of the newly synthesized compounds displayed mTAAR1 agonist activity higher than both SG2 and T1AM. Selected diphenylmethane analogues, namely 1 and 2, showed potent functional activity in in vitro and in vivo models.