Discovery of SYD5115, a novel orally active small molecule TSH-R antagonist.

The thyrotropin receptor (TSH-R) regulates the thyroid gland and is normally activated by thyrotropin. In patients with Graves' disease, TSH-R is also stimulated by stimulatory TSH-R autoantibodies leading to hyperthyroidism. In this paper, we describe the discovery of SYD5115 (67), a novel small molecule TSH-R antagonist with nanomolar potency. SYD5115 also blocks stimulating antibody induced synthesis of the thyroid hormone thyroxine (T4) in vivo, after a single oral dose. During optimization, several issues had to be addressed such as the low metabolic stability and the potential mutagenicity of our first series of compounds.

Thyrotropin receptor antagonists and inverse agonists, and their potential application to thyroid diseases.

The thyrotropin receptor (TSHR) plays critical roles in thyroid growth and function and in the pathogenesis of several thyroid diseases including Graves' hyperthyroidism and ophthalmopathy, non-autoimmune hyperthyroidism and thyroid cancer. Several low-molecular weight compounds (LMWCs) and anti-TSHR monoclonal antibodies (mAbs) with receptor antagonistic and inverse agonistic activities have been reported. The former binds to the pocket formed by the receptor transmembrane bundle, and the latter to the extracellular TSH binding site. Both are effective inhibitors of TSH/thyroid stimulating antibody-stimulated cAMP and/or hyaluronic acid production in TSHR-expressing cells. Anti-insulin-like growth factor 1 inhibitors are also found to inhibit TSHR signaling. Each agent has advantages and disadvantages; for example, mAbs have a higher affinity and longer half-life but are more costly than LMWCs. At present, mAbs appear most promising, yet the development of more efficacious LMWCs is desirable. These agents are anticipated to be efficacious not only for the above-mentioned diseases but also for resistance to thyroid hormone and have utility for thyroid cancer radionuclide scintigraphy/therapy as a new theranostic.

Blocking the Thyrotropin Receptor with K1-70 in a Patient with Follicular Thyroid Cancer, Graves' Disease, and Graves' Ophthalmopathy.

Background: We report the therapeutic use of K1-70™, a thyrotropin receptor (TSHR) antagonist monoclonal antibody, in a patient with follicular thyroid cancer (FTC), Graves' disease (GD), and Graves' ophthalmopathy (GO). Methods: A 51-year-old female patient, who smoked, presented in October 2014 with FTC complicated by GD, high levels of TSHR autoantibodies with high thyroid stimulating antibody (TSAb) activity, and severe GO. K1-70 was administered at 3 weekly intervals with the dose adjusted to block TSAb activity. Her cancer was managed with lenvatinib and radioiodine therapy. Results: Following initiation of K1-70 therapy, TSAb activity measured in serum decreased and GO (proptosis and inflammation) improved. On K1-70 monotherapy during the pause in lenvatinib, several metastatic lesions stabilized while others showed progression attenuation compared with that before lenvatinib therapy. Conclusions: These observations suggest that blocking TSHR stimulation with K1-70 can be an effective treatment for GO and may also benefit select patients with FTC and GD.

Treatment of moderate to severe orbitopathy: Current modalities and perspectives.

Graves' orbitopathy (GO) is the primary cause of exophthalmos in adults. It appears in 30 to 50% of patients with Graves' disease. About 5% are moderate-to-severe cases that might be see-threatening or lead to long term disabling sequelae. Recommendations have been established in 2016 by the European thyroid association (ETA) and the European group on Grave's orbitopathy (EUGOGO), suggesting a wide use of corticosteroids in moderate to severe forms. However, disappointing results have been reported in 20 to 30% of cases. Improved understanding of pathophysiological mechanisms has allowed the use of non-specific immunomodulatory agents, currently under evaluation, and which place in the therapeutic strategy remains to be determined. Very recently, new promising therapeutic advances have emerged with the identification of new therapeutic targets, such as the TSH receptor and IGF-1 receptor complex.

Implications of an Improved Model of the TSH Receptor Transmembrane Domain (TSHR-TMD-TRIO).

The thyroid-stimulating hormone receptor (TSHR) is a G-protein-coupled receptor group A family member with 7 transmembrane helices. We generated 3 new models of its entire transmembrane region using a 600 ns molecular simulation. The simulation started from our previously published model, which we have now revised by also modeling the intracellular loops and the C-terminal tail, adding internal waters and embedding it into a lipid bilayer with a water layer and with ions added to complete the system. We have named this model TSHR-TMD-TRIO since 3 representative dominant structures were then extracted from the simulation trajectory and compared with the original model. These structures each showed small but significant changes in the relative positions of the helices. The 3 models were also used as targets to dock a set of small molecules that are known active compounds including a new TSHR antagonist (BT362), which confirmed the appropriateness of the model with some small molecules showing significant preference for one or other of the structures.

New Thieno-[2,3-d]pyrimidine-Based Functional Antagonist for the Receptor of Thyroid Stimulating Hormone.

Thyroid stimulating hormone (TSH) receptor antagonists are required for the treatment of TSH-dependent tumors and Graves disease. We developed the compound 5-amino-N-(tert-butyl)-4-(4-iodophenyl)-2-(methylthio)thieno[2,3-d]pyrimidine-6-carboxamide (TP48) and showed that it reduces the TSH-stimulated adenylate cyclase activity in rat thyroid membranes. Pretreatment of rats with compound TP48 (ip, 40 mg/kg) reduced the increase in the levels of total and free thyroxin in blood and the increase in the expression of thyroglobulin and D2 deiodinase genes in the thyroid gland, which are responsible for the synthesis of thyroid hormones, which were caused by intranasal administration of thyroliberin to animals (300 µg/kg). These data indicate that compound TP48 is a functional antagonist of the TSH receptor and can be used to correct the thyroid status in hyperthyroidism.

Clinicopathologic characteristics and outcomes of papillary thyroid carcinoma in younger patients.

In the 7th edition of AJCC staging system, cervical lymph node metastases (LNM) in papillary thyroid carcinoma (PTC) is considered as a poorer prognostic indicator only in patients aged 45 years or older, but as a low-risk factor in patients younger than 45 years. The objective of this study is to investigate the influence of cervical LNM on prognostic outcomes of young patients (<45 years' old) with PTC.We carried out a retrospective analysis of 1896 PTC patients younger than 45 years' old at diagnosis, who were firstly treated in our department between January 2005 and December 2014. Clinicopathologic features, recurrences, disease-free survival (DFS) were recorded and analyzed.A total of 1896 consecutive patients were identified, comprising of 426 males and 1470 females after a median follow-up period of 40 months (3-129 months) from initial surgery to disease recurrence or to the end of follow-up. The rate of recurrence was 2.16% (n = 41). The DFS rates for a 1-year, 3-year, or 5-year team were 99.1%, 97.8%, or 97.4%, respectively. Univariate analysis showed that diagnosed age ≤30 years, tumor size >1.0 cm, extrathyroidal extension, multifocal lesions, lesions in bilateral lobes, central neck LNM, and lateral neck LNM were associated with a worse DFS. Multivariate analysis showed that only central neck LNM and lateral neck LNM were significant independent prognostic factors for DFS (P < .001). For patients with papillary thyroid microcarcinoma, cervical LNM were also identified as independent risk factors for DFS (P < .001).LNM have prognostic significance for DFS in PTC patients younger than 45 years. It indicated that PTC patients (<45 years old) with LNM, especially lateral neck LNM, were understaged by the 7th edition of AJCC staging system. Thus, radical resection of primary tumor and metastatic lymph nodes, frequent follow-up, and strict TSH suppression should be taken for young patients with PTC.

TSH/IGF1 receptor crosstalk: Mechanism and clinical implications.

Increasing evidence of interdependence between G protein-coupled receptors and receptor tyrosine kinase signaling pathways has prompted reevaluation of crosstalk between these receptors in disease and therapy. Investigations into thyroid-stimulating hormone (TSH) and insulin-like growth factor 1 (IGF1) receptor crosstalk, and its application to the clinic have in particular shown recent progress. In this review, we summarize current insights into the mechanism of TSH/IGF1 receptor crosstalk. We discuss evidence that crosstalk is one of the underlying causes of TSHR-based disease and the feasibility of using combinations of TSH receptor and IGF1 receptor antagonists to increase the therapeutic index for the treatment of Graves' hyperthyroidism and Graves' ophthalmopathy.

TSH inhibits eNOS expression in HMEC-1 cells through the TSHR/PI3K/AKT signaling pathway.

OBJECTIVE: To investigate the effects of thyroid-stimulating hormone (TSH) on the expression of endothelial nitric oxide synthase (eNOS) in human microvascular endothelial cells (HMEC-1) and explore the potential mechanism. MATERIALS AND METHODS: Expression of thyroid-stimulating hormone receptor (TSHR) in HMEC-1 cells was determined by immunofluorescence, reverse transcription-polymerase chain reaction (RT-PCR), and Western blotting. Cell proliferation and the production of nitric oxide (NO) and superoxide anion (SA) were measured after TSH treatment. eNOS expression and AKT phosphorylation were detected by Western blotting. RESULTS: TSHR was expressed in HMEC-1 cells. TSH promoted HMEC-1 cell proliferation and SA production, but inhibited NO generation by dose-dependent blocking of mRNA and protein expression of eNOS. Mechanism studies demonstrated that TSH promoted AKT phosphorylation (P<0.05), and that LY294002 inhibited the reduction of eNOS expression by TSH. Moreover, TSH activated the AKT signaling pathway through binding to TSHR on HMEC-1 cells. CONCLUSIONS: TSH inhibits NO production via the TSHR/AKT signaling pathway.

Comprehensive research on thyroid diseases associated with autoimmunity: autoimmune thyroid diseases, thyroid diseases during immune-checkpoint inhibitors therapy, and immunoglobulin-G4-associated thyroid diseases.

Various thyroid diseases are associated with autoimmunity. Major autoimmune thyroid diseases are Graves' disease (GD) and Hashimoto's thyroiditis (HT). Thyrotropin receptor is an autoantigen in GD, and its immunogenicity has been examined. Immune-checkpoint inhibitor (ICI) is recently widely used for treatment of malignant tumors, but cases of thyroid diseases during ICI treatment have been increasing. Thyroid diseases during ICI therapy have been investigated in immunological and clinical aspects, and their Japanese official diagnostic guidelines were established. In addition, serum and tissue immunoglobulin-G4 levels have been examined in association with clinicopathological characteristics in GD, HT, and Riedel's thyroiditis. We review these diseases associated with thyroid autoimmunity and comprehensively discuss their potential application in future research and therapeutic options.

Dihydrotestosterone regulates oxidative stress and immunosuppressive cytokines in a female BALB/c mouse model of Graves' disease.

Background: Graves' disease (GD) is an autoimmune disease that affects more women than men. In our previous study, a potent bioactive androgen, 5α-dihydrotestosterone (DHT) showed a protective effect against GD in female BALB/c mice. Evidence indicates that abnormal oxidative stress and immunosuppressive cytokines (TGF-ß, IL-35) play critical roles in the pathogenesis and development of GD. The purpose of this research is to measure these cytokines and oxidative stress markers to explore potential protective mechanisms of DHT in a BALB/c mouse model of GD. Methods: GD was induced in female BALB/c mice by intramuscular injection of an adenovirus expressing the A-subunit of the TSH receptor (Ad-TSHR289). DHT or a matching placebo was injected every 3 days. Mice were sacrificed four weeks after the third virus immunization to obtain blood, thyroid and spleen for further analysis. Results: Thyroid hormones were significantly reduced in DHT treated GD mice. In addition, DHT attenuated thyroid oxidative injuries in GD mice, as shown by decreased total antioxidation capability (TAOC), superoxide dismutase (SOD) and the level of malondialdehyde (MDA). The levels of immunosuppressive cytokines (TGF-ß, IL-35) in DHT group were significant higher compared with the GD group. Conclusions: The results demonstrated that DHT could reduce the severity of GD in female BALB/c mice by regulating oxidative stress. The upregulation of immunosuppressive cytokines might be another important protective mechanism.

A New Highly Thyrotropin Receptor-Selective Small-Molecule Antagonist with Potential for the Treatment of Graves' Orbitopathy.

BACKGROUND: The thyrotropin receptor (TSHR) is the target for autoimmune thyroid stimulating antibodies (TSAb) triggering hyperthyroidism. Whereas elevated thyroid hormone synthesis by the thyroid in Graves' disease can be treated by antithyroid agents, for the pathogenic activation of TSHR in retro-orbital fibroblasts of the eye, leading to Graves' orbitopathy (GO), no causal TSHR directed therapy is available. METHODS: Due to the therapeutic gap for severe GO, TSHR inhibitors were identified by high-throughput screening in Chinese hamster ovary cells expressing the TSHR. Stereo-selective synthesis of the screening hits led to the molecule S37, which contains seven chiral centers. Enantiomeric separation of the molecule S37 resulted in the enantiopure molecule S37a-a micro-molar antagonist of thyrotropin-induced cyclic adenosine monophosphate accumulation in HEK 293 cells expressing the TSHR. RESULTS: The unique rigid bent shape of molecule S37a may mediate the observed high TSHR selectivity. Most importantly, the closely related follitropin and lutropin receptors were not affected by this compound. S37a not only inhibits the TSHR activation by thyrotropin itself but also activation by monoclonal TSAb M22 (human), KSAb1 (murine), and the allosteric small-molecule agonist C2. Disease-related ex vivo studies in HEK 293 cells expressing the TSHR showed that S37a also inhibits cyclic adenosine monophosphate formation by oligoclonal TSAb, which are highly enriched in GO patients' sera. Initial in vivo pharmacokinetic studies revealed no toxicity of S37a and a remarkable 53% oral bioavailability in mice. CONCLUSION: In summary, a novel highly selective inhibitor for the TSHR is presented, which has promising potential for further development for the treatment of GO.

Aberrant Thyroid-Stimulating Hormone Receptor Signaling Increases VEGF-A and CXCL8 Secretion of Thyroid Cancer Cells, Contributing to Angiogenesis and Tumor Growth.

PURPOSE: Thyroid-stimulating hormone (TSH) suppression is widely used to treat well-differentiated thyroid cancer, whereas its role in poorly differentiated thyroid cancer (PDTC) is undetermined. Besides thyrocytes, TSH also binds to stromal cells, comprising tumor microenvironments. This study aimed to investigate the effects of TSH on tumor microenvironments in PDTC. EXPERIMENTAL DESIGN: An ectopic tumor model using PDTC cells (BHP10-3SCp and FRO), which exhibit TSH/cAMP-independent cell growth, was treated with TSH. IHC was performed using tissue microarrays from 13 PDTCs. RESULTS: TSH treatment significantly enhanced tumor growth of PDTCs with increased vascularity but not that of breast cancer cells, suggesting this effect is unique to thyroid cancer cells, not stromal cells. TSH significantly upregulated VEGF-A and CXCL8 expressions in BHP10-3SCp cells via AKT and ERK signaling, resulting in higher concentrations of VEGF-A and CXCL8 in conditioned medium of TSH-treated BHP10-3SCp cells (TSH-CM) compared with controls. TSH-CM treatment enhanced tube formation potentials of endothelial cells, and blocking VEGF and/or CXCL8 reduced them. Blocking VEGF and/or CXCL8 also reduced TSH-dependent tumor growth with reduced tumor vasculature in vivo. TSH-treated tumors showed increased macrophage densities, and macrophage inhibition reduced TSH-dependent tumor growth in vivo. In human PDTCs, preoperative TSH levels were positively associated with VEGF-A and tumor size, and the expression of VEGF-A was positively correlated with CD31, CD163, and CXCL8, and their clinical poor prognosis. CONCLUSIONS: Aberrant TSH receptor signaling modulates tumor angiogenesis by stimulating VEGF-A and CXCL8 secretion from PDTC cells and enhances tumor growth; thus, TSH suppression is beneficial for treating PDTCs.

Antigen-specific therapy of Graves´ disease and orbitopathy by induction of tolerance.

Graves´ disease is an autoimmune disorder, which is characterized by stimulatory antibodies targeting the human thyrotropin receptor (TSHR), resulting in hyperthyroidism and multiple organ damage. The disease can be modelled in mice using adenoviral immunizations with the extracellular A subunit of the TSHR, which induces a long-term stable disease state. TSHR binding cAMP-stimulatory antibodies, thyroid enlargement, elevated serum thyroxin levels, tachycardia, cardiac hypertrophy and orbitopathy are observed in these Ad-TSHR-immunized mice. T cell epitope-derived linear peptides have been identified using immunized HLA-DR3 transgenic mice, which may induce tolerance towards TSHR. A combination of such peptides are being investigated in a first clinical phase I trial in patients with Graves´ disease. Alternatively, intravenous administration of cyclic peptides derived from the interaction site of the TSHR A domain with stimulatory anti-TSHR antibodies can re-establish tolerance towards the antigen in immunized mice, improving symptoms of Graves´ disease within 3 - 4 months after starting these therapies. In immunologically naïve mice, administration of the cyclic peptides did not induce any immune response.

A Rapid CRISPR/Cas-based Mutagenesis Assay in Zebrafish for Identification of Genes Involved in Thyroid Morphogenesis and Function.

The foregut endoderm gives rise to several organs including liver, pancreas, lung and thyroid with important roles in human physiology. Understanding which genes and signalling pathways regulate their development is crucial for understanding developmental disorders as well as diseases in adulthood. We exploited unique advantages of the zebrafish model to develop a rapid and scalable CRISPR/Cas-based mutagenesis strategy aiming at the identification of genes involved in morphogenesis and function of the thyroid. Core elements of the mutagenesis assay comprise bi-allelic gene invalidation in somatic mutants, a non-invasive monitoring of thyroid development in live transgenic fish, complementary analyses of thyroid function in fixed specimens and quantitative analyses of mutagenesis efficiency by Illumina sequencing of individual fish. We successfully validated our mutagenesis-phenotyping strategy in experiments targeting genes with known functions in early thyroid morphogenesis (pax2a, nkx2.4b) and thyroid functional differentiation (duox, duoxa, tshr). We also demonstrate that duox and duoxa crispants phenocopy thyroid phenotypes previously observed in human patients with bi-allelic DUOX2 and DUOXA2 mutations. The proposed combination of efficient mutagenesis protocols, rapid non-invasive phenotyping and sensitive genotyping holds great potential to systematically characterize the function of larger candidate gene panels during thyroid development and is applicable to other organs and tissues.

Antithyroid Drugs Inactivate TSH Binding to the TSH Receptor by their Reducing Action.

Backgroud and Objective: Antithyroid drugs (ATDs) [methylmercaptoimidazole (MMI) and propylthiouracil (PTU) ] are used to treat hyperthyroidism in Graves' disease. The effect of ATDs and reducing agents (mercaptoethanol, dithiothreitol and cysteine) on bovine (b) TSH binding to human (h) and porcine (p) TSH receptor (R) was examined. METHODS AND RESULTS: (1) ATDs was pre-incubated with hTSHR coated tube for 1- 4 h, washed free of ATDs, and then 125I-bTSH binding to hTSHR after 1 h incubation was examined. MMI (10-40 mM) decreased 125I-bTSH binding in a dose-dependent manner and binding decreased proportionally as preincubation time increased from 1 to 4 h. PTU (10mM) slightly decreased binding, When reducing agents were pre-incubated with hTSHR for 2 h, 125I-bTSH binding similarly decreased. (2) Porcine thyroid membrane was pre-incubated with both agents for 2 h. Then, the washed or unwashed membrane was incubated with 125I-bTSH for 1 h. 125I-bTSH binding in both methods decreased. (3) When the effect of ATDs or reducing agents on the biological activity of 125I-bTSH and thyroid stimulating antibody (TSAb) was examined after gel-filtration of 125I-bTSH- and TSAb- treated with both reagents for 1 h, no inactivation was observed. (4) ATDs showed similar reducing action as reducing agents because iodine (I+) was reduced to I- by ATDs. CONCLUSION: ATDs inactivate the TSH-binding site of TSHR by reduction, although ATDs do not inactivate bTSH and TSAb activity. This suggests that TSAb would not stimulate the thyroid due to the inactivation of the TSHR when ATDs are administered to patients with Graves' disease.

Targeting the TSH receptor in thyroid cancer.

Recent advances in the arena of theranostics have necessitated a re-examining of previously established fields. The existing paradigm of therapeutic thyroid-stimulating hormone receptor (TSHR) targeting in the post-surgical management of differentiated thyroid cancer using levothyroxine and recombinant human thyroid-stimulating hormone (TSH) is well understood. However, in an era of personalized medicine, and with an increasing awareness of the risk profile of longstanding pharmacological hyperthyroidism, it is imperative clinicians understand the molecular basis and magnitude of benefit for individual patients. Furthermore, TSHR has been recently re-conceived as a selective target for residual metastatic thyroid cancer, with pilot data demonstrating effective targeting of nanoparticles to thyroid cancers using this receptor as a target. This review examines the evidence for TSHR signaling as an oncogenic pathway and assesses the evidence for ongoing TSHR expression in thyroid cancer metastases. Priorities for further research are highlighted.

Underlying Mechanisms of Pituitary-Thyroid Axis Function Disruption by Chronic Iodine Excess in Rats.

BACKGROUND: Iodine is essential for thyroid hormone synthesis and is an important regulator of thyroid function. Chronic iodine deficiency leads to hypothyroidism, but iodine excess also impairs thyroid function causing hyperthyroidism, hypothyroidism, and/or thyroiditis. This study aimed to investigate the underlying mechanisms by which exposure to chronic iodine excess impairs pituitary-thyroid axis function. METHODS: Male Wistar rats were treated for two months with NaI (0.05% and 0.005%) or NaI+NaClO4 (0.05%) dissolved in drinking water. Hormone levels, gene expression, and thyroid morphology were analyzed later. RESULTS: NaI-treated rats presented high levels of iodine in urine, increased serum thyrotropin levels, slightly decreased serum thyroxine/triiodothyronine levels, and a decreased expression of the sodium-iodide symporter, thyrotropin receptor, and thyroperoxidase mRNA and protein, suggesting a primary thyroid dysfunction. In contrast, thyroglobulin and pendrin mRNA and protein content were increased. Kidney and liver deiodinase type 1 mRNA expression was decreased in iodine-treated rats. Morphological studies showed larger thyroid follicles with higher amounts of colloid and increased amounts of connective tissue in the thyroid of iodine-treated animals. All these effects were prevented when perchlorate treatment was combined with iodine excess. CONCLUSIONS: The present data reinforce and add novel findings about the disruption of thyroid gland function and the compensatory action of increased thyrotropin levels in iodine-exposed animals. Moreover, they draw attention to the fact that iodine intake should be carefully monitored, since both deficient and excessive ingestion of this trace element may induce pituitary-thyroid axis dysfunction.

Clinical Value of Thyrotropin Receptor Antibodies for the Differential Diagnosis of Interferon Induced Thyroiditis.

OBJECTIVE: The clinical value of thyrotropin receptor antibodies for the differential diagnosis of thyrotoxicosis induced by pegylated interferon-alpha remains unknown. We analyzed the diagnostic accuracy of thyrotropin receptor antibodies in the differential diagnosis of thyrotoxicosis in patients with chronic hepatitis C (CHC) receiving pegylated interferon-alpha plus ribavirin. METHODS: Retrospective analysis of 274 patients with CHC receiving pegylated interferon-alpha plus ribavirin. Interferon-induced thyrotoxicosis was classified according to clinical guidelines as Graves disease, autoimmune and non- autoimmune destructive thyroiditis. RESULTS: 48 (17.5%) patients developed hypothyroidism, 17 (6.2%) thyrotoxicosis (6 non- autoimmune destructive thyroiditis, 8 autoimmune destructive thyroiditis and 3 Graves disease) and 22 "de novo" thyrotropin receptor antibodies (all Graves disease, 2 of the 8 autoimmune destructive thyroiditis and 17 with normal thyroid function). The sensitivity and specificity of thyrotropin receptor antibodies for Graves disease diagnosis in patients with thyrotoxicosis were 100 and 85%, respectively. Patients with destructive thyroiditis developed hypothyroidism in 87.5% of autoimmune cases and in none of those with a non- autoimmune etiology (p<0.001). CONCLUSION: Thyrotropin receptor antibodies determination cannot replace thyroid scintigraphy for the differential diagnosis of thyrotoxicosis in CHC patients treated with pegylated interferon.