Thyroid hormone receptors (TRs, nomenclature as agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors [13, 2]) are nuclear hormone receptors of the NR1A family, with diverse roles regulating macronutrient metabolism, cognition and cardiovascular homeostasis. TRs are activated by thyroxine (T4) and thyroid hormone (triiodothyronine). Once activated by a ligand, the receptor acts as a transcription factor either as a monomer, homodimer or heterodimer with members of the retinoid X receptor family. NH-3 has been described as an antagonist at TRs with modest selectivity for TRβ [43].

The NR superfamily comprises 48 transcription factors in humans that control a plethora of gene network programs involved in a wide range of physiologic processes. This review will summarize and discuss recent progress in NR biology and drug development derived from integrating various approaches, including biophysical techniques, structural studies, and translational investigation. We also highlight how defective NR signaling results in various diseases and disorders and how NRs can be targeted for therapeutic intervention via modulation via binding to synthetic lipophilic ligands. Furthermore, we also review recent studies that improved our understanding of NR structure and signaling. SIGNIFICANCE STATEMENT: Nuclear receptors (NRs) are ligand-regulated transcription factors that are critical regulators of myriad physiological processes. NRs serve as receptors for an array of drugs, and in this review, we provide an update on recent research into the roles of these drug targets.

Thyroid hormone receptors (TRs, nomenclature as agreed by the NC-IUPHAR Subcommittee on Nuclear Hormone Receptors [12, 2]) are nuclear hormone receptors of the NR1A family, with diverse roles regulating macronutrient metabolism, cognition and cardiovascular homeostasis. TRs are activated by thyroxine (T4) and thyroid hormone (triiodothyronine). Once activated by a ligand, the receptor acts as a transcription factor either as a monomer, homodimer or heterodimer with members of the retinoid X receptor family. NH-3 has been described as an antagonist at TRs with modest selectivity for TRβ [42].

Thyroid hormone receptors (TRs) are responsible for mediating thyroid hormone (T3 and T4) actions at a cellular level. They belong to the nuclear receptor (NR) superfamily and execute their main functions inside the cell nuclei as hormone-regulated transcription factors. These receptors also exhibit so-called “non-classic” actions, for which other cellular proteins, apart from coregulators inside nuclei, regulate their activity. Aiming to find alternative pathways of TR modulation, we searched for interacting proteins and found that PDIA1 interacts with TRβ in a yeast two-hybrid screening assay. The functional implications of PDIA1—TR interactions are still unclear; however, our co-immunoprecipitation (co-IP) and fluorescence assay results showed that PDI was able to bind both TR isoforms in vitro. Moreover, T3 appears to have no important role in these interactions in cellular assays, where PDIA1 was able to regulate transcription of TRα and TRβ-mediated genes in different ways depending on the promoter region and on the TR isoform involved. Although PDIA1 appears to act as a coregulator, it binds to a TR surface that does not interfere with coactivator binding. However, the TR:PDIA1 complex affinity and activation are different depending on the TR isoform. Such differences may reflect the structural organization of the PDIA1:TR complex, as shown by models depicting an interaction interface with exposed cysteines from both proteins, suggesting that PDIA1 might modulate TR by its thiol reductase/isomerase activity.

New approaches to treat ovarian cancer, the fifth leading cause of cancer mortality among women, are being sought, with the targeting of epigenetic modulators now receiving much attention. The histone acetyltransferase HBO1 functions in regulating diverse molecular processes, including DNA repair, transcription and replication, and is highly expressed in primary ovarian cancer. Here we define both the molecular function and a role in cell biomechanics for HBO1 in ovarian cancer. HBO1 preferentially acetylates histone H4 through the concomitant overexpression of co-regulator JADE2, and is required for the expression of YAP1, an ovarian cancer oncogene and mechano-transductor signaling factor. HBO1 appears therefore to have a role in determining the mechano-phenotype in ovarian cancer cells, through both signal transduction processes, and the modulation of cell elasticity as observed using direct measurements on live cells via atomic force microscopy.

Abstract Epithelial ovarian cancer (OC) is now the fifth leading cause of cancer death among women in the U.S. and the leading cause of death in gynecological cancers. OC comprises a variety of tumor types (histotypes) that differ in morphology, prognosis, etiology and molecular biology. Despite the knowledge of OC heterogeneity, current treatment strategies are very similar, and unfortunately, very ineffectual. New approaches, to inhibit OC growth and progression are therefore being sought, with few obvious targets for directed therapy. Currently, large–scale gene expression, DNA copy number and mutational screens have not been able to pinpoint new high frequency drugable targets. Epigenetic therapy conceives a whole genome approach, potentially circumventing the reliance on therapies that target infrequently mutated genes. Histone acetyl–transferases like GCN5 and PCAF have been targeted in cancer previously, and the main purpose of this study is to assess the role of one such acetyl–transferase, Histone acetyl–transferase binding to origin of replication complex 1” (HBO1, also known as KAT7 and MYST2) in the progression of OC. HBO1 is a histone acetyltransferase (HAT) that plays important roles in diverse molecular processes including DNA replication, transcription and DNA repair. Importantly, HBO1 is overexpressed in primary cancers (breast, testis, ovarian), although the molecular basis of its role is still unclear. To better understand HBO1's role in OC, we characterized its expression in well–defined OC cell lines both at the mRNA and protein level. Observed in vitro patterns of HBO1 expression correlate well with previous observations; HBO1 is overexpressed in several OC cell lines. Interestingly, the serous histotype generally shows the highest expression. Thus far, we have now analyzed HBO1's downstream regulatory pathways in several OC cell lines through use of siRNA knock–down coupled to microarray analysis. Comparisons of the various datasets have revealed pathways common to all cell lines studied, as well as distinction. Our approach aims to identify known epigenomic signatures that may be associated with HBO1 binding and downstream signaling which may drive differentiation toward the most advanced stages of the disease. Translating this new information through epigenetic targeting and structural inhibition may lead to novel, next–generation therapies for this most complex of disease presentations. Citation Format: M.Quintela, L.Francis, D.H.Sieglaff, P.Webb, S.Conlan. THE IMPACT OF HBO1 IN OVARIAN CANCER [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr MIP-070.

Resistance to clinical antiandrogens has plagued the evolution of effective therapeutics for advanced prostate cancer. As with the first-line therapeutic bicalutamide (Casodex), resistance to newer antiandrogens (enzalutamide, ARN-509) develops quickly in patients, despite the fact that these drugs have ∼10-fold better affinity for the androgen receptor than bicalutamide. Improving affinity alone is often not sufficient to prevent resistance, and alternative strategies are needed to improve antiandrogen efficacy. Covalent and reversible covalent drugs are being used to thwart drug resistance in other contexts, and activated aryl nitriles are among the moieties being exploited for this purpose. We capitalized on the presence of an aryl nitrile in bicalutamide, and the existence of a native cysteine residue (Cys784) in the androgen receptor ligand binding pocket, to develop 5N-bicalutamide, a cysteine-reactive antiandrogen. 5N-bicalutamide exhibits a 150-fold improvement in Ki and 20-fold improvement in IC50 over the parent compound. We attribute the marked improvement in affinity and activity to the formation of a covalent adduct with Cys784, a residue that is not among the more than 160 androgen receptor point mutations associated with prostate cancer. Increasing the residence time of bound antiandrogen via formation of a covalent adduct may forestall the drug resistance seen with current clinical antiandrogens.

BACKGROUND:Upregulation of estrogen receptor beta (ERβ) in breast cancer cells is associated with epithelial maintenance, decreased proliferation and invasion, and a reduction in the expression of the receptor has been observed in invasive breast tumors. However, proof of an association between loss of ERβ and breast carcinogenesis is still missing.METHODS:To study the role of ERβ in breast oncogenesis, we generated mouse conditional mutants with specific inactivation of ERβ and p53 in the mammary gland epithelium. For epithelium-specific knockout of ERβ and p53, ERβ F/F and p53 F/F mice were crossed to transgenic mice that express the Cre recombinase under the control of the human keratin 14 promoter.RESULTS:Somatic loss of ERβ significantly accelerated formation of p53-deficient mammary tumors. Loss of the receptor also resulted in the development of less differentiated carcinomas with stronger spindle cell morphology and decreased expression of luminal epithelial markers.CONCLUSIONS:Our results show that synergism between ERβ and p53 inactivation functions to determine important aspects of breast oncogenesis and cancer progression.

AIM:Thyroid hormones regulate metabolic response. While triiodothyronine (T3) is usually considered to be the active form of thyroid hormone, one form of diiodothyronine (3,5-T2) exerts T3-like effects on energy consumption and lipid metabolism. 3,5-T2 also improves glucose tolerance in rats and 3,5-T2 levels correlate with fasting glucose in humans. Presently, however, little is known about mechanisms of 3,5-T2 effects on glucose metabolism. Here, we set out to compare effects of T3, 3,5-T2 and another form of T2 (3,3-T2) in a mouse model of diet-induced obesity and determined effects of T3 and 3,5-T2 on markers of classical insulin sensitization to understand how diiodothyronines influence blood glucose.METHODS:Cell- and protein-based assays of thyroid hormone action. Assays of metabolic parameters in mice. Analysis of transcript and protein levels in different tissues by qRT-PCR and Western blot.RESULTS:T3 and 3,5-T2 both reduce body weight, adiposity and body temperature despite increased food intake. 3,3'-T2 lacks these effects. T3 and 3,5-T2 reduce blood glucose levels, whereas 3,3'-T2 worsens glucose tolerance. Neither T3 nor 3,5-T2 affects markers of insulin sensitization in skeletal muscle or white adipose tissue (WAT), but both reduce hepatic GLUT2 glucose transporter levels and glucose output. T3 and 3,5-T2 also induce expression of mitochondrial uncoupling proteins (UCPs) 3 and 1 in skeletal muscle and WAT respectively.CONCLUSIONS:3,5-T2 influences glucose metabolism in a manner that is distinct from insulin sensitization and involves reductions in hepatic glucose output and changes in energy utilization.

TLX (tailless receptor) is a member of the nuclear receptor superfamily and belongs to a class of nuclear receptors for which no endogenous or synthetic ligands have yet been identified. TLX is a promising therapeutic target in neurological disorders and brain tumors. Thus, regulatory ligands for TLX need to be identified to complete the validation of TLX as a useful target and would serve as chemical probes to pursue the study of this receptor in disease models. It has recently been proved that TLX is druggable. However, to identify potent and specific TLX ligands with desirable biological activity, a deeper understanding of where ligands bind, how they alter TLX conformation and of the mechanism by which TLX mediates the transcription of its target genes is needed. While TLX is in the process of escaping from orphanhood, future ligand design needs to progress in parallel with improved understanding of (i) the binding cavity or surfaces to target with small molecules on the TLX ligand binding domain and (ii) the nature of the TLX coregulators in particular cell and disease contexts. Both of these topics are discussed in this review.

This Special Issue on the topic of "Orphan Nuclear Receptors" should help to cement the long held view that orphan members of the Nuclear Receptor superfamily play crucial roles in development, physiology and multiple pathologies and that some are attractive druggable targets. Focusing on selected orphans, this issue highlights recent developments in orphan receptor action and addresses questions about function, ligand recognition, strategies for drug development and applications for such drugs. (C) 2016 Elsevier Ltd. All rights reserved.

Thyroid hormone (TH) receptors (TRs α and β) are homologous ligand-dependent transcription factors (TFs). While the TRs display distinct actions in development, metabolic regulation and other processes, comparisons of TRα and TRβ dependent gene regulation mostly reveal similar mechanisms of action and few TR subtype specific genes. Here, we show that TRα predominates in multipotent human adipose derived stem cells (hADSC) whereas TRβ is expressed at lower levels and is upregulated during hADSC differentiation. The TRs display several unusual properties in parental hADSC. First, TRs display predominantly cytoplasmic intracellular distribution and major TRα variants TRα1 and TRα2 colocalize with mitochondria. Second, knockdown experiments reveal that endogenous TRs influence hADSC cell morphology and expression of hundreds of genes in the absence of hormone, but do not respond to exogenous TH. Third, TRα and TRβ affect hADSC in completely distinct ways; TRα regulates cell cycle associated processes while TRβ may repress aspects of differentiation. TRα splice variant specific knockdown reveals that TRα1 and TRα2 both contribute to TRα-dependent gene expression in a gene specific manner. We propose that TRs work in a non-canonical and hormone independent manner in hADSC and that prominent subtype-specific activities emerge in the context of these unusual actions.

We report the pharmacophore of the peroxisome proliferator-activated receptor δ (PPARδ) agonist natural product phosphoiodyn A is the phosphonate core. Synthesis of simplified phosphonate esters 13 and 15 provide structurally novel, highly selective and potent PPARδ agonists (EC50=78 and 112nM, respectively). Further, both compounds demonstrate significant neuroprotective activity in an in vitro cellular model indicating that phosphonates may be an effective novel scaffold for the design of therapeutics for the treatment of neurodegenerative disorders.

Background/Objectives: Our objective was to assess the sustained, low-dose and constant administration of the thyroid receptor-β (TRβ)-selective agonist GC-1 (sobetirome) from a novel nanochannel membrane device (NMD) for drug delivery. As it known to speed up metabolism, accomplish weight loss, improve cholesterol levels and possess anti-diabetic effects, GC-1 was steadily administered by our NMD, consisting of an implantable nanochannel membrane, as an alternative to conventional daily administration, which is subject to compliance issues in clinical settings. Subjects/Methods: Diet-induced obese C57BL/J6 male mice were fed a very high-fat diet (VHFD) and received NMD implants subcutaneously. Ten mice per group received capsules containing GC-1 or phosphate-buffered saline (control). Weight, lean and fat mass, as well as cholesterol, triglycerides, insulin and glucose, were monitored for 24 days. After treatment, plasma levels of thyroid-stimulating hormone (TSH) and thyroxine were compared. mRNA levels of a panel of thermogenic markers were examined using real-time PCR in white adipose tissue (WAT) and brown adipose tissue (BAT). Adipose tissue, liver and local inflammatory response to the implant were examined histologically. Pancreatic islet number and β-cell area were assessed. Results: GC-1 released from the NMD reversed VHFD-induced obesity and normalized serum cholesterol and glycemia. Significant reductions in body weight and fat mass were observed within 10 days, whereas reductions in serum cholesterol and glucose levels were seen within 7 days. The significant decrease in TSH was consistent with TRβ selectivity for GC-1. Levels of transcript for Ucp1 and thermogenic genes PGC1a , Cidea , Dio2 and Cox5a showed significant upregulation in WAT in NMD–GC-1-treated mice, but decreased in BAT. Although mice treated by NMD–GC-1 showed a similar number of pancreatic islets, they exhibited significant increase in β-cell area. Conclusions: Our data demonstrate that the NMD implant achieves steady administration of GC-1, offering an effective and tightly controlled molecular delivery system for treatment of obesity and metabolic disease, thereby addressing compliance.

Aim: Thyroid hormones regulate metabolic response. While triiodothyronine (T3) is usually considered to be the active form of thyroid hormone, one form of diiodothyronine (3,5-T2) exerts T3-like effects on energy consumption and lipid metabolism. 3,5-T2 also improves glucose tolerance in rats and 3,5-T2 levels correlate with fasting glucose in humans. Presently, however, little is known about mechanisms of 3,5-T2 effects on glucose metabolism. Here, we set out to compare effects of T3, 3,5-T2 and another form of T2 (3,3-T2) in a mouse model of diet induced obesity and determined effects of T3 and 3,5-T2 on markers of classical insulin sensitization to understand how diiodothyronines influence blood glucose. Methods: Cell and protein based assays of thyroid hormone action. Assays of metabolic parameters in mice. Analysis of transcript and protein levels in different tissues by qRT-PCR and western blot. Results: T3 and 3,5-T2 both reduce body weight, adiposity and body temperature despite increased food intake. 3,3’-T2 lacks these effects. T3 and 3,5-T2 reduce blood glucose levels whereas 3,3’-T2 worsens glucose tolerance. Neither T3 or 3,5-T2 affects markers of insulin sensitization in skeletal muscle or white adipose tissue (WAT) but both reduce hepatic GLUT2 glucose transporter levels and glucose output. T3 and 3,5-T2 also induce expression of mitochondrial uncoupling proteins (UCPs) 3 and 1 in skeletal muscle and WAT, respectively. A cc ep te d A rt ic le This article is protected by copyright. All rights reserved. Conclusions: 3,5-T2 influences glucose metabolism in a manner that is distinct from insulin sensitization and involves reductions in hepatic glucose output and changes in energy utilization.

Loss-of-function mutation V290M in the ligand-binding domain of the peroxisome proliferator activated receptor γ (PPARγ) is associated with a ligand resistance syndrome (PLRS), characterized by partial lipodystrophy and severe insulin resistance. In this data article we discuss an X-ray diffraction dataset that yielded the structure of PPARγ LBD V290M mutant refined at 2.3 Å resolution, that allowed building of 3D model of the receptor mutant with high confidence and revealed continuous well-defined electron density for the partial agonist diclofenac bound to hydrophobic pocket of the PPARγ. These structural data provide significant insights into molecular basis of PLRS caused by V290M mutation and are correlated with the receptor disability of rosiglitazone binding and increased affinity for corepressors. Furthermore, our structural evidence helps to explain clinical observations which point out to a failure to restore receptor function by the treatment with a full agonist of PPARγ, rosiglitazone.

Sobetirome binds selectively to the main hepatic form of thyroid hormone (TH) receptor, TR beta 1, compared to TR alpha 1, which is principally responsible for thyrotoxic effects on heart, muscle and bone. Sobetirome also preferentially accumulates in liver. It was originally envisaged that sobetirome could be used to stimulate hepatic pathways that lower cholesterol without harmful side effects and might be used in conjunction with statins. Indeed, sobetirome progressed through preclinical animal studies and Phase I human clinical trials with excellent results and without obvious harmful side effects. Despite the fact that cardiovascular disease remains a major cause of mortality and that new therapies are desperately needed, it is unlikely that sobetirome will progress in further human clinical trials in the near future. The emergence of alternative cholesterol-lowering therapeutics may render selective thyromimetics redundant. Further, fears of thyrotoxic effects in the heart and emergence of cartilage defects in dogs after long-term use of eprotirome, a similar though not identical compound, has reduced enthusiasm for this strategy. We argue that it is nevertheless important to explore uses of sobetirome in humans; more treatment strategies would help patients with hard-to-treat dyslipidemias. Sobetirome may also have additional applications in orphan indications and short-term controlled weight loss.

Significance This work addresses a key question in the field of liver receptor homolog-1 (LRH-1) pathophysiology in colorectal cancer (CRC)—namely, does LRH-1 contribute exclusively to tumorigenesis, or does LRH-1 also drive established CRC tumor growth? These two models have widely different implications for pharmaceutical targeting in CRC. To our knowledge, our work is the first to demonstrate that silencing of LRH-1 in established human CRC cell lines impairs proliferation though G0/G1 phase prolongation. Our microarray gene expression analysis shows that loss of LRH-1 expression yields alterations in diverse cellular pathways consistent with the critical role of LRH-1 in CRC. Taken together, our study suggests that a subset of CRC patients could benefit from selective antagonism of LRH-1.

The estrogen receptor (ER) β variant ERβ2 is expressed in aggressive castration-resistant prostate cancer and has been shown to correlate with decreased overall survival. Genome-wide expression analysis after ERβ2 expression in prostate cancer cells revealed that hypoxia was an overrepresented theme. Here we show that ERβ2 interacts with and stabilizes HIF-1α protein in normoxia, thereby inducing a hypoxic gene expression signature. HIF-1α is known to stimulate metastasis by increasing expression of Twist1 and increasing vascularization by directly activating VEGF expression. We found that ERβ2 interacts with HIF-1α and piggybacks to the HIF-1α response element present on the proximal Twist1 and VEGF promoters. These findings suggest that at least part of the oncogenic effects of ERβ2 is mediated by HIF-1α and that targeting of this ERβ2 - HIF-1α interaction may be a strategy to treat prostate cancer.