The Concise Guide to PHARMACOLOGY 2023/24: Nuclear hormone receptors.

The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and nearly 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (https://www.guidetopharmacology.org/), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.16179. Nuclear hormone receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

International Union of Basic and Clinical Pharmacology CXIII: Nuclear Receptor Superfamily-Update 2023.

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.

THE CONCISE GUIDE TO PHARMACOLOGY 2021/22: Nuclear hormone receptors.

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15540. Nuclear hormone receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

A mutant form of ERα associated with estrogen insensitivity affects the coupling between ligand binding and coactivator recruitment.

A homozygous missense mutation in the gene encoding the estrogen receptor α (ERα) was previously identified in a female patient with estrogen insensitivity syndrome. We investigated the molecular features underlying the impaired transcriptional response of this mutant (ERα-Q375H) and four other missense mutations at this position designed to query alternative mechanisms. The identity of residue 375 greatly affected the sensitivity of the receptor to agonists without changing the ligand binding affinity. Instead, the mutations caused changes in the affinity of coactivator binding and alterations in the balance of coactivator and corepressor recruitment. Comparisons among the transcriptional regulatory responses of these six ERα genotypes to a set of ER agonists showed that both steric and electrostatic factors contributed to the functional deficits in gene regulatory activity of the mutant ERα proteins. ERα-coregulator peptide binding in vitro and RIME (rapid immunoprecipitation mass spectrometry of endogenous) analysis in cells showed that the degree of functional impairment paralleled changes in receptor-coregulator binding interactions. These findings uncover coupling between ligand binding and coregulator recruitment that affects the potency rather than the efficacy of the receptor response without substantially altering ligand binding affinity. This highlights a molecular mechanism for estrogen insensitivity syndrome involving mutations that perturb a bidirectional allosteric coupling between ligand binding and coregulator binding that determines receptor transcriptional output.

Long-Term Follow-Up and Treatment of a Female With Complete Estrogen Insensitivity.

CONTEXT: We previously reported the first female with a causative ESR1 gene variant, who exhibited absent puberty and high estrogens. At age 15 years, she presented with lower abdominal pain, absent breast development, primary amenorrhea, and multicystic ovaries. The natural history of complete estrogen insensitivity (CEI) in women is unknown. OBJECTIVE: The purpose of this report is to present the neuroendocrine phenotype of CEI, identify potential ligands, and determine the effect of targeted treatment. DESIGN: We have characterized gonadotropin pulsatility and followed this patient's endocrine profile and bone density over 8 years. Seventy-five different compounds were tested for transactivation of the variant receptor. A personalized medicine approach was tailored to our patient. SETTING: Academic medical center. PATIENT OR OTHER PARTICIPANTS: A 24-year-old adopted white female with CEI. INTERVENTION(S): The patient was treated with diethylstilbestrol (DES) for approximately 2.5 years. MAIN OUTCOME MEASURE(S): Induction of secondary sexual characteristics. RESULTS: Luteinizing hormone (LH) pulse studies demonstrated normal pulsatile LH secretion, elevated mean LH, and mildly elevated mean follicle-stimulating hormone (FSH) in the presence of markedly increased estrogens. DES transactivated the variant ESR1 in vitro. However, DES treatment did not induce secondary sexual characteristics in our patient. CONCLUSIONS: Treatment with DES was not successful in our patient. She remains hypoestrogenic despite the presence of ovarian cysts with a hypoestrogenic vaginal smear, absent breast development, and low bone mineral mass. Findings suggest additional receptor mechanistic actions are required to elicit clinical hormone responses.

Lavender Products Associated With Premature Thelarche and Prepubertal Gynecomastia: Case Reports and Endocrine-Disrupting Chemical Activities.

CONTEXT: Previous case reports associated prepubertal gynecomastia with lavender-containing fragrances, but there appear to be no reports of premature thelarche. OBJECTIVE: To add to a case series about lavender-fragranced product use and breast growth in children and to measure endocrine-disrupting chemical activity of essential oil components. DESIGN, SETTING, AND PATIENTS: Patients experiencing premature thelarche or prepubertal gynecomastia with continuous exposure to lavender-fragranced products were evaluated in the pediatric endocrinology departments of two institutions. Mechanistic in vitro experiments using eight components of lavender and other essential oils were performed at National Institute of Environmental Health Sciences. MAIN OUTCOME MEASURES: Case reports and in vitro estrogen and androgen receptor gene expression activities in human cell lines with essential oils. RESULTS: Three prepubertal girls and one boy with clinical evidence of estrogenic action and a history of continuous exposure to lavender-containing fragrances were studied. Breast growth dissipated in all patients with discontinuation of the fragranced products. Some of the components tested elicited estrogenic and antiandrogenic properties of varying degrees. CONCLUSION: We report cases of premature thelarche that resolved upon cessation of lavender-containing fragrance exposure commonly used in Hispanic communities. The precise developmental basis for such conditions could be multifactorial. In vitro demonstration of estrogenic and antiandrogenic properties of essential oil components suggests essential oils in these cases could be considered a possible source and supports a possible link with idiopathic prepubertal breast development. Whether the level of lavender oil estrogenic potency is sufficient to cause these effects is unknown.

Decoding the Inversion Symmetry Underlying Transcription Factor DNA-Binding Specificity and Functionality in the Genome.

Understanding why a transcription factor (TF) binds to a specific DNA element in the genome and whether that binding event affects transcriptional output remains a great challenge. In this study, we demonstrate that TF binding in the genome follows inversion symmetry (IS). In addition, the specific DNA elements where TFs bind in the genome are determined by internal IS within the DNA element. These DNA-binding rules quantitatively define how TFs select the appropriate regulatory targets from a large number of similar DNA elements in the genome to elicit specific transcriptional and cellular responses. Importantly, we also demonstrate that these DNA-binding rules extend to DNA elements that do not support transcriptional activity. That is, the DNA-binding rules are obeyed, but the retention time of the TF at these non-functional DNA elements is not long enough to initiate and/or maintain transcription. We further demonstrate that IS is universal within the genome. Thus, IS is the DNA code that TFs use to interact with the genome and dictates (in conjunction with known DNA sequence constraints) which of those interactions are functionally active.

Activation of hepatic estrogen receptor-α increases energy expenditure by stimulating the production of fibroblast growth factor 21 in female mice.

OBJECTIVE: The endogenous estrogen 17ß-estradiol (E2) promotes metabolic homeostasis in premenopausal women. In a mouse model of post-menopausal metabolic syndrome, we reported that estrogens increased energy expenditure, thus preventing estrogen deficiency-induced adiposity. Estrogens' prevention of fat accumulation was associated with increased serum concentrations of fibroblast growth factor 21 (FGF21), suggesting that FGF21 participates in estrogens' promotion of energy expenditure. METHODS: We studied the effect of E2 on FGF21 production and the role of FGF21 in E2 stimulation of energy expenditure and prevention of adiposity, using female estrogen receptor (ER)- and FGF21-deficient mice fed a normal chow and a cohort of ovariectomized women from the French E3N prospective cohort study. RESULTS: E2 acting on the hepatocyte ERα increases hepatic expression and production of FGF21 in female mice. In vivo activation of ERα increases the transcription of Fgf21 via an estrogen response element outside the promoter of Fgf21. Treatment with E2 increases oxygen consumption and energy expenditure and prevents whole body fat accumulation in ovariectomized female WT mice. The effect of E2 on energy expenditure is not observed in FGF21-deficient mice. While E2 treatment still prevents fat accumulation in FGF21-deficient mice, this effect is decreased compared to WT mice. In an observational cohort of ovariectomized women, E2 treatment was associated with lower serum FGF21 concentrations, which may reflect a healthier metabolic profile. CONCLUSIONS: In female mice, E2 action on the hepatocyte ERα increases Fgf21 transcription and FGF21 production, thus promoting energy expenditure and partially decreasing fat accumulation.

Estrogens Promote Misfolded Proinsulin Degradation to Protect Insulin Production and Delay Diabetes.

Conjugated estrogens (CE) delay the onset of type 2 diabetes (T2D) in postmenopausal women, but the mechanism is unclear. In T2D, the endoplasmic reticulum (ER) fails to promote proinsulin folding and, in failing to do so, promotes ER stress and ß cell dysfunction. We show that CE prevent insulin-deficient diabetes in male and in female Akita mice using a model of misfolded proinsulin. CE stabilize the ER-associated protein degradation (ERAD) system and promote misfolded proinsulin proteasomal degradation. This involves activation of nuclear and membrane estrogen receptor-α (ERα), promoting transcriptional repression and proteasomal degradation of the ubiquitin-conjugating enzyme and ERAD degrader, UBC6e. The selective ERα modulator bazedoxifene mimics CE protection of ß cells in females but not in males.

DNA methylation and transcriptome aberrations mediated by ERα in mouse seminal vesicles following developmental DES exposure.

Early transient developmental exposure to an endocrine active compound, diethylstilbestrol (DES), a synthetic estrogen, causes late-stage effects in the reproductive tract of adult mice. Estrogen receptor alpha (ERα) plays a role in mediating these developmental effects. However, the developmental mechanism is not well known in male tissues. Here, we present genome-wide transcriptome and DNA methylation profiling of the seminal vesicles (SVs) during normal development and after DES exposure. ERα mediates aberrations of the mRNA transcriptome in SVs of adult mice following neonatal DES exposure. This developmental exposure impacts differential diseases between male (SVs) and female (uterus) tissues when mice reach adulthood due to most DES-altered genes that appear to be tissue specific during mouse development. Certain estrogen-responsive gene changes in SVs are cell-type specific. DNA methylation dynamically changes during development in the SVs of wild-type (WT) and ERα-knockout (αERKO) mice, which increases both the loss and gain of differentially methylated regions (DMRs). There are more gains of DMRs in αERKO compared with WT. Interestingly, the methylation changes between the two genotypes are in different genomic loci. Additionally, the expression levels of a subset of DES-altered genes are associated with their DNA methylation status following developmental DES exposure. Taken together, these findings provide an important basis for understanding the molecular and cellular mechanism of endocrine-disrupting chemicals (EDCs), such as DES, during development in the male mouse tissues. This unique evidence contributes to our understanding of developmental actions of EDCs in human health.

Differential in Vitro Biological Action, Coregulator Interactions, and Molecular Dynamic Analysis of Bisphenol A (BPA), BPAF, and BPS Ligand-ERα Complexes.

BACKGROUND: Bisphenol A (BPA) is an endocrine-disrupting chemical (EDC) that might be harmful to human health. Recently, there has been widespread usage of bisphenol chemicals (BPs), such as bisphenol AF (BPAF) and bisphenol S (BPS), as replacements for BPA. However, the potential biological actions, toxicity, and the molecular mechanism of these compounds are still poorly understood. OBJECTIVES: Our objective was to examine the estrogenic effects of BPA, BPAF, and BPS and the molecular mechanisms of action in the estrogen receptor alpha (ERα) complex. METHODS: In vitro cell models were used to compare the estrogenic effects of BPA, BPAF, and BPS to estrogen. Microarray Assay for Real-Time Coregulator-Nuclear receptor Interaction (MARCoNI) analysis was used to identify coregulators of BPA, BPAF, and BPS, and molecular dynamic (MD) simulations were used to determine the compounds binding in the ERα complex. RESULTS: We demonstrated that BPA and BPAF have agonistic activity for both ERα and ERß, but BPS has ERα-selective specificity. We concluded that coregulators were differentially recruited in the presence of BPA, BPAF, or BPS. Interestingly, BPS recruited more corepressors when compared to BPA and BPAF. From a series of MD analysis, we concluded that BPA, BPAF, and BPS can bind to the ER-ligand-binding domain with differing energetics and conformations. In addition, the binding surface of coregulator interactions on ERα was characterized for the BPA, BPAF, and BPS complexes. CONCLUSION: These findings further our understanding of the molecular mechanisms of EDCs, such as BPs, in ER-mediated transcriptional activation, biological activity, and their effects on physiological functions in human health. https://doi.org/10.1289/EHP2505.

DNA Sequence Constraints Define Functionally Active Steroid Nuclear Receptor Binding Sites in Chromatin.

Gene regulatory programs are encoded in the sequence of the DNA. Since the completion of the Human Genome Project, millions of gene regulatory elements have been identified in the human genome. Understanding how each of those sites functionally contributes to gene regulation, however, remains a challenge for nearly every field of biology. Transcription factors influence cell function by interpreting information contained within cis-regulatory elements in chromatin. Whereas chromatin immunoprecipitation-sequencing has been used to identify and map transcription factor-DNA interactions, it has been difficult to assign functionality to the binding sites identified. Thus, in this study, we probed the transcriptional activity, DNA-binding competence, and functional activity of select nuclear receptor mutants in cellular and animal model systems and used this information to define the sequence constraints of functional steroid nuclear receptor cis-regulatory elements. Analysis of the architecture within sNR chromatin interacting sites revealed that only a small fraction of all sNR chromatin-interacting events is associated with transcriptional output and that this functionality is restricted to elements that vary from the consensus palindromic elements by one or two nucleotides. These findings define the transcriptional grammar necessary to predict functionality from regulatory sequences, with a multitude of future implications.

Binding of bisphenol A, bisphenol AF, and bisphenol S on the androgen receptor: Coregulator recruitment and stimulation of potential interaction sites.

Bisphenol A (BPA), bisphenol AF (BPAF), and bisphenol S (BPS) are well known endocrine disruptors. Previous in vitro studies showed that these compounds antagonize androgen receptor (AR) transcriptional activity; however, the mechanisms of action are unclear. In the present study, we investigated interactions of coregulator peptides with BPA, BPAF, or BPS at the AR complexes using Micro Array for Real-time Coregulator Nuclear Receptor Interaction (MARCoNI) assays and assessed the binding of these compounds on AR by molecular dynamics (MD) simulations. The set of coregulator peptides that are recruited by BPA-bound AR, either positively/or negatively, are different from those recruited by the agonist R1881-bound AR. Therefore, the data indicates that BPA shows no similarities to R1881 and suggests that it may recruit other coregulators to the AR complex. BPAF-bound AR recruits about 70-80% of the same coregulator peptides as BPA-bound AR. Meanwhile, BPS-bound AR interacts with only few peptides compared to BPA or BPAF-bound AR. MD results show that multiple binding sites with varying binding affinities are available on AR for BPA, BPAF, and BPS, indicating the availability of modified binding surfaces on AR for coregulator interactions. These findings help explain some of the distinct AR-related toxicities observed with bisphenol chemicals and raise concern for the use of substitutes for BPA in commercial products.

Differential Activation of a Mouse Estrogen Receptor ß Isoform (mERß2) with Endocrine-Disrupting Chemicals (EDCs).

BACKGROUND: Endocrine-disrupting chemicals (EDCs) are suspected of altering estrogenic signaling through estrogen receptor (ER) α or ß (mERß1 in mice). Several EDC effects have been reported in animal studies and extrapolated to human studies. Unlike humans, rodents express a novel isoform of ERß (mERß2) with a modified ligand-binding domain sequence. EDC activity through this isoform remains uncharacterized. OBJECTIVES: We identified the expression pattern of mERß2 in mouse tissues and assessed the estrogenic activity of EDCs through mERß2. METHODS: mERß2 mRNA expression was measured in mouse tissues. HepG2 cells were used to assess the transactivation activity of mERß isoforms with EDCs and ER co-activators. 293A cells transiently transfected with mER isoforms were used to detect EDC-mediated changes in endogenous ER target gene expression. RESULTS: Expression of mERß2 mRNA was detected in mouse reproductive tissues (ovary, testis, and prostate) and lung and colon tissues from both female and male mice. Five (E2, DES, DPN, BPAF, Coum, 1-BP) of 16 compounds tested by reporter assay had estrogenic activity through mERß2. mERß2 had a compound-specific negative effect on ERß/ligand-mediated activity and ER target genes when co-expressed with mERß1. mERß2 recruited coactivators SRC2 or SRC3 in the presence of EDCs, but showed less recruitment than mERß1. CONCLUSION: mERß2 showed weaker estrogenic activity than mERß1 in our in vitro system, and can dampen mERß1 activity. In vivo models of EDC activity and ER-mediated toxicity should consider the role of mERß2, as rodent tissue responses involving mERß2 may not be reproduced in human biology.

Transactivation Function-2 of Estrogen Receptor α Contains Transactivation Function-1-regulating Element.

ERα has a ligand-dependent transactivation function in the ligand binding domain of ERα C terminus (AF-2) and a ligand-independent activation function in the N terminus (AF-1). It is still not fully understood how AF-1 and AF-2 activities are regulated cooperatively by ligands. To evaluate the AF-1 involvement in the estrogenic activities of various compounds, we analyzed these transactivation functions using AF-1-truncated and AF-2-mutated ERα mutants. AF-2 is composed of two domains with flexible and static regions. We used an AF-2 flexible region mutant and an AF-2 static region mutant. Both mutants have been reported as non-E2 responsive due to disruption of E2-mediated coactivator recruitment to the AF-2. The AF-2 mutants were not activated by agonists, but surprisingly antagonists and selective estrogen receptor modulators (SERMs) activated the AF-2 mutants. This antagonist reversal activity was derived from AF-1. Furthermore, we demonstrated that the AF-2 contains an AF-1 suppression function using C-terminal-truncated ERα mutants. From these findings we hypothesized that the mutation of AF-2 disrupted its ability to suppress AF-1, causing the antagonist reversal. To assess the AF-2-mediated AF-1 suppression, we analyzed the transcription activity of physically separated AF-1 and AF-2 using a novel hybrid reporter assay. We observed that the AF-1 activity was not suppressed by the physically separated AF-2. Furthermore, SERMs did not induce the AF-1-mediated activity from the separated mutant AF-2, which differed from the intact protein. These results imply that SERM activity is dependent on a conformational change of the full-length ERα molecule, which allows for AF-1 activation.

Estrogen receptor α L543A,L544A mutation changes antagonists to agonists, correlating with the ligand binding domain dimerization associated with DNA binding activity.

A ligand-dependent nuclear transcription factor, ERα has two transactivating functional domains (AF), AF-1 and AF-2. AF-1 is localized in the N-terminal region, and AF-2 is distributed in the C-terminal ligand-binding domain (LBD) of the ERα protein. Helix 12 (H12) in the LBD is a component of the AF-2, and the configuration of H12 is ligand-inducible to an active or inactive form. We demonstrated previously that the ERα mutant (AF2ER) possessing L543A,L544A mutations in H12 disrupts AF-2 function and reverses antagonists such as fulvestrant/ICI182780 (ICI) or 4-hydoxytamoxifen (OHT) into agonists in the AF2ER knock-in mouse. Our previous in vitro studies suggested that the mode of AF2ER activation is similar to the partial agonist activity of OHT for WT-ERα. However, it is still unclear how antagonists activate ERα. To understand the molecular mechanism of antagonist reversal activity, we analyzed the correlation between the ICI-dependent estrogen-responsive element-mediated transcription activity of AF2ER and AF2ER-LBD dimerization activity. We report here that ICI-dependent AF2ER activation correlated with the activity of AF2ER-LBD homodimerization. Prevention of dimerization impaired the ICI-dependent ERE binding and transcription activity of AF2ER. The dislocation of H12 caused ICI-dependent LBD homodimerization involving the F-domain, the adjoining region of H12. Furthermore, F-domain truncation also strongly depressed the dimerization of WT-ERα-LBD with antagonists but not with E2. AF2ER activation levels with ICI, OHT, and raloxifene were parallel with the degree of AF2ER-LBD homodimerization, supporting a mechanism that antagonist-dependent LBD homodimerization involving the F-domain results in antagonist reversal activity of H12-mutated ERα.