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With the continuous progress in ultralarge virtual libraries which are readily accessible, it is of great interest to explore this large chemical space for hit identification and lead optimization using reliable structure-based approaches. In this work, a novel growth-based screening protocol has been designed and implemented in the structure-based design platform CONTOUR. The protocol was used to screen the ZINC database in silico and optimize hits to discover 11β-HSD1 inhibitors. In contrast to molecular docking, the virtual screening process makes significant improvements in computational efficiency without losing chemical equities through partitioning 1.8 million ZINC compounds into fragments, docking fragments to form key hydrogen bonds with anchor residues, reorganizing molecules into molecular fragment trees using matched fragments and common substructures, and then regrowing molecules with the help of developed intelligent growth features inside the protein binding site to find hits. The growth-base screening approach is validated by the high hit rate. A total of 50 compounds have been selected for testing; of these, 15 hits having diverse scaffolds are found to inhibit 11β-HSD1 with IC50 values of less than 1 μM in a biochemical enzyme assay. The best hit which exhibits an enzyme IC50 of 33 nM is further developed to a novel series of bicyclic 11β-HSD1 inhibitors with the best inhibition of enzyme IC50 of 3.1 nM. The final lead candidate exhibits IC50 values of 7.2 and 21 nM in enzyme and adipocyte assays, respectively, displayed greater than 1000-fold of selectivity over 11β-HSD2 and two other related hydroxysteroid dehydrogenases, and can serve as good starting points for further optimization to develop clinical candidates.

Mineralocorticoid receptor (MR) antagonists continue to be a prevalent area of research in the pharmaceutical industry. Herein we report the discovery of various spirooxindole and dibenzoxazepine constructs as potent MR antagonists. SAR analysis of our spirooxindole hit led to highly potent compounds containing polar solubilizing groups, which interact with the helix-11 region of the MR ligand binding domain (LBD). Various dibenzoxazepine moieties were also prepared in an effort to replace a known dibenzoxepane system which interacts with the hydrophobic region of the MR LBD. In addition, an X-ray crystal structure was obtained from a highly potent compound which was shown to exhibit both partial agonist and antagonist modes of action against MR.

To combat the increased morbidity and mortality associated with the developing diabetes epidemic new therapeutic interventions are desirable. Inhibition of intracellular cortisol generation from cortisone by blocking 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) has been shown to ameliorate the risk factors associated with the metabolic syndrome. A challenge in developing 11β-HSD1 inhibitors has been the species selectivity of small molecules, as many compounds are primate specific. Here we describe our strategy to identify potent selective 11β-HSD1 inhibitors while ensuring target engagement in key metabolic tissues, liver and fat. This strategy enabled the identification of the clinical candidate, BI 135585.

A potent, in vivo efficacious 11β hydroxysteroid dehydrogenase type 1 (11β HSD1) inhibitor (11j) has been identified. Compound 11j inhibited 11β HSD1 activity in human adipocytes with an IC50 of 4.3nM and in primary human adipose tissue with an IC80 of 53nM. Oral administration of 11j to cynomolgus monkey inhibited 11β HSD1 activity in adipose tissue. Compound 11j exhibited >1000× selectivity over other hydroxysteroid dehydrogenases, displays desirable pharmacodynamic properties and entered human clinical trials in 2011.

LXRs have been of interest as targets for the treatment of atherosclerosis for over a decade. In recent years, LXR modulators have also garnered interest for potential use in the treatment of inflammation, Alzheimer's disease (AD), dermatological conditions, hepatic steatosis, and oncology. To date, no LXR modulator has successfully progressed beyond phase I clinical trials. In this Perspective, we summarize published medicinal chemistry efforts in the context of the available crystallographic data, druglikeness, and isoform selectivity. In addition, we discuss the challenges that need to be overcome before an LXR modulator can reach clinical use.

Liver X receptor (LXR) agonists have been reported to lower brain amyloid beta (Aβ) and thus to have potential for the treatment of Alzheimer's disease. Structure and property based design led to the discovery of a series of orally bioavailable, brain penetrant LXR agonists. Oral administration of compound 18 to rats resulted in significant upregulation of the expression of the LXR target gene ABCA1 in brain tissue, but no significant effect on Aβ levels was detected.

This article describes the application of Contour to the design and discovery of a novel, potent, orally efficacious liver X receptor β (LXRβ) agonist (17). Contour technology is a structure-based drug design platform that generates molecules using a context perceptive growth algorithm guided by a contact sensitive scoring function. The growth engine uses binding site perception and programmable growth capability to create drug-like molecules by assembling fragments that naturally complement hydrophilic and hydrophobic features of the protein binding site. Starting with a crystal structure of LXRβ and a docked 2-(methylsulfonyl)benzyl alcohol fragment (6), Contour was used to design agonists containing a piperazine core. Compound 17 binds to LXRβ with high affinity and to LXRα to a lesser extent, and induces the expression of LXR target genes in vitro and in vivo. This molecule served as a starting point for further optimization and generation of a candidate which is currently in human clinical trials for treating atopic dermatitis.

A series of 2-(1H-pyrazol-1-yl)pyridines are described as inhibitors of ALK5 (TGFβ receptor I kinase). Modeling compounds in the ALK5 kinase domain enabled some optimization of potency via substitutions on the pyrazole core. One of these compounds PF-03671148 gave a dose dependent reduction in TGFβ induced fibrotic gene expression in human fibroblasts. A similar reduction in fibrotic gene expression was observed when PF-03671148 was applied topically in a rat wound repair model. Thus these compounds have potential utility for the prevention of dermal scarring.

Background Excessive dermal scarring is characterized by an overabundant deposition of extracellular matrix caused by fibrosis. The purpose of this study was to modify a rodent model of cutaneous healing for use in the development of compounds to minimize scarring, and to test the model with a small molecule inhibitor of transforming growth factor-β type I receptor, activin receptor-like kinase 5, because this class of inhibitors has been demonstrated to be effective in minimizing fibrosis in other organs. Methods The rodent model of cutaneous healing consists of uniform full-thickness incisional dermal wounds in rats. Wounds were allowed to heal by secondary intention, generally over a 14-day period. The usefulness of the model was tested by the application of an activin receptor-like kinase 5 inhibitor, CP-639180. Activin receptor-like kinase 5 inhibition antagonizes the transforming growth factor-β pathway, and was used to determine whether there was an effect on collagen deposition in wounds. The compound was applied once per day for 7 days starting at postwounding day 0 or 7 (early or late treatment regimens). Wounds were analyzed histologically for collagen deposition and biochemically for quantification of collagen changes. Results Early and late treatment regimens with the activin receptor-like kinase 5 inhibitor significantly reduced collagen deposition without impairing wound healing. Conclusions Application of a small molecular inhibitor of activin receptor-like kinase 5 appears to significantly reduce collagen deposition in rat dermal wounds as reported here for the first time. Activin receptor-like kinase 5 inhibition may offer a novel approach to reducing proliferative scars in humans because collagen accumulation is a core event in scarring.

Background Liver X receptors (LXRs) are involved in maintaining epidermal barrier and suppressing inflammatory responses in model systems. The LXR agonist VTP-38543 showed promising results in improving barrier function and inflammatory responses in model systems. Objective To assess the safety, tolerability, cellular and molecular changes, and clinical efficacy of the topical VTP-38543 in adults with mild to moderate atopic dermatitis (AD). Methods A total of 104 ambulatory patients with mild to moderate AD were enrolled in this randomized, double-blind, vehicle-controlled trial between December 2015 and September 2016. VTP-38543 cream in 3 concentrations (0.05%, 0.15%, and 1.0%) or placebo was applied twice daily for 28 days. Pretreatment and posttreatment skin biopsy specimens were obtained from a subset of 33 patients. Changes in SCORing of Atopic Dermatitis, Eczema Area and Severity Index, Investigator's Global Assessment, and tissue biomarkers (by real-time polymerase chain reaction and immunostaining) were evaluated. Results Topical VTP-38543 was safe and well tolerated. VTP-38543 significantly increased messenger RNA (mRNA) expression of epidermal barrier differentiation (loricrin and filaggrin, P = .02) and lipid (adenosine triphosphate–binding cassette subfamily G member 1 and sterol regulatory element binding protein 1c, P H 17/T H 22-related (phosphatidylinositol 3, S100 calcium-binding protein A12) and innate immunity (interleukin 6) markers. Conclusion Topical VTP-38543 is safe and well tolerated. Its application led to improvement in barrier differentiation and lipids. Longer-term studies are needed to clarify whether a barrier-based approach can induce meaningful suppression of immune abnormalities. Trial Registration clinicaltrials.gov Identifier: NCT02655679.

Inhibition of local cortisol regeneration from circulating cortisone by blocking 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) has been shown to ameliorate the risk factors associated with the metabolic syndrome. Chronic modulation of glucocorticoid homeostasis may result in hypothalamic-pituitary-adrenal (HPA) axis stimulation. HPA axis over-activation leading androgen excess would be undesirable in a therapeutic intervention designed to treat a chronic condition such as the metabolic syndrome. To address whether 11β-HSD1 inhibition would lead to excess androgens, we treated female cynomolgus monkeys with a selective inhibitor, BI 135558, for 4 weeks. Continual action of the compound over the dosing period was confirmed by constant plasma exposure, and a maintained change in urinary glucocorticoid metabolites consistent with 11β-HSD1 inhibition. No significant changes in adrenal function, as evidenced by an adrenocorticotropic hormone (ATCH) challenge, were observed. An examination of androgenic hormones revealed a slight increase in dehydroepiandrosterone sulfate (DHEA-S), while other hormones such as testosterone remained within reference values. Overall, treatment with BI 135558 in monkeys did not result in obvious over-activation of the HPA axis.

The ligand-activated transcription factors LXRα and LXRβ are important regulators of cholesterol metabolism and inflammation. Activation of LXR was previously shown to inhibit atherosclerosis in animal models through activation of reverse cholesterol transport (RCT) and suppression of vascular inflammation. VTP-38443 is a potent selective and orally bioavailable modulator of LXRβ that is being pursued for the prevention of subsequent vascular events following an acute coronary syndrome (ACS) episode. VTP-38443 is a potent binder (Ki=12 nM) and activator (EC50=17 nM) of LXRβ. It is ~20x selective for LXRβ versus LXRβ in both binding and activation. In primary human cells, it induces the expression of the cholesterol efflux pumps ABCA1 and ABCG1, markers of RCT, and promotes cholesterol efflux from human fibroblasts at low concentrations (EC50 = 14 nM). Orally dosed VTP-38443 demonstrates robust induction of ABCA1 and ABCG1 in mice (ED50 < 0.3 mg/kg) and primates (ED50 < 0.1 mg/kg). Based on this robust activity, VTP-38443 was tested in the apoE -/- carotid artery ligated mouse model, an accelerated atherosclerosis model. ApoE-/- mice (9 week) were fed a Western diet for 2 weeks at which point the left common carotid artery was ligated. Mice remained on the Western diet for 2 weeks post-surgery and were dosed BID with VTP-38443 (0.05, 0.2 and 1 mg/kg/dose) or a non-selective LXR agonist, TO90137 (20 mg/kg/day) during this time. Plasma cholesterol and TGs were measured as were cholesterol esters and FDG-6-phosphate, a biomarker of plaque inflammation, in the carotid plaques. VTP-38443 gave a dose-dependent reduction in plasma cholesterol (35% at 1 mg/kg) and a modest dose-dependent increase in plasma TGs ranging from 5% to 45% of the TG increase seen with TO90137. Plaque analysis showed a highly significant decrease in carotid cholesterol ester content at all doses, achieving >90% reduction at the highest dose. In addition, there was a significant reduction (~40-50%) in FDG-6 phosphate at all doses of VTP-38443, indicating a decrease in vascular inflammation. These data indicate that LXRβ activation may decrease plaque cholesterol content and reduce plaque inflammation in ACS.

Mineralocorticoid receptor (MR) antagonism has proven to effectively attenuate the pathophysiological effects of aldosterone in clinical and experimental settings of hypertension and heart failure. MR activates transcription of target genes upon aldosterone binding, and eplerenone selectively binds to MR and blocks aldosterone- mediated activation. In this review, we summarize the preclinical and clinical evidence supporting the beneficial effects of eplerenone (INSPRATM), a selective aldosterone blocker, in the treatment of hypertension and heart failure. We also review the current status in understanding the molecular mechanisms of action of the MR and its ligand. In addition, we compare the effects of eplerenone and spironolactone, a nonselective aldosterone blocker, on the transcriptional activity of MR and provide a molecular explanation for the improved side-effect profile of eplerenone compared with spironolactone.

We investigated the coregulator (coactivator and corepressor) interactions with the mineralocorticoid receptor (MR) that lead to activation and inhibition of the receptor in the presence of agonist and/or antagonist. Our results indicate that MR ligand binding domain (LBD) interacts strongly with only a few specific coactivator peptides in the presence of the agonist aldosterone and that these interactions are blocked by the antagonist eplerenone. We also discovered that cortisol, the preferred physiological ligand for the glucocorticoid receptor in humans, is a partial MR agonist/antagonist, providing a possible molecular explanation of the tissue-selective effects of glucocorticoids on MR. However, when we examined the coactivator and corepressor peptide interactions in the presence of cortisol, we found that MR bound with cortisol or aldosterone interacted with the same set of peptides. Thus, the partial agonism shown by cortisol is unlikely to be the result of differential interaction with known coactivators and corepressors. On the other hand, we have identified coactivator binding groove mutations that are critical for cortisol activation but not for aldosterone activation, suggesting that the two steroids induce different MR LBD conformations. In addition, we also show that cortisol becomes full agonist when S810L mutation is introduced in the LBD of MR. Interestingly, MR antagonists, such as eplerenone and progesterone, become partial agonist/antagonist of S810L but are still able to recruit LXXLL peptides to the mutant receptor. Together, these findings suggest a model to explain the MR activation by various ligands.

Glucocorticoids are among the most potent antiinflammatory agents, acting through the glucocorticoid receptor (GR) to suppress gene expression of a variety of cytokines. This appears to be via transcriptional interference (or transrepression) of key regulatory factors such as nuclear factor-kappaB and activator protein 1. Ligand-bound GR can also activate gene transcription (transactivation) via direct binding to glucocorticoid response elements. Transactivation by GR is potentiated by accessory coactivators such as steroid receptor coactivator 1 and peroxisome proliferator-activated receptor gamma coactivator 1, whereas the role of these proteins in transrepression is unclear.Here, we show that GR can recruit several coactivator receptor interacting domains in a ligand-dependent manner. All interactions require the charge clamp defined by K579/E755, while a subset also requires a second charge clamp defined by R585/D590, within the GR ligand-binding domain. A point mutation, E755A, abolished all GR-receptor interacting domain interactions and led to a decrease in GR-mediated transactivation, but did not significantly affect GR-mediated transrepression of Gal4-p65 activity. Overexpression of a GR-interacting coactivator peptide blocked transactivation but did not affect transrepression of p65 or TNFalpha-induced IL-6 promoter activity. Finally, the GR antagonist RU486 did not recruit coactivators to GR but maintained the ability to transrepress p65 activity. Our data suggest that different coactivators utilize distinct contact points to interact with GR. Although GR interactions with specific coactivators are critical for transactivation, they appear to be dispensable for at least certain aspects of GR-mediated transrepression of nuclear factor-kappaB. This is consistent with the notion that all GR- mediated repression is not intrinsically linked to activation and can be separated mechanistically.

Liver X receptors (LXRs) are members of the nuclear receptor superfamily that regulate gene expression in response to oxysterols and play a critical role in cholesterol homeostasis by regulating genes that are involved in cholesterol transport, catabolism, and triglyceride synthesis. Oxysterols and synthetic agonists bind LXRs and activate transcription by recruiting coactivator proteins. The role of LXRs in regulating target gene expression in the absence of ligand is unknown. Here we show that LXRs interact with corepressors, N-CoR (nuclear receptor corepressor) and SMRT (silent mediator of retinoic acid receptor and thyroid receptor), which are released upon binding agonists. The LXR-corepressor interaction is isoform selective, wherein LXRα has a very strong interaction with corepressors and LXRβ only shows weak interaction. LXRs also exhibit a preference for interacting with N-CoR vs. SMRT. Similar to other nuclear receptors, mutations in the LXR helix 3 and 4 region abolish corepressor interaction. Using a transient transfection assay, we demonstrate that LXR represses transcription that can be further increased by cotransfecting N-CoR into cells. Chromatin immunoprecipitation experiments further indicated that N-CoR is recruited onto endogenous LXR target genes, and addition of LXR agonists releases N-CoR from their promoters. Collectively, these results suggest that corepressors play an important role in regulating LXR target gene expression.

Ayurveda, the ancient Indian system of health care and medicine, has a well-organized materia medica in which plants form a dominant part. A key illustration of the exploitation of this knowledge toward the development of a modern drug is the isolation and characterization of two antihyperlipidemic compounds, Z-, and E-guggulsterone from the tree Commiphora mukul, the exudate of which has been traditionally used for mitigating lipid disorders. Here, we demonstrate that Z-guggulsterone and an analog, 80-574 currently in clinical trials, act as antagonists of the bile acid receptor (BAR), a member of the intracellular receptor superfamily. These compounds antagonize the activity of BAR in vitro, and in cell culture systems on promoters and endogenous target genes. In biochemical assays, they are able to displace coactivator peptides from the receptor in a dose-dependent manner. The mechanism by which they act as BAR antagonists is likely through their inability to recruit coactivator proteins, failure to release corepressor proteins from unliganded receptor, and ability to compete with BAR agonists to block coactivator recruitment. Our data suggest these compounds may mediate at least some of their effects via the BAR.

The orphan nuclear receptor steroidogenic factor 1 (SF-1, also called Ad4BP and officially designated NR5A1) has emerged as an essential regulator of endocrine development and function. Initially identified as a tissue-specific transcriptional regulator of the cytochrome P450 steroid hydroxylases, SF-1 has considerably broader roles, as evidenced from studies in knockout mice lacking SF-1. The SF-1-knockout mice lacked adrenal glands and gonads and therefore died from adrenal insufficiency within the first week after birth. In addition, SF-1 knockout mice exhibited male-to-female sex reversal of their internal and external genitalia, impaired expression of multiple markers of pituitary gonadotropes, and agenesis of the ventromedial hypothalamic nucleus (VMH). These studies delineated essential roles of SF-I in regulating endocrine differentiation and function at multiple levels, particularly with respect to reproduction. This chapter will review the experiments that established SF-1 as a pivotal, global determinant of endocrine differentiation and function. We next discuss recent insights into the mechanisms controlling the expression and function of SF-1 as well as the current status of research aimed at delineating its roles in specific tissues. Finally, we highlight areas where additional studies are needed to expand our understanding of SF-1 action.

Steroidogenic factor 1 (SF-1), an orphan nuclear receptor, initially was isolated as a key regulator of the tissue-specific expression of the cytochrome P450 steroid hydroxylases. Thereafter, analyses of sites of SF-1 expression during mouse embryological development hinted at considerably expanded roles for SF-1, roles that were strikingly confirmed through the analyses of SF-1 knockout mice. These SF-1 knockout mice exhibited adrenal and gonadal agenesis, associated with male-to-female sex reversal of their internal and external genitalia and death from adrenocortical insufficiency. These findings showed unequivocally that SF-1 is essential for the embryonic survival of the primary steroidogenic organs. SF-1 knockout mice also had impaired pituitary expression of gonadotropins and agenesis of the ventromedial hypothalamic nucleus (VMH), establishing that SF-1 regulates reproductive function at all three levels of the hypothalamic-pituitary gonadal axis. This article reviews the experiments that have defined these essential roles of SF-1 in endocrine development and highlights important areas for future studies.

Retinoid X receptor (RXR) plays a central role in the regulation of many intracellular receptor signalling pathways and can mediate ligand-dependent transcription, acting as a homodimer or as a heterodimer. Here we identify an antagonist towards RXR homodimers which also functions as an agonist when RXR is paired as a heterodimer to specific partners, including peroxisome proliferator-activated receptor and retinoic acid receptor. This dimer-selective ligand confers differential interactions on the transcription machinery: the antagonist promotes association with TAF110 (TATA-binding protein (TBP)-associated factor 110) and the co-repressor SMRT, but not with TBP, and these properties are distinct from pure RXR agonists. This unique class of RXR ligands will provide a means to control distinct target genes at the level of transcription and allow the development of retinoids with a new pharmacological action.

VTP-43742 is an orally active RORγt inhibitor being developed for the treatment of autoimmune disorders, including psoriasis, through inhibition of IL-17A production and down regulation of the IL-23 receptor. Safety, tolerability, pharmacokinetic (PK) and pharmacodynamic (PD) profiles of VTP-43742 were assessed in two, randomized, double-blind, placebo-controlled studies in which VTP-43742 was administered to normal healthy volunteers. The first study was a single ascending dose (SAD) study (N = 53) in which participants were randomized to a one-time administration of VTP-43742 (7 dose levels, 30–2000 mg) or placebo. The second study (NCT02555709) was a multiple ascending dose (MAD) study in which participants (N = 40) received VTP-43742 (5 dose levels, 100–1400 mg/d) or placebo once daily for 10 days. VTP-43742 was shown to be safe and generally well tolerated at all dose levels in both studies. No serious adverse events were reported, and all study subjects completed dosing. No clinically significant clinical chemistry, hematologic or ECG abnormalities were observed, and dose proportionality was demonstrated across all dose levels. In the SAD study, VTP-43742 had a terminal plasma half-life of ~30 hours. In an ex vivo whole blood assay (WBA) of IL-17A secretion, VTP-43742 suppressed RORγt dependent secretion of IL-17A in a dose-responsive manner; >90% inhibition was observed at the higher doses which was mostly maintained over 24 hours. In the MAD study, those receiving VTP-43742 showed >90% inhibition of RORγt dependent IL-17A secretion in the WBA for a full 24hrs in all but the lowest dose cohort. These data support the further development of VTP-43742 for the treatment of autoimmune disorders.

Liver X receptor (LXR) α and LXRβ function as physiological sensors of cholesterol metabolites (oxysterols), regulating key genes involved in cholesterol and lipid metabolism. LXRs have been extensively studied in both human and rodent cell systems, revealing their potential therapeutic value in the contexts of atherosclerosis and inflammatory diseases. The LXR genome landscape has been investigated in murine macrophages but not in human THP-1 cells, which represent one of the frequently used monocyte/macrophage cell systems to study immune responses. We used a whole-genome screen to detect direct LXR target genes in THP-1 cells treated with two widely used LXR ligands [N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)-ethyl]phenyl]-benzenesulfonamide (T0901317) and 3-[3-[N-(2-chloro-3-trifluoromethylbenzyl)-(2,2-diphenylethyl)amino]propyloxy] phenylacetic acid hydrochloride (GW3965)]. This screen identified the sphingomyelin phosphodiesterase acid-like 3A (SMPDL3A) gene as a novel LXR-regulated gene, with an LXR response element within its promoter. We investigated the regulation of SMPDL3A gene expression by LXRs across several human and mouse cell types. These studies indicate that the induction of SMPDL3A is LXR-dependent and is restricted to human blood cells with no induction observed in mouse cellular systems.

The cholesterol side-chain cleavage cytochrome P450 (CYP11A; P450scc) gene is expressed in rat ovarian follicles in response to gonadotropins (FSH/LH) and cAMP. To identify functional regions within the rat P450scc promoter, 894 basepairs (bp) of 5'-flanking sequence and 5'-deletions (at -379, -101, -73, and -38 bp) were linked to the human GH reporter gene and transfected into cultured rat granulosa cells. cAMP inducibility of the rat promoter was localized to a region (between -73/-38 bp) that contains one of two AGGT/CC/TA motifs, designated SCC1 (-51/-43 bp) and SCC2 (-79/-71 bp), within the rat promoter. One of the nuclear proteins in granulosa cells that binds to SCC1 was identified as the orphan receptor, steroidogenic factor-1 (SF-1). In contrast, multiple protein-DNA complexes formed with SCC2, only one of which was clearly identified as SF-1. Nuclear extract binding was sequence specific; SCC1 bound SF-1 more strongly than did SCC2. Thus, the two AGGT/CC/TA motifs of the rat promoter appear to differ structurally and functionally. Furthermore, because the expression of SF-1 mRNA precedes hormonal/cAMP induction of P450scc mRNA and is not regulated in vitro by cAMP, the functional role of SF-1 in transcriptional regulation of the P450scc gene, including its induction by cAMP, is not entirely clear and is probably dependent on other factors and/or the modification (phosphorylation?) of SF-1.

Publisher Summary This chapter focuses on recent advances in mineralocorticoid receptor (MR) antagonists. MR is a member of the nuclear hormone receptor superfamily and is structurally related to the progesterone receptor, androgen receptor, estrogen receptor, and glucocorticoid receptor. There is extensive clinical validation for treating hypertension and congestive heart failure with spironolactone and eplerenone, both of which are steroid-based antagonists of MR. These agents have side effects—such as gynecomastia, hyperkalemia, and drug–drug interactions—that limit their safety and effectiveness, thus providing a need for MR antagonists with superior profiles. Recent evidence suggests that MR blockade, when given in combination with standard therapy, reduces proteinuria in patients with renal disorders, such as diabetic nephropathy and chronic kidney disease. The past several years have seen a resurgence in targeting MR for intervention in cardiovascular disease. Given the high rate of occurrence of type 2 diabetes in an aging, overweight population, these diseases represent a potentially huge unmet medical need. MR antagonism is also shown to be cardioprotective, reducing mortality rates among patients recovering from acute myocardial infarction.

Bovine adrenocortical cells undergo a process in which expression of steroid hydroxylases is lost progressively as a function of population doubling level (PDL) in culture. Each cytochrome P450 shows a characteristic rate of loss of expression as a function of PDL (in order of rates of loss: CYP11B >CYP21 >CYP17 >CYP11A). CYP11B and CYP21 require insulin-like growth factor I as well as cyclic AMP; these are the only factors required for induction in the primary culture. Middle- and later passage cells do not express CYP11B and CYP21 under the same conditions, but will do so when cells are grown in extracellular matrix Matrigel. In late-passage cells neither CYP17, CYP21, nor CYP11B are expressed, even in the presence of Matrigel; only CYP11A is expressed in late-passage cultures. When the different environmental factors required for induction of CYP11B and CYP21 are taken into account, induction of these genes disappears with the same kinetics as previously shown for CYP17 as a function of PDL. The primary cause of the loss of expression of these genes is likely to be a phenotypic switching event similar to that previously demonstrated for CYP17 by in situ hybridization. The mechanism of phenotypic switching is unknown. However, one HpaII site at −2.3 kb of CYP17 was methylated in the bovine adrenal cortex in vivo but showed rapid and complete demethylation when adrenocortical cells were placed in culture. This indicates a unique, reproducible, environmentally determined change in methylation, with as yet undetermined consequences. However, data from reporter constructs suggest that phenotypic switching does not result from a simple loss of regulatory factors that act within 2.5 kb of the promoter. Previous data suggested that SV40 T antigen may affect phenotypic switching, and thus that SV40 may be useful for the derivation of functional adrenocortical cell lines. Adaptation of methods previously used for bovine cells to human adrenocortical cells to produce SV40 T antigen-transfected clones yielded data indicating preservation of essential aspects of the human adrenocortical cell differentiated phenotype.

We proposed that a cell-selective regulatory protein coordinately regulates the expression of three enzymes that are required for the biosynthesis of corticosteroids: cholesterol side chain cleavage enzyme, steroid 21-hydroxylase, and the aldosterone synthase isozyme of steroid 11 beta-hydroxylase. In this report, we identify a 53-kilodalton protein, termed steroidogenic factor 1 (SF-1), that interacts with the related promoter elements from these steroidogenic enzymes, and we isolate and characterize a cDNA that very likely encodes this protein. We first showed that nuclear extracts from bovine adrenal glands interact with the mouse steroidogenic regulatory elements, forming complexes indistinguishable from those produced by nuclear extracts from mouse Y1 adrenocortical cells. These bovine adrenal extracts were subjected to sequential ion exchange and affinity chromatography to yield a highly enriched preparation of SF-1. The predominant protein in the affinity-purified preparation comigrated with shift activity and had a mol wt of 53,000; UV cross-linking experiments demonstrated directly that this 53-kilodalton protein interacted with the steroidogenic regulatory element. Even with this marked enrichment, affinity-purified SF-1 bound six steroidogenic regulatory elements. These results support strongly the model that a steroidogenic cell-selective protein interacts with related promoter elements from three steroidogenic enzymes to regulate their coordinate expression. The recognition sequence of SF-1 closely resembles those of nuclear hormone receptor family members, suggesting that SF-1 may belong to this supergene family. By screening a Y1 cell cDNA library with the DNA-binding region of the H-2RIIBP nuclear hormone receptor cDNA, we isolated a cDNA that is selectively expressed in steroidogenic cells. When expressed as a glutathione S-transferase fusion protein in Escherichia. coli, the protein encoded by this cDNA interacts with all six related steroidogenic regulatory elements with a binding specificity indistinguishable from that of SF-1. Surprisingly, the sequence of the putative DNA-binding domain of this cDNA matches exactly the corresponding sequence of the mouse homolog of the Drosophila transcription factor fushi tarazu-factor I. The demonstration that a member of the nuclear hormone receptor family interacts with the steroidogenic regulatory elements provides intriguing insights into possible mechanisms by which these essential genes are regulated.

The liver X receptors (LXRs), members of the nuclear receptor superfamily, are potential targets for a variety of diseases. While there are many opportunities for the development of LXR-based therapeutics, there are some major hurdles, such as the ability of LXRs to cause hypertriglyceridemia, as well as some species-dependent aspects of LXR-mediated gene regulation. In addition to classical pharmacological approaches using relevant cellular and animal models, systematic molecular-based strategies will be important in overcoming these obstacles.

Cardiovascular disease, the primary cause of death and illness in the industrialized world, is typically due to complications of atherosclerosis, a multifactorial disease of the arterial intima. The liver X receptors (LXRs), LXRalpha and LXRbeta, are intracellular receptors that appear to play an important role in protection against atherosclerosis; however, LXR activation also leads to a dramatic increase in liver and serum triglycerides. This presents a challenge to developing drugs via these targets. This article discusses the role of LXRs in atherosclerosis and lipid regulation and the possibility of designing LXR ligands that may be anti-atherogenic without side effects.

1 High-throughput screening (HTS) has become an essential part of the drug discovery process. Due to the rising requirements for both data quality and quantity, along with increased screening cost and the demand to shorten the time for lead identifica- tion, increasing throughput and cost-effectiveness has become a necessity in the hit identification process. The authors present a multiplexed HTS for 2 nuclear receptors, the farnesoid X-activated receptor and the peroxisome proliferator-activated recep- tor delta in a viable cell-based reporter gene assay. The 2 nuclear receptors were individually transfected into human hepatoma cells, and the transient transfected cell lines were pooled for the multiplexed screen. Hits identified by the multiplexed screen are similar to those identified by the individual receptor screens. Furthermore, the multiplexed screen provides selectivity in- formation if ligands selective for one and not the other receptor are one of the hit criteria. The data demonstrate that multiplexing nuclear receptors can be a simple, efficient, cost-effective, and reliable alternative to traditional HTS of individ- ual targets without compromising data quality. (Journal of Biomolecular Screening 2003:239-246)

The liver X receptors (LXRs), members of the nuclear receptor superfamily, play an important role in controlling lipid homeostasis by activating several genes involved in reverse cholesterol transport. These include members of the ATP binding cassette (ABC) superfamily of transporter proteins ABCA1 and ABCG1, surface constituents of plasma lipoproteins like apolipoprotein E, and cholesterol ester transport protein. They also play an important role in fatty acid metabolism by activating the sterol regulatory element-binding protein 1c gene. Here, we identify human LXRα (hLXRα) as an autoinducible gene. Induction in response to LXR ligands is observed in multiple human cell types including macrophages and occurs within 2–4 h. Analysis of the hLXRα promoter revealed three LXR response elements (LXREs); one exhibits strong affinity for both LXRα:RXR and LXRβ:RXR (a type I LXRE), and deletion and mutational studies indicate it plays a critical role in LXR-mediated induction. The other two LXREs are identical to each other, exist within highly conserved Alu repeats, and exhibit selective binding to LXRα:RXR (type II LXREs). In transfections, the type I LXRE acts as a strong mediator of both LXRα and LXRβ activity, whereas the type II LXRE acts as a weaker and selective mediator of LXRα activity. Our data suggest a model in which LXR ligands trigger an autoregulatory loop leading to selective induction of hLXRα gene expression. This would lead to increased hLXRα levels and transcription of its downstream target genes such as ABCA1, providing a simple yet exquisite mechanism for cells to respond to LXR ligands and cholesterol loading.

The RXR serves as a heterodimer partner for the PPARgamma and the dimer is a molecular target for insulin sensitizers such as the thiazolidinediones. Ligands for either receptor can activate PPAR-dependent pathways via PPAR response elements. Unlike PPARgamma agonists, however, RXR agonists like LG100268 are promiscuous and activate multiple RXR heterodimers. Here, we demonstrate that LG100754, a RXR:RXR antagonist and RXR:PPARalpha agonist, also functions as a RXR:PPARgamma agonist. It does not activate other LG100268 responsive heterodimers like RXR:liver X receptoralpha, RXR:liver X receptorbeta, RXR:bile acid receptor/farnesoid X receptor and RXR:nerve growth factor induced gene B. This unique RXR ligand triggers cellular RXR:PPARgamma-dependent pathways including adipocyte differentiation and inhibition of TNFalpha-mediated hypophosphorylation of the insulin receptor, but does not activate key farnesoid X receptor and liver X receptor target genes. Also, LG100754 treatment of db/db animals leads to an improvement in insulin resistance in vivo. Interestingly, activation of RXR:PPARgamma by LG100268 and LG100754 occurs through different mechanisms. Therefore, LG100754 represents a novel class of insulin sensitizers that functions through RXR but exhibits greater heterodimer selectivity compared with LG100268. These results establish an approach to the design of novel RXR-based insulin sensitizers with greater specificity.

The nuclear receptor superfamily consists of a class of transcription factors comprising more than 100 different proteins. In contrast to membrane-bound receptors, the nuclear receptors are intracellular and act by controlling the activity of genes directly. Most members of this family bind directly to small lipidsoluble signaling molecules, or ligands, which owing to their lipophilic nature can easily enter the target cell. This superfamily includes known receptors for steroid hormones, such as the glucocorticoid receptor (GR), estrogen receptor (ER), progesterone receptor (PR) (see Chapter 20) and nonsteroid hormone receptors such as the vitamin D receptor (VDR), thyroid hormone receptor (TR), retinoic acid receptor (RAR), peroxisome-proliferator activated receptor (PPAR), and retinoid X receptor (RXR), all of which play key roles in animal development, physiology and human disease. The steroid hormones are derived from cholesterol, share a common chemical structural motif, and act in a classic endocrine manner. In contrast, ligands for the nonsteroid receptors are chemically diverse, including vitamin D, thyroid hormone, retinoids, and prostanoids. Furthermore, the source of the ligands for this class of receptors may be either endocrine, or generated intracellularly and not secreted (intracrine), or may be modified within the cell from an apohormone (Fig. 1).

Steroidogenic factor 1 (SF-1), an orphan member of the intracellular receptor superfamily, plays an essential role in the development and function of multiple endocrine organs. It is expressed in all steroidogenic tissues where it regulates the P450 steroidogenic genes to generate physiologically active steroids. Although many of the functions of SF-1 in vivo have been defined, an unresolved question is whether a ligand modulates its transcriptional activity. Here, we show that 25-, 26-, or 27-hydroxycholesterol, known suppressors of cholesterol biosynthesis, enhance SF-1-dependent transcriptional activity. This activation is dependent upon the SF-1 activation function domain, and, is specific for SF-1 as several other receptors do not respond to these molecules. The oxysterols activate at concentrations comparable to those previously shown to inhibit cholesterol biosynthesis, and, can be derived from cholesterol by P450c27, an enzyme expressed within steroidogenic tissues. Recent studies have shown that the nuclear receptor LXR also is activated by oxysterols. We demonstrate that different oxysterols differ in their rank order potency for these two receptors, with 25-hydroxycholesterol preferentially activating SF-1 and 22(R)-hydroxycholesterol preferentially activating LXR. These results suggest that specific oxysterols may mediate transcriptional activation via different intracellular receptors. Finally, ligand-dependent transactivation of SF-1 by oxysterols may play an important role in enhancing steroidogenesis in vivo .

Recent analyses of the cytochrome P450 steroid hydroxylases have established a key role for an orphan nuclear receptor, designated steroidogenic factor 1 (SF-1), in their coordinate, cell-selective expression. SF-1 was proposed to regulate the steroid hydroxylases by interacting with shared promoter elements in their 5′-flanking regions. During mouse embryonic development, SF-1 was expressed from the earliest stages of organogenesis of the steroidogenic tissues, suggesting a key role in steroidogenic cell differentiation. Finally, disruption of the gene encoding SF-1 revealed its essential function in the development of the adrenal glands and gonads and in pituitary gonadotrope function. These studies suggest that SF-1 acts at multiple levels of the reproductive axis to maintain reproductive competence.

Recent studies of the gene regulation of the cytochrome P450 steroid hydroxylases have established a key role for an orphan nuclear receptor, designated steroidogenic factor 1 (SF-1). SF-1 binds to shared promoter elements upstream of the steroid hydroxylases to mediate their coordinate expression in steroidogenic cells. Analyses of SF-1 expression during mouse embryonic development showed that SF-1 is expressed from the earliest stages of organogenesis of the steroidogenic tissues, suggesting an intimate link between SF-1 and steroidogenic cell differentiation. Finally, in gene disruption experiments, the gene encoding SF-1 was shown to be essential for development of the adrenal glands and gonads. These results establish the essential role of this orphan nuclear receptor in the development and function of the primary steroidogenic tissues.

Steroidogenic factor 1 (SF-1), an orphan nuclear receptor, regulates the enzymes that produce sex steroids, and disruption of the Ftz-F1 gene encoding SF-1 precludes adrenal and gonadal development. We now study the role of SF-1 at other levels of the hypothalamic/pituitary/gonadal axis. In Ftz-F1-disrupted mice, immunohistochemical analyses with antibodies against pituitary trophic hormones showed a selective loss of gonadotrope-specific markers, supporting the role of SF-1 in gonadotrope function. In situ hybridization analyses confirmed these results; pituitaries from Ftz-F1-disrupted mice lacked transcripts for three gonadotrope-specific markers (LH beta, FSH beta, and the receptor for gonadotropin-releasing hormone), whereas they exhibited decreased but detectable expression of the alpha-subunit of glycoprotein hormones. SF-1 transcripts in the developing mouse pituitary, which first became detectable at embryonic day 13.5-14.5, preceded the appearance of FSH beta and LH beta transcripts. In adult rat pituitary cells, SF-1 transcripts colocalized with immunoreactivity for the gonadotrope-specific LH. Finally, SF-1 interacted with a previously defined promoter element in the glycoprotein hormone alpha-subunit gene, providing a possible mechanism for the impaired gonadotropin expression in Ftz-F1-disrupted mice. These studies establish novel roles of this orphan nuclear receptor in reproductive function.

The cytochrome P450 steroid hydroxylases are coordinately regulated by steroidogenic factor 1 (SF-1), a protein expressed selectively in steroidogenic cells. Based on its expression in steroidogenic tissues and DNA-binding specificity, we isolated a putative SF-1 cDNA from an adrenocortical cDNA library. As evidence that this cDNA encodes SF-1, we now show that it is selectively expressed in steroidogenic cells, that an antiserum against its protein product specifically abolishes the SF-1-related gel-shift complex, and that its coexpression increases promoter activity of the 21-hydroxylase 5'-flanking region in transfection experiments. Sequence analyses of the SF-1 cDNA revealed that it is the mouse homolog of fushi tarazu factor I (FTZ-F1), a nuclear receptor that regulates the fushi tarazu homeobox gene in Drosophila. A second FTZ-F1 homolog, embryonal long terminal repeat-binding protein (ELP), was recently isolated from embryonal carcinoma cells. SF-1 and ELP cDNAs are virtually identical for 1017 base pairs, including putative DNA-binding domains, but diverge at their 5'- and 3'-ends. One genomic clone contained both SF-1- and ELP-specific sequences, confirming their origin from a single gene. Characterization of this gene defined shared exons encoding common regions and alternative promoters and 3'-exons leading to differences between the two FTZ-F1 transcripts. We used in situ hybridization with transcript-specific probes to study the ontogeny of SF-1 and ELP expression. ELP transcripts were not detected from embryonic day 8 to adult, consistent with its previous isolation from embryonal carcinoma cells and its postulated role in early embryonic development.(ABSTRACT TRUNCATED AT 250 WORDS)

Cultured bovine adrenocortical cells reach replicative senescence after 100–120 population doublings in culture. Before reaching senescence, cells undergo high frequency phenotypic switching from CYP17 + to CYP17 −, where ‘+’ and ‘−’ refer to the ability of intracellular cyclic AMP to induce expression of CYP17 (steroid 17α-hydroxylase). We used luciferase reporter constructs to assess the activity of the CYP17 promoter in bovine adrenocortical cells before and after phenotypic switching. We constructed two plasmids containing −2544 to + 29 and −488 to + 29 of the 5' region of CYP17 linked to a promoterless luciferase gene. Because of technical difficulties with transient transfection of late-passage bovine adrenocortical cells, these experiments were performed using stable transfection. Cells at early passage (PDL 10) and late passage (PDL 55) were cotransfected with either of these two plasmids ligated to pSV3neo, and G418-resistant pools of clones were derived. The activity of the CYP17 promoter in these transfectants was tested by growing cells in complete medium until semiconfluent and then transferring them into defined medium with cholera toxin and insulin-like growth factor I for 6 h. Luciferase activity was consistently induced by cholera toxin/IGF-I over five passages in pooled clones derived by transfection of early passage cells with the −488 construct. Despite the lack of expression of the endogenous CYP17 gene in transfectants from late-passage cells, induced luciferase activity was higher in late-passage transfectants than early-passage transfectants for both the −2544 and −488 constructs. However, the −2544 construct had a lower response to cholera toxin than the −488 construct in cells from both PDLs, indicating the potential existence of a negative regulatory element. The lower expression of the −2544 construct was confirmed in assays on individual clones of cells as well as pooled clones. These data indicate that the loss of cyclic AMP-inducible CYP17 expression during bovine adrenocortical cell senescence likely does not result from a loss of positive regulatory factors that act within 2.5 kb upstream of the CYP17 promoter.