PRINCIPLES FOR MODULATION OF THE NUCLEAR RECEPTOR SUPERFAMILY

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1 PRINCIPLES FOR MODULATION OF THE NUCLEAR RECEPTOR SUPERFAMILY Hinrich Gronemeyer*, Jan-Åke Gustafsson and Vincent Laudet Abstract Nuclear receptors are major targets for drug discovery and have key roles in development and homeostasis, as well as in many diseases such as obesity, diabetes and cancer. This review provides a general overview of the mechanism of action of nuclear receptors and explores the various factors that are instrumental in modulating their pharmacology. In most cases, the response of a given receptor to a particular ligand in a specific tissue will be dictated by the set of proteins with which the receptor is able to interact. One of the most promising aspects of nuclear receptor pharmacology is that it is now possible to develop ligands with a large spectrum of full, partial or inverse agonist or antagonist activities, but also compounds, called selective nuclear receptor modulators, that activate only a subset of the functions induced by the cognate ligand or that act in a cell-type-selective manner. *Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC) CNRS/INSERM/ULP, 1 rue Laurent Fries, BP 10142, Illkirch Cedex, C.U. de Strasbourg, France. Karolinska Institute, Department of Medical Nutrition and Center of Biotechnology, Novum, S Huddinge, Sweden. Laboratoire de Biologie Moléculaire de la Cellule, CNRS UMR 5161, INRA LA 1237, Ecole Normale Supérieure de Lyon, 46, Allée d Italie, Lyon Cedex 07, France. Correspondence to V.L. Vincent.Laudet@ens-lyon.fr doi: /nrd1551 Nuclear receptors are transcription factors that are essential in embryonic development, maintenance of differentiated cellular phenotypes, metabolism and cell death. Dysfunction of nuclear receptor signalling leads to proliferative, reproductive and metabolic diseases such as cancer, infertility, obesity and diabetes 1. Pharmaceutical nuclear receptor agonists or antagonists, such as tamoxifen for oestrogen receptors (targeted in breast cancer), thiazolidinediones for peroxisome proliferator-activated receptor-γ (PPARγ) (targeted in type II diabetes) or dexamethasone for the glucocorticoid receptor (targeted in inflammatory diseases), are among the most commonly used drugs. One of the most exciting messages for nuclear receptor-based drug development emanates from the observation that chemistry can provide us not only with receptor-selective 2 and various types of full, partial and INVERSE (ANT)AGONISTS 3,but also with compounds that activate only a subset of the functions induced by the cognate ligand 4 6 or with ligands that act in a celltype-selective manner (SELECTIVE NUCLEAR RECEPTOR MODU- LATORS (SNuRMs)) 7,8.Importantly, recent studies have shed light on the molecular mechanisms underlying SNuRM action, which has made it possible to design screening and target validation 9.Moreover, recent data indicate that several other factors and mechanisms have to be considered for a full understanding of nuclear receptor pharmacology. For example, we have just begun to recognize the importance of the so-called NON-GENOMIC ACTION of nuclear receptor ligands 10,11 and the impact of factors that re-direct nuclear receptor signalling 12.In all of these cases, molecular biology has provided us with a variety of concepts by which nuclear receptors and/or their ligands can induce or modulate important physiological programs (see REF. 13 for a recent example). The challenge now is to generate chemical compounds that address one or only some of these functions to achieve the pharmacologically desired effect. It has to be emphasized that the nuclear receptor superfamily contains many liganded receptors (24 among the 48 known nuclear receptors in the human genome) (TABLE 1) but also orphan receptors, for which no ligand has yet been discovered. It is not known whether all orphan receptors have the potential to bind natural or synthetic ligands or whether they are true orphans that do not possess a bona fide 950 NOVEMBER 2004 VOLUME 3

2 INVERSE (ANT)AGONIST A ligand that stabilizes an inactive conformation of a receptor for example, by increasing corepressor interaction, thereby decreasing signalling below basal levels. SELECTIVE NUCLEAR RECEPTOR MODULATORS (SNuRMS). Ligands that selectively modulate different receptor subtypes and/or act in a cell-selective manner. NON-GENOMIC ACTION tion of a ligand that does not involve the activation of the target genes of its cognate receptor. Table 1 Human nuclear receptors Name Abbreviation Nomenclature Ligand Thyroid hormone receptor TRα NR1A1 Thyroid hormone TRβ NR1A2 Thyroid hormone Retinoic acid receptor RARα NR1B1 Retinoic acid RARβ NR1B2 Retinoic acid RARγ NR1B3 Retinoic acid Peroxisome proliferator- PPARα NR1C1 Fatty acids, leukotriene B4, activated receptor fibrates PPARβ NR1C2 Fatty acids PPARγ NR1C3 Fatty acids, prostaglandin J2, Reverse erba Rev-erbα NR1D1 Orphan Rev-erbβ NR1D1 Orphan RAR-related orphan receptor RORα NR1F1 Cholesterol, cholesteryl sulphate RORβ NR1F2 Retinoic acid RORγ NR1F3 Retinoic acid Liver X receptor LXRα NR1H3 Oxysterols, T , GW3965 LXRβ NR1H2 Oxysterols, T , GW3965 Farnesoid X receptor FXRα NR1H4 Bile acids, Fexaramine FXRβ* NR1H5 Lanosterol Vitamin D receptor VDR NR1I1 1,25-dihydroxy vitamin D 3, litocholic acid Pregnane X receptor PXR NR1I2 Xenobiotics, PCN Constitutive androstane receptor CAR NR1I3 Xenobiotics, phenobarbital Human nuclear factor 4 HNF4α NR2A1 Orphan HNF4γ NR2A2 Orphan Retnoid X receptor RXRα NR2B1 Retinoic acid RXRβ NR2B2 Retinoic acid RXRγ NR2B3 Retinoic acid Testis receptor TR2 NR2C1 Orphan TR4 NR2C2 Orphan Tailless TLL NR2E2 Orphan Photoreceptor-specific nuclear receptor PNR NR2E3 Orphan Chicken ovalbumin upstream COUP-TFI NR2F1 Orphan promoter-transcription factor COUP-TFII NR2F2 Orphan ErbA2-related gene-2 EAR2 NR2F6 Orphan Oestrogen receptor ERα NR3A1 Oestradiol-17β, tamoxifen, raloxifene ERβ NR3A2 Oestradiol-17β, various synthetic compounds Oestrogen receptor-related receptor ERRα NR3B1 Orphan ERRβ NR3B2 DES, 4-OH tamoxifen ERRγ NR3B3 DES, 4-OH tamoxifen Glucocorticoid receptor GR NR3C1 Cortisol, dexamethasone, RU486 Mineralocorticoid receptor MR NR3C2 Aldosterone, spirolactone Progesterone receptor PR NR3C3 Progesterone, medroxyprogesterone acetate, RU486 Androgen receptor AR NR3C4 Testosterone, flutamide NGF-induced factor B NGFIB NR4A1 Orphan Nur related factor 1 NURR1 NR4A2 Orphan Neuron-derived orphan receptor 1 NOR1 NR4A3 Orphan Steroidogenic factor 1 SF1 NR5A1 Orphan Liver receptor homologous protein 1 LRH1 NR5A2 Orphan Germ cell nuclear factor GCNF NR6A1 Orphan DSS-AHC critical region on the DAX1 NR0B1 Orphan chromosome, gene 1 Short heterodimeric partner SHP NR0B2 Orphan *FXRβ is a pseudogene in humans and does not encode a functional receptor. DES, diethylstilbestrol; DSS-AHC, dosage-sensitive sex reversal-adrenal hypoplasia congenita; NGF, nerve growth factor; PCN, pregnenolone 16α-carbonitrile. NATURE REVIEWS DRUG DISCOVERY VOLUME 3 NOVEMBER

3 TRANSACTIVATION tivation of transcription by the binding of a transcription factor to a DNA regulatory sequence. ligand-binding pocket and might be regulated by alternative mechanisms. Undoubtedly, the existence of orphan receptors with apparent (patho)physiological activities constitutes both a major challenge for nuclear receptor pharmacology and a potential opportunity for drug discovery. Subsequent to a general introduction describing the basic principles of the mode of action of nuclear receptors, this review will focus on the various factors that are instrumental in modulating the pharmacology of nuclear receptors. One of the most important mechanistic aspects that we have learnt over recent years is that the response of a given nuclear receptor to a particular ligand in a given tissue is dictated by the set of proteins with which this nuclear receptor interacts following ligand-induced allosteric alterations that generate, expose or remove interaction surfaces. The pharmacology of nuclear receptors therefore involves a plethora of other proteins, ranging from other nuclear receptors and other transactivators to transcriptional cofactors. Nuclear receptors in action Nuclear receptors are understood primarily as ligandregulated transcription factors that modulate target gene transcription (FIG. 1a).Even before the first genes encoding nuclear receptors were cloned it was known that they are modular proteins, with three major domains 14 (FIG. 1b).The amino terminus (also known as the A/B region) contains a TRANSACTIVATION domain (AF-1), which is of variable length and sequence in the different family members, and is recognized by coactivators and/or other transcription factors. The central DNA-binding domain (DBD) has two zinc-finger motifs that are common to the entire family, with the exception of two divergent members: dosage-specific sex reversal-adrenal hypoplasia congenita critical region on the X chromosome-1 (DAX1) and short heterodimeric partner (SHP). The carboxy-terminal ligand-binding domain (LBD), whose overall architecture is well conserved between the various family members, nonetheless diverges sufficiently to guarantee selective ligand recognition. This domain also has the ligand-induced activation function (known as AF2) that is crucially involved in transcriptional coregulator interaction. Nuclear receptors can form monomers (for example, steroidogenic factor-1 (SF1)), homodimers (for example, steroid receptors), or heterodimers with the promiscuous retinoid X receptor (RXR) (for example, retinoic acid receptor (RAR), thyroid hormone receptor (THR), vitamin D receptor (VDR) and several orphan nuclear receptors); the nuclear receptors then bind response elements within the regulatory region(s) of target genes. Nuclear receptor response elements are derivatives of the canonical sequence RGGTCA (in which R is a purine), termed hormone response elements (HREs). Modification, extension and duplication (including alternate relative orientations of the repeat (direct, inverted, everted)) of this sequence generate response elements that are selective for a given receptor(s) or class of receptors (for example, oestrogen response elements (EREs) for oestrogen receptors or steroidogenic factor response elements (SFREs) for the orphan receptors SF1 and oestrogen receptor-related receptor (ERR); see REF. 1 for a review). The three-dimensional structure of the LBD has been determined for few unliganded (apo) and many liganded (holo) nuclear receptors, which has provided a good understanding of the mechanisms involved in ligand binding and transactivation (see supplementary information S1 (table)) 15.The LDBs of all nuclear receptors have a common overall three-dimensional structure 16.There are 11 helices, which form a compact structure comprising a ligand-binding pocket, the size of which varies significantly between family members (FIG. 2a).The entrance to the pocket is guarded by a twelfth helix (H12), which forms a movable lid over the pocket and contains residues that are crucial for the function of AF2. The orientation of H12 is a consequence of allosteric effects induced by the particular chemical structure of the specific ligand that is binding. In the absence of ligand, the LBD of many but not all nuclear receptors (for example, not steroid receptors) is bound to a set of transcriptional corepressors, such as nuclear receptor corepressor 1 (NCoR1) or silencing mediator for retinoid and thyroid hormone receptor (NCoR2, also known as SMRT), which recruit transcriptional complexes that contain specific histone deacetylases (HDACs). These deacetylases generate a condensed chromatin structure over the target promoter, which results in gene repression (FIG. 1a). Corepressors contain a region called the corepressor nuclear-receptor box (CoRNR box), which docks to a hydrophobic groove in the surface of the LBD encompassing helices H3 and H4. In most cases, an agonistic ligand promotes complex allosteric effects that lead to an alternative positioning of H12 on the LBD core, which disrupts the hydrophobic groove and leads to corepressor complex dissociation. This holo-positioning of H12 allows co-activators to interact with short LxxLL-like motifs (where L is leucine and x is any amino acid) called nuclear-receptor boxes that are present in most co-activators and are common motifs for interaction with nuclear receptor LBDs (FIG. 2b). The LxxLL motif recognizes a hydrophobic cleft formed by H3, H4 and H12 that is similar but nonidentical to that involved in corepressor interaction and has a decisive role in supporting co-activator interaction. The agonist-induced conformational change of the LBD is therefore a structural manifestation of a nuclear receptor transactivation function. Note that the allosteric ligand effect on the LBD conformation can also modulate the activity of the N-terminal AF1 for example, through intramolecular crosstalk between N- and C-terminal domains. The precise structural basis of this N C-terminal domain interaction is unknown, as no three-dimensional structure of an entire nuclear receptor has been obtained, but in the case of the androgen receptor (AR), evidence for a direct interaction between the N- and C-terminal regions has been obtained NOVEMBER 2004 VOLUME 3

4 A Ligand (auto-, para- or intracrine) PI(3) kinase Cytoplasm a NR? ERK Nuclear translocation c b HDAC Nucleus HAT CRM d Pol II holoenzyme TAF mediator e tivation B NLS (ligand-dependent) Function Dimerization (strong) Isoform-specific NLS Helix H12 Region AD A/B AD AD AD C D E AD F AF1 (cell- and promoter-specific) DNA binding Hormone binding Function Dimerization (weak; DNA binding induced) AF1 (ligand-dependent, cell- and promoterspecific) Figure 1 Nuclear receptors in action. A Mode of action of nuclear receptors (NRs). After diffusion through the cytoplasmic membrane, the ligand can interact with its cognate receptor where it can exert a non-genomic effect by interacting directly, for example, with kinases (a). The ratio between cytoplasmic and nuclear location can vary between different receptors and is affected by the nature of a ligand. Ligand binding modulates the interaction of the receptor with a plethora of factors. In the absence of ligand, several nuclear receptors are believed to be bound to the regulatory regions of target genes as a corepressor or histone deacetylase (HDAC) complex (b). Histone deacetylation is responsible for the chromatin condensation that accounts for the gene-silencing effect of apo receptors. Ligand binding releases the HDAC complex (c) and results in the recruitment of histone acetyltransferase (HAT) and chromatin-remodelling (CRM) complexes (d). The temporal order and requirement of these complexes can occur in a receptor-, target-gene- and cell-specific manner. In the last step (e), the polymerase II holoenzyme, which comprises the pol II enzyme, TAF (TATA-binding protein-associated factor) and mediator complexes, is recruited and increases the frequency of transcription initiation. B Schematic illustration of the structural and functional organization of nuclear receptors. The evolutionarily conserved regions C and E are indicated as boxes (green and orange, respectively), and a black bar represents the divergent regions A/B, D and F. Domain functions are depicted above and below the scheme. AD, activation domain; AF1, activation function 1; NLS, nuclear localization signal. NATURE REVIEWS DRUG DISCOVERY VOLUME 3 NOVEMBER

5 a H9 b H1 H8 H10 H7 H4 H5 H3 H4 GRIP1/TIF2/ SRC2 peptide K362 9C-RA H3 H11 H12 H11 H6 H2 H12 Ω-loop H3 E542 Figure 2 Ligand binding induces a conformational change of the ligand-binding domain structure of nuclear receptors. a A comparison of the crystal structures of the apo-retinoid X receptor-α (RXRα) ligand-binding domain (LBD) (PDB: 1lbd) with the holo-rxrα LBD complexed with 9-cis retinoic acid (PDB: 1fby). The figure reveals the ligand-induced trans-conformation that generates the transcriptionally active form of the receptor. The coloured helices H2, H3, H11 and H12 (purple in the apo-form; red in the holo form) are relocalized during the conformational change. In this model, ligand binding induces a structural transition that triggers a mousetrap-like mechanism: pushed by the ligand, H11 is repositioned in alignment with H10 and the concomitant swinging of H12 unleashes the omega-loop, which flips underneath H6, carrying along the amino-terminal part of H3. In its final position, H12 seals the ligand-binding cavity as a lid and further stabilizes ligand binding by contributing to the hydrophobic pocket. b The co-activator nuclear-receptor box LxxLL peptide-binding surface on the ERα diethylstilbestrol (DES) complex is shown in white (PDB: 3erd; see supplementary information S1 (table). A charge clamp (that is, charged amino acids that interact with both ends of the peptide and increase the strength of the interaction; the two ends of the clamp are indicated by the arrow) controls the binding of the LxxLL-containing nuclear-receptor-box peptide of co-activator proteins. The two residues constituting the charge clamp are indicated by blue and red surfaces. Oestrogen receptor (ER) residues E542 (red) and K362 (blue) stabilize co-activator binding in addition to the hydrophobic interactions established by the leucines. The regions of the surface that correspond to helices H3 and H4 of the receptor LBD are indicated. Genetic and/or biochemical data have revealed a plethora of factors generally components of multiprotein transcriptional complexes that mediate nuclear receptor function. These include the p160 transcription intermediary factor/nuclear receptor coactivator/steroid receptor co-activator (TIF/NCoA/ SRC) family of proteins; coactivator-associated arginine methyltransferase-1 (CARM1); E1A-binding protein 300 (EP300; also known as p300/cbp); p300/cbpassociated factor (PCAF); histone acetyl-transferases (HATs), some of which can form stable complexes on responsive promoters but not necessarily in solution; ATP-dependent chromatin-remodelling proteins, such as Switch factor (SWI SNF); and the RNA polymerase holoenzyme, which comprises the polymerase-iiassociated transcription factor D (TFIID) and the mediator complexes. In addition, numerous other individual proteins have been reported to regulate nuclear receptor function 18.Consequently, models of transcriptional regulation have become increasingly complex and case-specific 19.This is illustrated by a recent study of the oestrogen-regulated ps2 promoter, which revealed an amazing pattern of cyclo-temporal association of a multitude of transcription factors 20.It is beyond the scope of this article to review all aspects of current models of the roles for different factors/complexes in transcriptional regulation by nuclear receptors. Below we will primarily discuss effects that are relevant for nuclear receptor pharmacology. The advantage of multiplicity It is frequently the case that several paralogous genes that originated by gene duplications characteristic of the vertebrate lineage 21,22 encode the receptor for a given ligand (TABLE 1). The two oestrogen receptors ERα and ERβ,which originate from two separate genes on different chromosomes, show distinct pharmacological profiles and expression patterns. Similarly, three paralogues exist for the retinoic acid receptor (RARα,-β and -γ), and peroxisome proliferator-activated receptor 954 NOVEMBER 2004 VOLUME 3

6 Glu353 Wat Arg384 BIO-ISOSTERIC REPLACEMENT The creation of a new compound with similar biological properties to the parent compound by exchanging an atom or a group of atoms with another, broadly similar atom or group of atoms. ALL-TRANS RETINOIC ACID SYNDROME A side effect that occurs in 10 15% of patients that is preceded by increasing leukocyte count and that includes fever, respiratory distress, weight gain and oedema of the lower extremities and which is fatal in at least 10% of cases. 8-β-vinyl-estradiol Leu384 Met336 Met421 Ile373 His524 Figure 3 Comparative view of the ligand-binding pockets of the oestrogen receptor-α and -β. Modelled complex of the β-selective 8-β-vinyl oestradiol ligand bound to oestrogen receptor (ER)-α and ERβ. The model is based on the ERα oestradiol crystallographic structure (PDB: 1ERE). The protein is represented by green tubes, as are crucial conserved residues (Glu353, Arg394 and His524), which make hydrogen-bonding interactions (dashed yellow lines) with the ligand. Residues that differ between ERα (Leu384 and Met421) and ERβ (Met336 and Ile373) are represented by magenta and orange tubes, respectively. In the ligand, the carbon atoms are white and the oxygen atoms are red. There is a repulsive steric interaction between the vinyl group of the ligand (yellow star) and Leu384 in ERα that accounts for the ERβ-selectivity of this ligand. This unfavourable steric interaction is reinforced by the bulky Met421 residue of ERα. This figure was created with PyMol molecular graphics system. (PPARα, β/δ and -γ). This multiplicity of nuclear receptors is an important factor that contributes to both signal diversification and specification. However, whereas paralogue-selective ligands have been synthesized, it is not known whether endogenous ligands with such selectivities are operative in vivo.the three-dimensional structures of many nuclear receptor LBDs and of various ligand complexes have been determined, and have provided an understanding of the structural basis of selectivity and mode of action of receptor agonists and antagonists 15.These data will be discussed below in the case of the LBD of ERs and RARs. Following the discovery of ERβ 23, it has become clear that oestrogen signalling can be viewed as a balancing act between ERα and ERβ:ERα is often an activating factor, whereas ERβ suppresses the effects of ERα. For instance, oestradiol-17β-activated ERα stimulates the proliferation of MCF-7 breast cancer cells, whereas ligand-activated ERβ acts as an antiproliferative factor and prevents the effects of ERα 24.The antiproliferative effects of ERβ are also seen in colon and prostate cancer cells, which indicates that ERβ agonists should be considered in the prevention/treatment of these cancers 24. Several novel functions of ERβ have been revealed from studies of phenotypes of mice in which the gene that encodes ERβ has been deleted. For instance, these mice show neuronal degeneration in the central nervous system, proliferation of the prostate, a haematological syndrome that mimics chronic myeloid leukaemia, follicular arrest in the ovaries, uninflated lungs, as well as several other pathological characteristics that are under investigation 24.All these findings indicate that ERβ is central to the regulation of many important physiological functions in the organism. This expands the number of possible pharmaceutical targets for intervention with ERβ agonists or antagonists. In structural terms, the binding of oestrogens to ERα and ERβ is well known (FIG. 3): in both cases the architecture of the region of the ligand-binding pocket where the A ring of oestradiol-17β lodges is rigid and will only accommodate planar structures. There are also key amino acids within the pocket (Glu353, Arg394 and His524), which, on binding, interact with the two hydroxyl groups of 17β-oestradiol. Interaction of Glu353 and Arg394 with a hydroxyl group (for example, the 3-hydroxyl group of oestradiol) or BIO- ISOSTERIC REPLACEMENT is required for high-affinity binding, whereas interaction with His524 is less important for affinity but does contribute to the specificity of binding. The region of the pocket that accommodates the D ring of oestradiol is flexible, which means that bulky side chains can be added to this part of the ligand to make an antagonistic compound. ERα- and ERβ-specific agonists and antagonists are being developed, and differences in the size and shape of the ligand-binding pocket of the two receptors permit some educated guesses about how molecules might be modified to produce the desired specificity. The volume of the binding cavity of ERβ is smaller than that of ERα, and Katzenellenbogen and colleagues 25 have shown that by increasing the size of substituent groups on an ERβ agonist, the agonism on ERβ disappears and the ligands act as pure ERβ-selective antagonists. Genetic studies have revealed that the three RAR isoforms α, β and γ have distinct, albeit sometimes partially redundant, functions during embryogenesis 26, after birth 27 and in the adult Moreover, in vitro studies have revealed that certain cellular phenomena, such as differentiation, proliferation and apoptosis, require the activation of a specific RAR isotype, even though others are also expressed. Examples include the differentiation of leukaemic cells and the inhibition of proliferation of certain breast-cancer cell lines by RARα agonists 31.It is believed that in these cases RARα responds to its ligand as a heterodimer with an RXR partner, as RXR-selective ligands ( rexinoids ), which are inactive on their own, synergize with RAR-selective ligands 32.But some phenomena might require that both heterodimeric partners are activated by ligand, such as the post-maturation apoptosis of HL60 leukaemia cells that requires both retinoids and rexinoids 33. In an attempt to limit the side effects of retinoids, such as teratogenicity and the so-called ALL-TRANS RETINOIC ACID SYNDROME,RAR-isotype-selective synthetic agonists and antagonists have been generated. Studies with these compounds demonstrated a surprising potential for drug design and fine-tuning of receptor activities: not only was it possible to design entirely isotype-selective ligands, but also ligands that had complex activities (for NATURE REVIEWS DRUG DISCOVERY VOLUME 3 NOVEMBER

7 Table 2 Three divergent residues in the RAR ligand-binding pockets Receptor Helix 3 Helix 5 Helix 11 RARα Ser232 Ile270 Val395 RARβ Ala225 Ile263 Val388 RARγ Ala234 Met272 Ala397 RAR, retinoic acid receptor. example, BMS453, which is an RARα antagonist and an RARβ agonist in vitro 4 and in vivo,respectively 26 ). The structural basis for isotype-selectivity of RAR ligands is reasonably well understood and principles for the rational design of such compounds have been established. Two aspects are of major importance in this context. First, analyses of crystal structures have revealed that in human receptors only three residues have diverged between the ligand-binding pockets of human RARα,RARβ and RARγ (TABLE 2).Swapping experiments in which the LBD of a given receptor was mutated to acquire the ligand-binding characteristics of another subtype have revealed that these residues confer ligand isotype-selectivity irrespective of the RAR subtype into which they are integrated and irrespective of whether the ligand acts as agonist or antagonist 2,34. (A detailed structural discussion of the impact of the divergent residues in mediating isotype selectivity has been presented elsewhere 35.) Second, for RARs, in contrast to a number of other nuclear receptors, it seems that the ligand adapts to the ligand-binding cavity. This is illustrated by the crystal structures of the RARγ LBD in the presence of all-trans and 9-cis retinoic acids 36.Bending and torsion of the 9-cis isomer in the receptor-bound state causes significant deviation from its classic structure and displays a structure resembling the all-trans isomer. This conformational adaptation is apparently the consequence of steric constraints imposed by the highly conserved ligand-binding pocket on ligands with a great degree of inherent flexibility. The structure of the receptor-bound retinoid can therefore serve as a fingerprint of the ligand-binding pocket of the receptor and can be used as a template for the design of alternative retinoids. These distinct ligand-binding selectivities of paralogous receptors is a common theme within the superfamily; however, the structural features underlying ligand-binding selectivity are best understood for oestrogen receptors and RARs. Steroid receptors (androgen receptor, glucocorticoid receptor, mineralocorticoid receptor and progesterone receptor) represent extreme cases, as do the three PPARs and pregnane X receptor/constitutive androstane receptor (PXR/CAR), in which paralogous receptors show major differences with respect to ligand selectivity and to physiological functions. Interestingly, the family also comprises examples (such as RXRs) for which no (synthetic) isotype-selective ligand has been described; this is probably due to the identity of the residues lining the ligand cavity, which are highly conserved between the three receptors. These observations indicate that the structural and functional variation of the ligand-binding pocket and, more broadly, the entire LBD can drastically change from one group of receptors to another. The plasticity of the LBD has recently been emphasized by two observations for the insect ecdysone receptor-ultraspiracle (EcR-USP) heterodimer as well as for its vertebrate orthologue, liver X receptor (LXRβ) 37,38. In the case of EcR, the three-dimensional structure determination of the LBD complexed with different ligands (an ecdysteroid agonist, ponasterone A and the non-steroidal agonist BYI06830) revealed radically different and only partially overlapping ligand-binding pockets 37.The same is true of the LXR ligand-binding pocket, which can adjust to accommodate fundamentally different ligands 38.These observations also highlight the complexity of ligand receptor interactions, because none of these ligand-binding pockets was predicted by molecular modelling or docking studies. The molecular basis of antagonism Tamoxifen was the first selective oestrogen-receptor modulator (SERM) to be developed and used for clinical applications, most frequently for the treatment of breast cancer 7,8.It behaves as an agonist or as an antagonist depending on the promoter (that is, on the other transcription factors that are recruited to the promoter, as well as the architecture of the ERE within the promoter) and cellular contexts. Raloxifene is another classic SERM that was developed for its bone-protective and non-uterotrophic effects, and its antiproliferative effect on breast-cancer cells. The agonistic effect of tamoxifen is often mediated through indirect binding of the receptor to a promoter that does not contain classic EREs 3 (see REF. 40 for an alternative view). Indeed, in response to ligands, both ER isoforms, ERα and ERβ, can regulate gene transcription either by binding to specific EREs in the promoters of target genes, or by binding to other transcription factors acting at activator protein 1 (AP1) 41 and SP1 42 sites. Binding of ER to an ERE is clearly a pathway through which oestradiol modulates gene transcription, including the functions of oestradiol that are involved in cellular differentiation. The interaction of ER with AP1 and SP1 sites might influence pathways involved in proliferation. It could be proposed that oestradiol mediates differentiation and proliferation, respectively, through two distinct pathways, and that, as selective ER ligands are developed in the future, the aim will not only be to achieve ERα and ERβ selectivity, but also selectivity for interaction at EREs (oestradiolinduced differentiation) versus AP1 sites (oestradiolinduced proliferation functions). This attractive scheme nevertheless remains to be firmly substantiated. Of great interest is the fact that, on AP1 sites, the tamoxifen ERβ complex has opposite effects to those of tamoxifen ERα complex 43.Ifthe ERα complex inhibits proliferation, then the ERβ complex enhances it. In such a situation, the possibility cannot be excluded that, paradoxically, the presence of ERβ in a breast cancer might well impede the antiproliferative effects of tamoxifen. ER-mediated transcriptional activation involves the recruitment of several co-activators and 956 NOVEMBER 2004 VOLUME 3

8 a H10 9C-RA (ago.) BMS614 (ant.) b H11 I412 H1 H3 H12 (ago.) Figure 4 Structural basis of antagonist action. a Superposition of the ligand-binding sites in the complex between retinoic acid receptor-α (RARα) and the RARα-selective antagonist BMS649 (grey; PDB: 1dkf) and the RARγ-retinoic acid complex (pink; PDB: 2lbd). The superposition illustrates the steric clash between the antagonist BMS614 (white arrow) and Ile412 (yellow) of the receptor. b Depiction of the human ERα ligand-binding domain complexed with the agonist diethyl stilbesterol (left; PDB: 3erd) and 4-hydroxytamoxifen (right; PDB: 3ert). The protein is depicted in green, except for helix 12, which is coloured cyan and the GRIP co-activator peptide, which is coloured red. Ligand atoms are coloured white (carbon), red (oxygen) and blue (nitrogen). This figure was created with PyMol molecular graphics system. subsequent histone acetylation 18,44.Antagonist-bound ERs, on the other hand, recruit corepressors such as NCoR1 and NCoR2 and a subset of HDACs 45.It seems, therefore, that cell-type- and promoter-specific differences in coregulator recruitment determine the cellular response to SERMs. In mammary cells, both tamoxifen and raloxifene induce the recruitment of corepressors to target gene promoters; however, in endometrial cells, tamoxifen, but not raloxifene, acts like oestradiol that is, it recruits co-activators onto some genes. The oestradiol-like activity of tamoxifen in the uterus requires a high level of nuclear receptor coactivator 1 (NCOA1, also know as steroid receptor co-activator 1 (SRC1)) expression 9. ERs regulate transcription through association with coregulators, and so the overall balance and relative concentrations of co-activators and corepressors can influence the oestrogenic activity of tamoxifen. It is also likely that as-yet-unidentified tissue-specific cofactors have a crucial role. Using the yeast two-hybrid screen, one of our laboratories found that Tat-interactive protein 60 kda (Tip60) a coactivator with histone acetyl transferase activity that interacts with several other transcription factors 46 interacts with tamoxifen-bound ERβ (J.-Å.G., unpublished observations), which could explain the agonistic effects of tamoxifen. How these results are to be integrated into the complex cyclo-dynamic binding pattern of a large number of factors, including HATs and HDACs, to a given promoter 20 will be the subject of intense research over the coming years. But how do SERMs recruit different co-activator or corepressor complexes? When raloxifene is in the binding pocket of ER, its side chain is bulky enough to protrude and prevent H12 from forming a lid over the pocket (FIG. 4). Instead, H12 occupies a position on the surface of the receptor that is reserved for co-activator binding, as it is the site where the LxxLL sequence (nuclear receptorbox, see above) in co-activators docks onto the nuclear receptor LBD (FIG. 4b). When raloxifene is in the pocket, co-activators are therefore prevented from binding. One important conclusion to be drawn from studies of the crystal structure of ER LBD with various ligands is that H12 does not only have two positions ( on and off ), but can also adopt several intermediary positions. The implication of this is that ligands can be designed to have differing degrees of agonism or antagonism. Can we generalize the concept of SERMs to other nuclear receptors? Are there, for other members of the superfamily, ligands that are not pure antagonists but that have a tissue-selective pharmacology (agonists in some tissues, antagonists in others and mixed agonists/ antagonists in yet others)? As discussed above, it has been demonstrated in the case of ERs that the contradictory actions of tamoxifen in breast (antagonist) and uterus (agonist) depend, at least partially, on conditional interactions between ERα and the specific co-activator SRC1, which is present at higher levels in uterine cells than in mammary gland cells 9.In this example, the selectivity of the compounds depends on the LBD conformation of the SERM-receptor complex; the promoter context of the target genes; and the availability of a specific coregulator 47. In principle, such a mechanism might work for other nuclear receptors, and evidence for such a mechanism can be seen in early experiments that characterized the cell-type-specific effects of AF1 and AF2 48 (see also REF. 1 for a review). We can therefore generalize from the concept of a SERM and define a selective nuclear receptor modulator (SNuRM) as a nuclear receptor ligand that induces tissue-selective agonist or antagonist activity of a nuclear receptor. Indeed, research in this area is very active for PPARs 49, androgen receptors 50 and progesterone receptors 51.A striking case of a selective PPARγ modulator (SPARM) is N-(9-fluorenylmethyloxycarbonyl)-L-leucine (F-L-Leu). Interestingly, two molecules of this compound bind to the LBD of PPARγ and induce a particular allosteric conformation of this domain that results in differential cofactor recruitment, distinct pharmacological properties and a specific pattern of target gene activation 52.Although the basis for its NATURE REVIEWS DRUG DISCOVERY VOLUME 3 NOVEMBER

9 LYMPHOMATOID PAPULOSIS A chronic lymphoproliferative disease of the skin. action is still incompletely understood, F-L-Leu improves insulin sensitivity in normal diet-induced diabetic mice and in the classic diabetic db/db mice, in which it has a lower adipogenic activity. Selective progesterone receptor modulators (SPRMs), which have both agonist and antagonist activities depending on the site of action, are currently being studied for their effects on endometrial growth, endometrial vascular development, the hypothalamic pituitary ovarian axis and cervical integrity. SPRMs could prove useful in the treatment of endometriosis and fibroids, as well as for postmenopausal hormone replacement therapy and the treatment of dysfunctional uterine bleeding 53.Even if much work still has to be done, SNuRMs might represent the Holy Grail of nuclear receptor pharmacology. The results of some recent epidemiological studies on the health risks and benefits of combined oestrogen and progestin replacement therapy for post-menopausal women, which indicate that it could increase the risk of invasive breast cancer, will provide a strong impetus for the development of even more selective SNuRMs 54. RXR heterodimers Numerous nuclear receptors bind to DNA as heterodimers with RXRs, which are believed to be cognate receptors for 9-cis retinoic acid (although evidence for alternative natural RXR ligands has recently been provided 55 ). In contrast to homodimerization, heterodimerization allows, in principle, fine-tuning of nuclear receptor action by using combinatorial sets of ligands, and regulation of alternative target-gene repertoires, and therefore provides interesting pharmacological opportunities. However, although RAR agonists can autonomously activate transcription through RAR RXR heterodimers, RXR is unable to respond to RXRselective agonists in the absence of an RAR ligand. Consequently, RXR-selective ligands alone could not trigger RXR RAR heterodimer-mediated retinoic-acidinduced events in various cell systems 3,32.Similarly, RXR cannot autonomously respond to its ligand in the corresponding THR and VDR heterodimers, unless those heterodimeric partners are liganded. The biological significance of this RXR subordination or silencing is presumably to avoid confusion between retinoic acid, thyroid hormone and vitamin D 3 signalling pathways. RXR subordination is, however, not due to an inability of RXR to bind its cognate ligand in DNA-bound heterodimers, as suggested 56,because RXR ligand binding has been demonstrated to occur in such complexes A recent study showed that although RXR can bind its ligand and recruit co-activators in heterodimers with aporar, in the usual cellular environment corepressors do not dissociate from heterodimeric aporar but compete with co-activators for binding 3.Notably, in certain cells and for complexes with the RARβ isotype, RXR subordination can be weak and consequently rexinoids can activate transcription through aporar RXR. Whether and how this distinct characteristic of RARβ is linked to its presumptive role as a tumour supressor remains to be established (see REF. 60 for a review). The recent elucidation of the RARβ LBD crystal structure will facilitate the development of selective agonists and antagonists to study its tumour-suppressive role in relevant animal models 61. The only way for RXR to modulate transactivation in response to its own ligand in RXR RAR heterodimers is, therefore, through synergy between RXR ligands and RAR ligands due to increased interaction efficiency of two nuclear-receptor boxes in a single coactivator molecule with both holorar and holorxr of the heterodimer. Notably, RAR antagonists can also synergize with rexinoid agonists and can activate transcription of endogenous target genes 32.All of these observations demonstrate that RXR is a transcriptionally competent partner in RAR RXR heterodimers. However, RXR subordination might not apply to all apo-nuclear receptor partners, as the ligand-induced RXR activity was apparently permissive in heterodimers with farnesoid X receptor (FXR), LXR, PPAR or NR4A family members 62.Yet another interesting and so far unique mechanism by which RXR can regulate transcription is seen with the RXR LXR heterodimer; in this example, apo-rxr can change the conformation of apo-lxr, which leads to a transcriptionally active RXR LXR heterodimer 63.The precise nature of the signal governing this mechanism is not known. The possibility of modulating RXR heterodimer activity through rexinoids has inspired significant research in this direction, not least because the toxicity of rexinoids is lower that that of pan-retinoids (that is, retinoids that are not selective for specific subtypes) 64.Interestingly, by acting through the PPARγ RXR heterodimer, rexinoids are insulin sensitizers in rodent models of non-insulin-dependent diabetes mellitus 65.Although the initial rexinoid bexarotene (LGD1069), which has residual RAR agonist activity, produces significant side effects in this rodent model (including pronounced increases in triglycerides and profound suppression of the thyroid hormone axis), recently synthesized rexinoids have been reported to retain the insulin-sensitizing activity but with substantially reduced side effects 66. As with retinoids, rexinoids are also active as anticancer drugs; they can be used either alone, as in the therapy of cutaneous T-cell lymphoma 67 and LYMPHOMATOID PAPULOSIS 68,or as the panrar RXR agonist alitretinoin (9-cis retinoic acid) for the therapy of Kaposi sarcoma 69. In addition, there is initial evidence that rexinoids can extend the therapeutic profile of existing signalling drugs when used in combination 60,70 (see REF. 71 for a recent review on synthetic retinoids). Retinoids and rexinoids have been discussed as potential candidates for cancer chemoprevention in at-risk patients, particularly with respect to the prevention of oral cancers, and cancers of the head and neck, and lung 31,72 74.Moreover, in a randomized placebo-controlled study, the loss of RARβ expression, which is considered to be a biomarker of pre-neoplasia 75, could be significantly reversed in former smokers by treatment with 9-cis retinoic acid, but not with 13-cis retinoic acid 76.The recent finding that retinoic acid can induce a cancer-cell-selective apoptosis program 13, and 958 NOVEMBER 2004 VOLUME 3

10 Box 1 Nomenclature of nuclear receptors Given the complexity of the nuclear receptor superfamily, how can we define a logical and useful classification of nuclear receptors in terms of pharmacology? Using a pure pharmacological perspective based on the action of ligands would have been difficult owing to heterodimer formation with several types of monomeric receptor (for example, RXR RAR or RXR THR) that are pharmacologically active in binding ligands. Rather, it has proven useful to construct a classification on the basis of nuclear receptor sequences using molecular phylogeny. The phylogeny of the nuclear receptor superfamily allows the definition of 6 subfamilies of receptors that can be subdivided into 28 groups, each of which clusters several paralogous genes. This is also useful for the establishment of a nomenclature that can avoid the use of numerous different names for the same receptor 112. The systemis based on the nomenclature system that was developed for cytochrome P450 by Daniel W. Nebert and his colleagues. It has turned out to be convenient and flexible, and allows the inclusion of an ever-increasing number of cytochrome P450 genes and proteins. Each receptor is described by the letters NR (for nuclear receptor ) and a three-digit identifier: this denotes the subfamily to which a given receptor belongs (indicated by the first digit, an Arabic numeral), the group (denoted by capital letters) and the individual gene (again denoted by Arabic numerals). For example, human thyroid hormone receptor-α,which belongs to subfamily I, group A and which is the first described gene in that group, is called NR1A1. This system is flexible enough to integrate nuclear receptors from invertebrates as well as sequences generated from genome projects for which no biological data are yet available.(readers interested in following the evolution of this nomenclature should consult the Nuclear Receptor Nomenclature Homepage and Nurebase, listed in the further information.) This new nomenclature, which was proposed in 1999, has been endorsed by the major groups in the field and by the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR) as a provisional working nomenclature. The nomenclature is being assessed within NC-IUPHAR to refine the nomenclature for the human nuclear receptors. This will be useful for generating a classification system relevant for pharmacologists, despite the fact that it contains both real receptors, in a pharmacological sense, and orphan members of the superfamily. To start this merging of the two different but complementary nomenclature approaches, several NC-IUPHAR sub-committees have been formed for the main types of nuclear receptor to further clarify receptor nomenclature in an effort to integrate both structure and function. the observation that the tumour suppressor interferon regulatory factor-1 mediates the action of retinoic acid on the death ligand TNF-related apoptosis-inducing ligand (TRAIL) 77, further underscores the potential of retinoids in chemotherapy and chemoprevention. Orphan receptors and endogenous ligands Orphan receptors are important for nuclear receptor pharmacology for three main reasons: they modulate the action of other nuclear receptors and can therefore modify their pharmacology; they might become liganded receptors because synthetic/endogenous ligands could be discovered; and studies of their biology have refined nuclear receptor ligand signalling paradigms. ERRs were the first identified orphan receptors 78.As their name indicates, they are closely related at the sequence level to the oestrogen receptors, yet they do not bind to, and are not activated by, oestrogens. Recent studies have indicated that oestrogenic compounds, such as diethylstilbestrol and 4-hydroxytamoxifen, can bind to ERRβ and ERBγ,but not ERBα, and inhibit their transcriptional activity 79,80.Interestingly, several lines of evidence indicate that ERRs interact both physically and functionally with ERs and modulate their action 81 : ERRs and ERs share overlapping but not identical sets of HREs; ERRs bind to half-site sequences called SFREs, whereas ERs bind as homodimers to the well-known EREs 82 ; and it has been shown that ERRs can bind to EREs and, more strikingly, that ERα,but not ERβ, can also bind to the SFRE site. Taken together, these observations indicate that complex crosstalk might exist between the two types of receptor and that oestrogens can regulate target genes from promoters that are devoid of EREs. Given that tamoxifen binds and represses both ERs and ERRs (albeit not with the same efficiency), it is clear that its action in a given tissue will depend on many factors. This could be relevant to the pharmacology of the drug, because ERRs are strongly expressed in bone, a classic oestrogen target tissue with low ER levels 83,84. In some cases, specific ligands have been identified for orphan receptors. Since the first cloned receptors (for example, ER, GR and TR) are receptors for hydrophobic hormones that bind with a high affinity (K d in the nanomolar range) and specificity, ligands for orphan receptors were searched by screens designed to find molecules with similar characteristics. Nevertheless, most of the orphan receptors for which ligands were found (for example, PPARs, PXR/CAR and so on) bind these molecules with poor affinity (K d in the micromolar range) and have a broad specificity. PPARs, for example, can accommodate many natural and synthetic molecules that fill only a very small portion (20%) of a very large pocket (1,600 Å 3 for PPARγ). As mentioned above, even two molecules of F-L-Leu can bind in two different regions of the PPARγ LBD. A similar situation holds true for PXR and CAR, which are activated by a wide variety of xenobiotics and promote their degradation by activating cytochrome P450 genes in the liver. To distinguish them from the classic high-affinity receptors such as steroid or thyroid hormone receptors, these receptors are now referred to as sensors because their function is probably to monitor a given physiological status of the organism (for example, the amount of fatty acids or cholesterol) and to finely tune homeostasis 85,86. NATURE REVIEWS DRUG DISCOVERY VOLUME 3 NOVEMBER