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The Therapeutic Potential of Nuclear Receptor Modulators for Treatment of Metabolic Disorders: PPARg,RORs,andRev-erbs

David P. Marciano,1 Mi Ra Chang,1 Cesar A. Corzo,1 Devrishi Goswami,1 Vinh Q. Lam,1 Bruce D. Pascal,1 and Patrick R. Griffin1,* 1Department of Molecular Therapeutics, The Scripps Research Institute, Scripps Florida, Jupiter, FL 33458, USA *Correspondence: pgriffi[email protected] http://dx.doi.org/10.1016/j.cmet.2013.12.009

Nuclear receptors (NRs) play central roles in metabolic syndrome, making them attractive drug targets despite the challenge of achieving functional selectivity. For instance, members of the thiazolidinedione class of insulin sensitizers offer robust efficacy but have been limited due to adverse effects linked to activation of genes not involved in insulin sensitization. Studies reviewed here provide strategies for targeting subsets of PPARg target genes, enabling development of next-generation modulators with improved therapeutic index. Additionally, emerging evidence suggests that targeting the NRs ROR and Rev-erb holds promise for treating metabolic syndrome based on their involvement in circadian rhythm and metabolism.

Metabolic Disorders 2006). Therapeutic strategies to directly combat obesity have The percentage of the global population categorized as obese demonstrated successful outcomes in the clinic by suppressing has skyrocketed over the last two decades. This trend is pre- either appetite or absorption of calories (Berlie and Hurren, dicted to continue as developing nations increasingly adopt 2013; Carter et al., 2012; Hung et al., 2010); however, adverse more sedentary lifestyles and gain easier access to high-calorie effects and other challenges persist with these approaches diets. As recently as 10 years ago in the United States, obese (Bloom et al., 2008; Heal et al., 2009; Jain et al., 2011). Recent adults (defined as BMI >30) made up less than 12% of the pop- efforts to target obesity have expanded to therapeutic ap- ulation. Now more than 20% of the adult population meets the proaches that include restoring dysregulated circadian rhythms CDC criteria for obesity, and greater than 40% are considered that are associated with obesity (Maury et al., 2010), and induc- overweight. Metabolic syndrome is characterized as a clus- tion of brown adipose tissue (BAT) or browning of white adi- tering of factors associated with an increased risk of cardiovas- pose tissue (WAT) resulting in dissipation of energy as heat cular disease and stroke, and is becoming more common (Seale and Lazar, 2009). Many of these strategies evolved (Moller and Kaufman, 2005). The essential risk factors that from our increased understanding of nuclear receptor (NR) constitute metabolic syndrome are age, atherogenic dyslipide- function, and they represent opportunities for therapeutic mia (high triglycerides and low HDL-C), hypertension, elevated development. plasma glucose, a prothrombotic state, and a proinflammatory state. According to current views, there are two major underly- Nuclear Receptors as Therapeutic Targets for Metabolic ing causes of metabolic syndrome: obesity and type 2 diabetes Syndrome mellitus (T2DM) (Grundy et al., 2005). Obesity is conceptually NRs are a unique superfamily of ligand-dependent transcription defined as an excess of body lipid of sufficient magnitude to factors that control a diverse set of biological activities by trans- impair health and longevity. T2DM is a chronic metabolic disor- lating dietary and endocrine signals into changes in expression der that results partly in the inability of the body to respond of gene networks. NRs are attractive therapeutic targets for the adequately to circulating insulin, a condition termed insulin treatment of metabolic syndrome because their dysfunction resistance. The comorbidities of metabolic syndrome will due to naturally occurring mutations can result in metabolic dis- continue to strain global health care systems and require the orders (Gurnell, 2003; Hegele et al., 2002; Savage et al., 2003), development of alternative strategies to combat this epidemic. and their activity can be robustly modulated by small lipophilic The current standard of care for treating metabolic syndrome molecules that can be substituted by exogenous synthetic includes pharmacologic intervention and lifestyle modification small molecules. NRs are the molecular target of approximately such as nutritional consultation and modification of diet, 10%–15% of drugs currently approved by the FDA, highlighting rigorous weight control, and increased regular exercise. their tractability for therapeutic intervention (Overington et al., Modest weight loss can result in an improvement in metabolic 2006). parameters (Grundy et al., 2004). However, weight loss and ex- There are 48 NRs in the human genome that are all thought to ercise often are not sufficient due to poor compliance and share a common evolutionary origin as evidenced by the signif- perhaps confounding genetic factors (Bouchard, 1988; Moller icant sequence homology and conserved cellular function et al., 1996). Pharmacologic approaches have focused on the across the superfamily (Figure 1). NRs are characterized by a use of combination therapy for diabetes treatment in an effort multidomain architecture comprised of an N-terminal ligand- to postpone the inevitable need for insulin therapy (Gavin, independent Activating Function 1 (AF1) domain, DNA-binding

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Figure 1. Sequence Alignment of Nuclear Receptor Superfamily Alignment of all 48 human NR receptors demonstrates significant conservation of the DBD and LBD across the superfamily, with significant divergence of the A/B, Hinge, and F domains. All receptors are aligned by their DNA binding domains and are drawn to scale based on length of amino acid sequence.

domain (DBD), hinge, and ligand-binding domain (LBD) contain- ing recruitment of the basal transcription complex to the initiation ing the ligand-dependent AF2 (Evans, 1988). The AF1 and hinge site of NR target genes (Spencer et al., 1997). In contrast, are the most divergent in sequence and length across the super- corepressor proteins like the NR corepressor (NCoR) and family and are considered intrinsically disordered (Krasowski silencing mediators of retinoid and thyroid (SMRT) contain an et al., 2008), and their function and significance have been L/IxxI/VI motif referred to as ‘‘CoRNR boxes,’’ which interact reviewed previously (Clinckemalie et al., 2012; Moore et al., with high affinity at AF2 when the receptor is in the inactive 2006; Tremblay et al., 1999; Wa¨ rnmark et al., 2003; Zwart conformation (Hu and Lazar, 1999). SMRT and NCoR recruit his- et al., 2010). The DBD is the most highly conserved sequence tone deacetylase 3 (HDAC3), which keeps chromatin compact among NRs and contains two zinc-finger motifs to bind distinct leading to repression of basal transcriptional activity (Privalsky, DNA response elements. NR response elements are commonly 2004). arranged as either direct or inverted repeats of a consensus half-site (RGGTCA; R = purine). NRs can bind DNA as mono- NR Ligand Binding mers, homodimers, or heterodimers with a member of the NRs are classified as ligand-dependent transcription factors, as retinoid X receptor (RXR) subfamily. changes in their conformational dynamics induced by binding The LBD is often the focus of drug-discovery efforts and is ligand ultimately drive downstream transcriptional events. In structurally conserved across the superfamily and is described the apo state, NRs typically have high affinity for corepressor as having three stacked a-helical sheets that create an internal leading to transcriptional repression. Activation of NRs by hydrophobic cavity to which small-molecule ligands can bind agonist ligands occurs primarily through conformational (Moore et al., 2006). The ligand-dependent AF2 structural changes in AF2 that displace corepressor and recruit coactiva- element contained in the LBD is the surface of the receptor tors. Alternatively, ligands that repress NR transcriptional directly involved in interactions with coactivator and corepressor activity below basal level through recruitment of corepressor proteins that either have intrinsic chromatin remodeling activity are termed inverse agonists (Germain et al., 2006). The or tether in such enzymes. Coactivator proteins contain a com- Rev-erb subfamily of ‘‘constitutive repressors’’ is an exception, mon LXXLL motif known as ‘‘NR boxes,’’ which interact at AF2 as repressive ligands that induce increased affinity for NCoR when the receptor is in an active conformation (Heery et al., are considered agonists (Woo et al., 2007). 1997). Coactivators like steroid receptor coactivator 1 (SRC-1) Over the past two decades it has become clear that there is can act locally to acetylate histones, unraveling DNA and allow- significantly more complexity to the functional regulation of

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PPARa is a major regulator of lipid metabolism in the liver and is activated under conditions of energy deprivation and pro- longed fasting to initiate ketogenesis (Kersten et al., 1999). Acti- vation of PPARa by synthetic ligands, including the fibrate drugs used to treat hyperlipidemia, promote uptake, utilization, and catabolism of fatty acids via increased expression of classic target genes PDK4, ACOX1, and CPT1 (Rakhshandehroo et al., 2010; Staels et al., 1998). The therapeutic potential of PPARa for the treatment of metabolic syndrome has been reviewed previously (Cheng and Leiter, 2008; Hiukka et al., 2010; Staels and Fruchart, 2005). PPARd (also called PPARb) is highly expressed in intestinal epithelium, liver, and keratinocytes, consistent with its significant biological role in these tissues, and is also detected at lower levels in the brain, skin, kidney, lung, and testes (Girroir et al., 2008). It has been reported that forced PPARd activation in trans- Figure 2. Mechanisms of PPARg Functional Control genic models protects against obesity and induces an exercise- Ligand-mediated control of PPARg can be achieved through classical AF2 activation, modulation of PTM status, RXRa, cofactor, and DNA affinity. like metabolic status, spurring interest in the development of PPARd modulators for the treatment of metabolic syndrome (Wang et al., 2004). Studies in animals and humans demonstrate NRs than simply a ligand-induced on/off switch. Ligands not only that PPARd activation with synthetic ligands like GW501516 differentially affect the conformational dynamics of AF2 leading exerts desirable effects that include reduced weight gain, to various degrees of activation but can also impact NR affinity increased skeletal muscle metabolic rate and endurance, for DNA (Thuillier et al., 1998; Zhang et al., 2011), receptor degra- improved insulin sensitivity, and reduced atherogenic inflamma- dation (Hauser et al., 2000), interaction with cofactor proteins tion (Rise´ rus et al., 2008; Tanaka et al., 2003). The potential of (Ohno et al., 2012), and modulation of posttranslational modifi- PPARd as a therapeutic target for the treatment of metabolic cation (PTM) status (Choi et al., 2010; Choi et al., 2011), a central syndrome has been reviewed previously (Reilly and Lee, 2008). focus of this review (Figure 2). NR ligands can also demonstrate PPARg is the most well-characterized member of the PPAR tissue-specific effects as a result of differing cofactor popula- subfamily, as its central role in glucose metabolism and fatty tions, NR subtypes, and isoforms (Stossi et al., 2004; Tee acid storage has made it a desirable pharmacological target. et al., 2004). This exceptional biological complexity in response There are two PPARg isoforms, PPARg1, which is expressed to ligand binding is the primary challenge of translating readily ubiquitously except for muscle, and PPARg2, which is ex- identifiable, high-affinity NR ligands into clinical therapeutics. pressed primarily in WAT under normal metabolic conditions Understanding this complexity may afford the opportunity to (Fajas et al., 1997). PPARg2 is often called the ‘‘master regulator develop functional selective modulators that translate into of adipogenesis’’ because it is required to drive the terminal dif- improved drugs. Here we review the evolution of PPARg tar- ferentiation of preadipocytes toward maturation (Chandra et al., geted ligands for the treatment of T2DM from blunt 2008). Fat-specific PPARg knockout mice fail to generate adi- instruments (super agonists) that carry significant adverse pose tissue in response to high-fat diet, demonstrating the effects, to next-generation ligand classes that selectively modu- receptors’ critical role in regulating fatty acid storage and late receptor function with improved therapeutic index. Similar glucose metabolism (Jones et al., 2005). The proper function of strategies will likely apply to therapeutic development efforts PPARg is critical to metabolic homeostasis, as genetic variants for ROR and Rev-erb targeted ligands, for which recent studies in humans can lead to familial partial lipodystrophy, an extreme reviewed here demonstrate promise for the treatment of meta- monogenic form of metabolic syndrome (Jeninga et al., 2009). bolic syndrome. Interestingly, no general defects are observed in transgenic het- erozygote PPARg knockout mice that demonstrate protection The PPAR Subfamily of NRs from high-fat-diet-induced obesity, fatty liver, and insulin resis- The peroxisome proliferator-activated receptor (PPAR) NR sub- tance as a result of reduced triglyceride content in WAT, skeletal family was identified with the discovery that a diverse class of muscle, and liver (Akune et al., 2004; Kubota et al., 1999; rodent hepatocarcinogens induced proliferation of peroxisomes Yamauchi et al., 2001). Activation of PPARg by endogenous (Issemann and Green, 1990). Three PPAR subtypes have been ligands which may include polyunsaturated fatty acids (Kliewer identified—PPARa (NR1C1), PPARb/d (NR1C2), and PPARg et al., 1997; Krey et al., 1997), prostanoids like 15-deoxy- (NR1C3)—each with unique expression profiles, ligand speci- D12,14 prostaglandin J2 (15-dPGJ2) (Forman et al., 1995; ficity, and functional roles (Berger and Moller, 2002). These Kliewer et al., 1995), and oxidized fatty acids like 9-HODE and receptors regulate transcriptional activity by forming obligate 13-HODE (Nagy et al., 1998) leads to increased expression of heterodimers with RXR and binding to specific DNA response many target genes including aP2, GLUT4, PEPCK, C/EBP, elements (PPREs) that consist of two repeats of the core Adipsin, and adiponectin (Perera et al., 2006; Tontonoz et al., AGGTCA sequence separated by one nucleotide (DR-1) in the 1994a, 1994b, 1995). The promiscuity of PPARg activation is a promoter or enhancer regions of their target genes (Kliewer major challenge of avoiding adverse effects when pharmaco- et al., 1992; Mangelsdorf and Evans, 1995). logically targeting the receptor, and therapeutic development

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efforts now aim to selectively modulate subsets of PPARg target retain comparable antidiabetic efficacy to full agonist TZDs? genes by harnessing tissue-specific effects (Festuccia et al., And to the broader point, why is synthetic activation of PPARg 2009; Montague, 2002; Zı´dek et al., 2013) and nonclassical so effective at insulin sensitization, when many obese and AF2 mechanisms of receptor control (Ahmadian et al., 2013; diabetic patients that respond to TZDs have no defects in the Cariou et al., 2012; Rosenson et al., 2012). receptor?

PPARg Is the Pharmacological Target of the TZDs Posttranslational Modification of PPARg PPARg is the pharmacological target of the antidiabetic thiazoli- Some aspects of the PPARg paradox were addressed by the dinediones (TZDs) class of drugs that include rosiglitazone discovery that inflammatory cytokines associated with obesity (Avandia) and pioglitazone (Actos) (Lehmann et al., 1995). The activate the protein kinase cyclin-dependent kinase 5 (Cdk5), precursor lead to the TZDs, 2-chloro-3-phenylpropanoic acid resulting in phosphorylation of PPARg at S273 (pS273) (Choi ethyl ester (AL-294), was discovered through in vivo screening et al., 2010). This modification of PPARg correlated with repres- efforts against insulin-resistant rodent models (Ikeda et al., sion of a subset of target genes shown to be dysregulated in 1981; Iwatsuka et al., 1970; Kawamatsu et al., 1980). As such, obesity, including repression of the insulin-sensitizing adipokine, the molecular target of the TZDs and their mechanism of action adiponectin. The efficacy of antidiabetic PPARg ligands such as were unknown until several years after their discovery (Lehmann rosiglitazone and the partial agonist MRL24 were shown to et al., 1995). TZDs have proven robust as insulin sensitizers and correlate with their ability to block pS273, independent of clas- work synergistically with other T2DM therapeutic approaches sical receptor transcriptional agonism (Choi et al., 2010; Rocchi (Elte and Blickle´ , 2007; Garber et al., 2007; Hanefeld, 2007; et al., 2001). Based on these findings, an alternative class of Zinman et al., 2007, 2009). The mechanism by which TZDs derive high-affinity PPARg-targeting insulin sensitizers was developed efficacy is not fully understood but is thought to be through a that effectively block pS273 while avoiding classical AF2-driven combination of induction of insulin-sensitizing adipokines, anti- receptor activation, termed functional selective PPARg modula- inflammatory effects, and fatty acid partitioning from muscle to tors (FSPPARMs). SR1664 is a representative compound from adipocytes (Girard, 2001; Martin et al., 1998; Saraf et al., this class that, in terms of insulin sensitization, was shown to 2012). These drugs have also been shown to have positive be equally efficacious as rosiglitazone, without causing weight effects on cardiovascular disease (Hamblin et al., 2009; Plutzky, gain or hemodilution in vivo (Choi et al., 2011). SR1664 was 2011), Alzheimer’s disease (Heneka et al., 2011), Parkinson’s derived from the potent partial agonist SPPARM GSK538 disease (Carta et al., 2011), cancer (Blanquicett et al., 2008), (Lamotte et al., 2010) and was designed to actively antagonize and the browning of fat (Ohno et al., 2012) through various the receptor through an AF2 clash to ablate classical agonism PPARg-mediated mechanisms. Unfortunately, concerns over as previously described for the estrogen receptor (Brzozowski adverse side effects including weight gain (Fonseca, 2003), et al., 1997; Shiau et al., 1998). Thus, this class of compounds edema (Nesto et al., 2003), plasma volume expansion (PVE) is functionally selective in that they are devoid of classical AF2 (Staels, 2005), increased risk of congestive heart failure (Nesto agonism (do not drive expression of adipogenic genes) yet et al., 2003), and bone fracture (Aubert et al., 2010) have limited potently modulate a specific PTM to control expression of a their utility. In particular, rosiglitazone has been restricted or unique subset of PPARg target genes. withdrawn from most markets due to the increased incidence The picture that emerges from these findings is that the design of myocardial infarction, likely a result of increased backpressure of compounds targeting PPARg must move beyond the tradi- on the heart due to edema and PVE (Hsiao et al., 2009; Lips- tional paradigm of agonist/antagonist and include a focus on combe et al., 2007; Loke et al., 2011). Pioglitazone, a compara- the modulation of PTMs. Recent work from the Manglesdorf tively less potent PPARg agonist with modest PPARa activity, and Kliewer labs suggesting a path toward more selective mod- has demonstrated improved cardiovascular outcomes, ulators has demonstrated that both beneficial and adverse providing a potential mechanistic explanation for this improved effects of TZDs are a product of the induced expression of Fibro- therapeutic index (Juurlink et al., 2009; Winkelmayer et al., blast Growth Factor-21 (FGF21) and its ability to modulate 2008). Based on these findings, efforts have focused on the receptor activity through SUMOylation of K107 (Dutchak et al., pharmacological development of a class of ‘‘selective PPARg 2012; Qiang and Accili, 2012). A thorough review of PPARg modulators’ (SPPARMs). PTMs to AF1 has previously been published (van Beekum et al., 2009). Here we focus on PTMs to the ligand binding SPPARMs and the PPARg Paradox domain that have been demonstrated to be modulated by SPPARMs display reduced transcriptional activity in reporter synthetic ligand binding as strategies for targeting metabolic assays (partial agonists), exhibit potent insulin sensitization on syndrome. the same order as TZDs, and are anitiadipogenic with reduced adverse effects in animal models (Rangwala and Lazar, 2002). SUMOylation of the PPARg LBD Leads to Repression Importantly, SPPARMs demonstrate that the insulin-sensitizing of NFkB Inflammatory Genes effects of PPARg-targeted ligands can be separated from the Efforts to elucidate the mechanism by which PPARg agonists are adverse adipogenic effects. Several SPPARMs have advanced capable of simultaneously activating PPARg and repressing into clinical trials and may hold therapeutic promise (Gregoire NFkB target genes led to the discovery of a SUMOylation site et al., 2009; Motani et al., 2009); however, mechanistically they at lysine 365 on the ligand binding domain (Pascual et al., create a paradox. If PPARg-targeted ligands derive antidiabetic 2005). Rosiglitazone and the SPPARM GW00072 were reported efficacy by activating the receptor, why do these poor agonists to promote SUMOylation of the receptor, targeting PPARg to the

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NCoR/HDAC3 complex on NFkB inflammatory gene promoters. tative synthetic ligands have been reported, leading to the obser- This association prevents ubiquitylation and proteosomal degra- vation that full agonist TZDs and non-TZDs like MRL20 make a dation of the repressor complex that would typically lead to conserved hydrogen bond with Y473 on helix 12, while partial target gene activation, instead maintaining a repressed state agonist SPPARMs do not (Bruning et al., 2007; Einstein et al., (Pascual et al., 2005). Ligand-induced repression of these 2008; Nolte et al., 1998). These crystal structures have been NFkB inflammatory target genes is associated with the desirable valuable in identifying differences between TZDs and SPPARMs; antidiabetic and antiatherogenic efficacy of rosiglitazone. This however, no large-scale changes in the global protein fold of finding highlights the multiple roles of PPARg, which acts not PPARg have been observed to explain the differential effects only as a transcription factor to activate its own target genes on corepressor and coactivator affinities (Bruning et al., 2007). but also as a cofactor capable of regulating the activity of other The full-length crystal structure of PPARg in complex with transcription factors. RXRa and DNA was a significant achievement and provided the first insight into the interplay between various receptor Acetylation of the PPARg LBD Controls Induction domains, DNA, and ligands (Chandra et al., 2008). However, of Brown Adipose Tissue solution structures of the same full-length PPARg complex Adipose tissue in mammals can be distinguished as either WAT obtained using SAXS, SANS, and FRET demonstrate an used to store excess energy or BAT that acts to dissipate energy extended asymmetric shape highlighting the dynamic nature of in the form of heat. The strategy to promote the development of these interactions and the importance of utilizing complimentary BAT pharmacologically is conceptually appealing as a defense structural approaches when developing structure activity rela- against obesity and has been demonstrated viable in animal tionships (SAR) (Rochel et al., 2011). It is important to note that models (Enerba¨ ck, 2010; Fukui et al., 2000; Sell et al., 2004; none of these structures have been solved for the various Wilson-Fritch et al., 2003) and in humans (Bogacka et al., PTMs of the receptor mentioned above. 2005). PPARg ligands have been shown to drive the conversion In efforts to better understand the complexity of functional of WAT to BAT by modulating association with PRDM16, a factor response induced by ligand binding, hydrogen/deuterium that controls the development of classical brown fat (Ohno et al., exchange mass spectrometry (HDX-MS) has been applied to 2012). Despite the observation that full agonist TZDs are characterize changes in protein dynamics (Bruning et al., 2007; most efficacious at driving the ‘‘browning’’ effect, efforts to Dai et al., 2008, 2009; Wright et al., 2011; Zhang et al., 2010, develop functional selective ligands with improved therapeutic 2011). Some of these studies have demonstrated that the degree index are buoyed by the finding that the association of to which ligand binding stabilizes the AF2 domain correlates with PPARg:PRDM16 is regulated by recently identified PTMs (Qiang receptor activation (Bruning et al., 2007; Choi et al., 2011), while et al., 2012). other modulators stabilize different regions of the LBD. This Two acetylation sites on the PPARg ligand binding domain approach has been used to demonstrate that at least some (lysine 268 and lysine 293) were recently identified in a report partial-agonist SPPARMs activate PPARg using a helix 12- addressing the mechanism by which NAD-dependent deacety- independent mechanism (Bruning et al., 2007), a finding now lase SirT1 gain of function mimics the insulin-sensitizing and supported by other reports (Klein et al., 2005; Puigserver et al., ‘‘browning’’-of-WAT effects associated with TZDs (Qiang et al., 1998; Waku et al., 2010). NMR studies have also demonstrated 2012). The acetylation state of these sites was found to be asso- that ligand and receptor dynamics on a subsecond timescale ciated with physiological cues such as low temperature, which affect the graded transcriptional output of PPARg modulators triggers reduced acetylation and increased browning of WAT, (Hughes et al., 2012). The application of complimentary struc- while high-fat diet led to increased acetylation, reduced SirT1 tural approaches will continue to be critical in understanding association, and decreased insulin sensitivity. The TZDs rosigli- the complex functional control of PPARg and other NRs by tazone and troglitazone were both reported to increase associa- endogenous and synthetic ligands. tion of PPARg:SirT1, reducing acetylation of both K268 and K293. Site-directed mutagenesis studies revealed that deacety- Rev-erbs and RORs: Regulators of Circadian Rhythm, lation of K293 was requisite for association of PPARg with Lipid Homeostasis, and Metabolism PRDM16, while K268 PTM status had no effect. However, Circadian rhythm is a biological process that displays a pattern acetylation of both lysines was required to associate with of daily oscillation, is controlled by endogenous factors, and is NCoR, while deacetylation of either site was sufficient to entrainable. Circadian oscillations are necessary for an organ- displace. These findings provide a potential strategy for devel- ism’s survival, as they allow the anticipation and adaptation to oping functional SPPARMs that drive the conversion of WAT to predictable changes in light/dark cycles, activity/rest cycles, BAT by modulating acetylation of the receptor while avoiding and feeding times. The central circadian clock is located in the AF2 activation that would lead to induction of adipogenesis suprachiasmatic nucleus within the hypothalamus and directly and the associated adverse effects of TZDs. receives environmental cues from ganglion cells in the retina (Green et al., 2008). Disturbances of the circadian clock have Development of PPARg Structure Activity Relationship been associated with increased incidence of obesity, diabetes, Significant efforts have been made to structurally characterize and other disorders (Gachon et al., 2004; Sahar and Sassone- the interaction between PPARg and synthetic ligands to develop Corsi, 2009; Weldemichael and Grossberg, 2010). Hormones optimized therapeutics and better characterize the functional and adipokines are expressed and secreted in a circadian rhyth- differences between TZDs, SPPARMs, and FSPPARMs. Several mic fashion; however, their expression patterns in obese animals cocrystal structures of isolated PPARg LBD bound to represen- and humans are distorted (Ando et al., 2005; Yildiz et al., 2004).

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Figure 3. Nuclear Receptors and Circadian Rhythm Members of the PPAR, ROR, and Rev-erb NR subfamilies regulate circadian rhythms and expression of core clock genes. Therapeutic targeting of these NRs to normalize dysregulated circadian rhythm may afford a treatment strategy for metabolic syndrome.

constitutive repressors and RORs as constitutive activators to fine-tune the expression of BMAL1, CLOCK, and several other oscillating gene networks involved in glucose and lipid metabolism (Cho et al., 2012; Guillaumond et al., 2005; Jetten, 2009; Sato et al., 2004; Solt et al., 2010). Here we review the cur- rent understanding of the ROR and Rev- The observation that shift workers, who typically have irregular erb NR subfamilies’ role in metabolic disorder, and the most sleep and eating times, disproportionately display impaired insu- recent efforts to develop synthetic ligand modulators to serve lin sensitivity, higher body mass, and hypertension highlights the as chemical probes and therapeutics. correlation between proper circadian clock oscillation and meta- bolic homeostasis (Gangwisch et al., 2005; Rudic et al., 2004). Therapeutic Targeting of RORs for Metabolic Syndrome Metabolic control of the circadian rhythm starts with the olig- The first member of the ROR subfamily of receptors (RORa) was omerization and binding of core clock activators brain and identified based on sequence similarities to the retinoic acid re- muscle aryl hydrocarbon receptor nuclear translocator (ARNT)- ceptor (RAR) and the RXR, yielding the name ‘‘retinoic acid like protein 1 (BMAL1), circadian locomotor output cycles kaput receptor-related orphan receptor’’ (Gigue` re et al., 1994). Two (CLOCK), and neuronal PAS domain protein 2 (NPAS2) to E box other members of this NR subfamily, RORb and RORg, were (50-CACGTC-30) enhancer elements (Church et al., 1985; subsequently identified (Hirose et al., 1994). The RORs display DeBruyne et al., 2007; Gachon et al., 2004; Hao et al., 1997). distinct patterns of tissue expression and are involved in the This event drives the activation of several clock genes, including regulation of various physiological processes. RORa is predom- Per and Cry, which heterodimerize, and at critical concentrations inantly expressed in lung, muscle, brain, heart, peripheral blood can translocate into the nucleus repressing BMAL1/CLOCK, leukocytes, spleen, liver, and ovary, whereas expression of completing the negative feedback loop in a circadian pattern RORb is limited to the central nervous system (Andre´ et al., (Ko and Takahashi, 2006). Environmental cues such as light, 1998a, 1998b). Two isoforms of RORg are found in both humans temperature, and food can induce the rapid activation of cyclic and mice (RORg1 and RORg2), with RORg2 commonly referred AMP production, Ca2+ signaling, and MAPK pathway, leading to as RORgt, as it was originally identified in the thymus (Jetten to induction of Per and Cry genes and demonstrating the com- et al., 2001), although its expression is not limited to T cells. plex interplay of circadian factors that control energy balance, RORgt, specifically RORgt2, is highly expressed in immune tis- feeding behavior, and metabolic processes (Hirota and Fukada, sue including the thymus, but there is also significant expression 2004; Panda et al., 2002; Storch et al., 2002). of RORg in the liver, skeletal muscle, adipose tissue, and kidney The RORs and Rev-erbs are considered core clock machinery (Hirose et al., 1994; Jetten, 2009). Given the specific tissue dis- because they regulate the cyclic expression of BMAL1 and tribution of each ROR receptor and their potential role in CLOCK, providing an essential link between the positive and pathophysiological conditions, there is considerable interest in negative loops of the circadian clock (Figure 3)(Charoensuksai developing synthetic ligand modulators as therapeutics. and Xu, 2010; Solt et al., 2011a). The PPARs and core clock RORa has been shown to regulate the expression of several genes have been demonstrated to crossregulate transcriptional components of the circadian clock system. These include genes outputs in peripheral tissues (Canaple et al., 2006; Gatfield et al., encoding enzymes and transporters involved in nutrient trans- 2009; Nakamura et al., 2008; Oishi et al., 2005; Wang et al., port and metabolism, cellular cholesterol homeostasis, xenobi- 2008), and CLOCK mutant mice have been shown to disrupt otic detoxification, and energy balance (Panda et al., 2002). the circadian expression of PPARa in the liver (Lemberger Furthermore, RORa regulates the expression of apolipoprotein et al., 1996; Oishi et al., 2005). Rev-erbs (a and b isoforms) and CIII, a component of HDL and very-low-density lipoprotein that RORs (a and g isoforms) are coexpressed in adipose tissue, liver, plays a role in regulation of triglyceride levels and lipoprotein skeletal muscle, and brain (Bonnelye et al., 1994; Dumas et al., lipase (LPL) activity (Raspe´ et al., 2001). Genetic, molecular, 1994; Forman et al., 1994; Moore et al., 2006). As monomers, and biochemical studies have demonstrated that RORa- both Rev-erbs and RORs occupy the same RORE ‘‘half-site’’ deficient mice develop severe atherosclerosis and have low with a 50 AT-rich region preceding the response element (Har- HDL-C, hypo-a-lipoproteinemia, muscular atrophy, and height- ding and Lazar, 1993, 1995; Moraitis and Gigue` re, 1999). The oc- ened inflammatory response (Jarvis et al., 2002; Lau et al., cupancy of the RORE is synergized between Rev-erbs acting as 1999; Steinmayr et al., 1998). More recently, RORa has been

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shown to control the expression of genes involved in lipid (Kallen et al., 2002, 2004). It is interesting to note that in spite absorption, b-oxidation, cholesterol efflux, and energy expendi- of the high sequence similarity and similar-sized ligand binding ture in skeletal muscle cells (C2C12) (Lau et al., 2004). RORa has pockets (722 A˚ 3 and 766 A˚ 3, respectively) of the RORa and also been shown to regulate the expression of FGF21, a potential RORb LBDs, cholesterol has no effect on RORb activity. pharmacological target gene for treatment of metabolic disease More recently, the crystal structures of RORg in complex with (Wang et al., 2010). 20a-, 22R-, and 25- hydroxycholesterol were reported (Jin et al., Observations in the RORasg/sg stagger mouse provide insight 2010). These oxysterols were shown to have agonist activity on into the role of the receptor in metabolic homeostasis. These the receptor driving an active AF2 confirmation that facilitates mice, which express nonfunctional RORa, are resistant to diet- recruitment of coactivator to the conserved charge clamp induced obesity (Lau et al., 2008). Analysis of these mice showed comprised of helices 3, 4, 5, and 12 of the LBD. This finding a significant reduction in the levels of expression of SREBP1c served as a critical first step toward designing synthetic RORg and FAS. SREBP1 is a critical transcriptional regulator that con- modulators and further suggests the receptors’ role as a central trols the expression of genes involved in fatty acid biogenesis, mediator of metabolism and lipid homeostasis. In contrast, the and directly regulates lipogenic enzymes such as FAS (Shimano crystal structure of RORg in complex with antagonist digoxin, et al., 1999). Interestingly, genes related to the thermogenic pro- an extract from the foxglove plant (Digitalis lanata), highlights gram such as b2-AR, UCP-1, NOR-1, and PGC1(a/b) were the molecular interactions critical for antagonizing coactivator increased in BAT in these mice (Bertin et al., 1990; Crunkhorn interaction (Fujita-Sato et al., 2011). This structure reveals that et al., 2007; Lau et al., 2008). These findings suggest that the digoxin protrudes between helices 3 and 11 of the LBD and pre- development of synthetic RORa agonist/inverse agonist ligands vents positioning of helix 12 into an active conformation. These may hold therapeutic potential for the treatment of metabolic observations provide a template by which to design synthetic disorders and in parallel will serve as useful chemical probes to small-molecule modulators of RORg. better understand the receptors’ functional role. Due to the emerging role of RORs in several human diseases, RORg, along with RORa, is expressed in skeletal muscle, a tis- considerable efforts have been made to develop synthetic exog- sue that accounts for approximately 40% of total body mass and enous ligands with improved potency, specificity, and pharma- 50% of energy expenditure and is a major site of fatty acid and cokinetics. The synthetic agonist of the liver X receptor (LXR) glucose oxidation (Rasmussen and Wolfe, 1999). It has been T0901317 was the first modest affinity synthetic small molecule shown that RORg controls expression of genes that regulate to be identified as a dual RORa/g antagonist/inverse agonist muscle activity, fat mass, and lipid homeostasis (FABP4, (Figure 4)(Houck et al., 2004; Kumar et al., 2010b; Li et al., CD36, and LPL) and plays a role in the regulation of reactive ox- 2006; Mitro et al., 2007). Removal of the sulfonamide alkyl group ygen species (ROS) (Raichur et al., 2007). Microarray analyses of from the T0901317 scaffold resulted in RORa/g antagonists/ liver tissue from RORasg/sg, RORg/, and RORsg/sg RORg/ inverse agonist devoid of LXR activity (Kumar et al., 2010a; double knockout mice (Kang et al., 2007) revealed that RORa Solt et al., 2011b). One such example, SR1001, repressed both and RORg are critical regulators of hepatic genes encoding RORa-Gal4 and RORg-Gal4 transcriptional activity with several phase I and phase II metabolic enzymes, including EC50’s 4–6 mm(Solt et al., 2011b). SR1001 inhibited the devel- 3b-hydroxysteroid dehydrogenases, cytochrome P450 en- opment of murine TH17 cells, as demonstrated by inhibition of zymes, and sulfotransferases. Mice deficient in RORg also interleukin-17A gene expression and protein production. exhibit reduced blood glucose levels (Kang et al., 2007). In dou- Furthermore, SR1001 inhibited the expression of cytokines ble knockout mice, a similar reduction in cholesterol, triglyceride, (IL17a, IL17f, IL21, and IL22) when added to differentiated and blood glucose levels was observed as compared to single murine or human TH17 cells. Finally, SR1001 effectively delayed gene knockout (Kang et al., 2007; Meissburger et al., 2011). As the onset and clinical severity of autoimmune disease in a MOG- muscle and liver are critical mediators of insulin sensitivity, lipid induced mouse model (EAE) of multiple sclerosis. HDX ex- metabolism, and energy balance (Lau et al., 2004; Ramakrishnan periments have provided the first structural insight into the et al., 2005), the pharmacological targeting of RORa and RORg mechanism of transcriptional repression, demonstrating that holds promise for the treatment of metabolic syndrome and SR1001 disrupts interaction with the receptor interacting domain associated diseases. (RID) of coactivator SRC2 (Solt et al., 2011b). Further optimization of the T091317 scaffold led to the identi- Ligand Modulation of the RORs fication of SR3335, a RORa selective antagonist/inverse agonist RORa, RORb, and RORg have long been considered orphan (Kumar et al., 2010a; Solt et al., 2012a). SR3335 significantly receptors, as endogenous ligands have yet to be unanimously inhibited the constitutive activity of RORa in a cell-based trans- agreed upon. Regardless, crystal structures of these receptors activation assay (IC50 = 480 nM) but had no effect on the activity bound to naturally occurring ligands have provided insight into of LXRa, RORb, and RORg. Diet-induced obese (DIO) mice were molecules that may be functionally relevant. The first ROR LBD treated with SR3335 for 6 days, and a pyruvate tolerance test subtype to be crystallized was RORb in complex with stearic revealed reduced plasma glucose levels compared with acid, a prevalent saturated fatty acid (Stehlin et al., 2001). Sub- vehicle-treated cohorts. Importantly, mice treated with SR3335 sequently, all-trans retinoic acid (ATRA) and a synthetic analog displayed no difference in body weight or food intake after (ALRT 1550) were identified as functional ligands (Stehlin-Gaon 7 days of treatment, indicating the effects on glucose homeosta- et al., 2003). RORa LBD crystal structures identified cholesterol sis are not secondary to weight loss and represent a metabolic and cholesterol sulfate as potential ligands and served as early response. These data suggest SR3335 is a useful chemical indicators that RORa played a central role in lipid metabolism probe for evaluating the in vitro and in vivo functions of RORa

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Figure 4. Evolution of Synthetic RORa/g Inverse Agonists Next-generation selective RORa/g duel agonist and subtype-specific inverse agonists from T0901317, a synthetic dual RORa/g inverse agonist with LXR agonist activity. T0901317 RORa IC50 0.132 mM, RORg 0.051 mM; SR1078 IC50 RORa 1–3 mM, RORg 1–3 mM; SR1001 IC50 RORa 0.172 mM, RORg 0.111 mM; SR3335 IC50 RORa 0.480 mM; SR2211 IC50 RORg 0.320 mM; SR1555 IC50 RORg 1.5 mM.

Rev-erb proteins are expressed in multi- ple tissues including adipose, liver, brain, and skeletal muscle. The orchestrated expression pattern of Rev-erba in adi- pose tissue has been demonstrated to be a critical regulator of adipogenesis (Chawla and Lazar, 1993; Laitinen et al., 2005; Wang and Lazar, 2008) and can be modulated by TZD-induced activation of PPARg (Fontaine et al., 2003). The Rev- erbs’ role in lipid metabolism and glucose homeostasis and involvement in choles- terol trafficking are also a result of their ability to modulate key enzymes in each pathway. Rev-erbs have been reported to regulate the expression of LPL, an enzyme involved in the uptake and accu- mulation of lipids in multiple organs and cell types (Babaev et al., 1999; Kern, 1997; Ong et al., 1994; Pulinilkunnil and Rodrigues, 2006). Rev-erba deficiency in mice elevates LPL levels in peripheral tis- sues (liver, muscle, and adipose tissue) correlating with increases in body weight and overall adiposity (Delezie et al., 2012). Further, the sterol regulatory element- binding protein 1c (Srebp-1c) and its and suggest that compounds like SR3335 may hold utility in the target gene fatty acid synthase (FAS), key regulators of lipogen- treatment of type 2 diabetes. Optimization of synthetic ligands esis, were reduced upon silencing of Rev-erba (Le Martelot et al., for the RORs has recently been reviewed (Kamenecka et al., 2009). Rev-erba deficiency in skeletal muscle leads to reduced 2013). It is particularly interesting that subtle changes to the mitochondrial content and oxidative function resulting in same chemical scaffold can yield ROR subtype selective ligands compromised exercise capacity, while overexpression and and alter their pharmacology from antagonists/inverse agonists pharmacological activation of the receptor led to an improve- to agonists. ment (Woldt et al., 2013). Cholesterol trafficking is partly regulated by Rev-erb proteins Therapeutic Targeting of the Rev-Erbs for Metabolic through transcriptional regulation of various lipoproteins. Syndrome APOC3, a component of HDL and VLDL that regulates triglycer- The Rev-erb subfamily of NRs consists of two members, Rev- ide levels, is present in high levels in Rev-erba null mice (Coste erba (NR1D1) (Lazar et al., 1989) and Rev-erbb (NR1D2) (Dumas and Rodrı´guez, 2002). These same mice also suffer from abnor- et al., 1994), similar in protein structure and function. The Rev- mally high VLDL and triglyceride levels. ApoA1, a significant erbs are unique NRs in that they have a truncated C terminus component of HDL necessary for the transport of cholesterol (devoid of helix 12 of AF2) which prevents association with coac- to the liver for excretion into the bile, is repressed by fibrates tivator proteins (Woo et al., 2007). Importantly, the Rev-erbs (Lefebvre et al., 2009). Fibrates, a class of hypolipidemic drugs retain the ability to complex with corepressor proteins like and known PPARa activators, increased Rev-erb mRNA levels NCoR (Zamir et al., 1996), forming a potent transcriptional 10-fold in rat livers, likely mediated through PPARa transactiva- repressor complex that is critical for proper metabolic function. tion (Gervois et al., 1999). Rev-erbs also can modulate bile acid

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a physiological ligand capable of regulating receptor function provided further molecular and mechanistic insights into the role of Rev-erbs (Burris, 2008; Raghuram et al., 2007; Yin et al., 2007). Heme binding modulates the repressive activity of Rev-erb by increasing recruitment of the corepressor NCoR and HDAC3 to DNA response elements in promoter regions of Rev-erb target genes (Guillaumond et al., 2005; Kumar et al., 2010c; Raspe´ et al., 2002; Yin et al., 2007). Heme has also been shown to promote proteasomal degradation of the re- ceptor, a critical event for adipogenesis (Kumar et al., 2010c; Wang and Lazar, 2008). The cocrystal structure of Rev-erbb in complex with heme shows that a single molecule of the por- phorin is bound, and it has been demonstrated that the receptor can adopt multiple conformational states depending on the iron oxidation state (Marvin et al., 2009; Pardee et al., 2009). Despite these findings, the utility of heme as a chemical probe to study the functional effects of Rev-erb ligand binding is limited due to its cell toxicity at high concentrations and the lack of selectivity for Rev-erb (Alayash, 2004; Kim et al., 2004). To address these shortcomings, efforts to develop synthetic Rev-erb ligands were initiated with the development of a fluores- cence resonance energy transfer (FRET) biochemical assay monitoring the interaction between the Rev-erb LBD and a pep- tide representing the first interaction domain of NCoR (NCoR 1D1) (Grant et al., 2010). Using this assay to screen targeted chemical libraries, the first synthetic agonist GSK4112 was iden- tified, and it was shown that this compound enhanced the inter- action of Rev-erbs with NCoR (Figure 5)(Grant et al., 2010; Kumar et al., 2010c; Meng et al., 2008). Importantly, GSK4112 Figure 5. Evolution of Synthetic Rev-Erb Modulators was compatible with cellular studies and was shown to inhibit Generation of synthetic Rev-erb ligands started from the discovery of heme as the expression of the circadian target gene bmal1, to repress an endogenous ligand. To date, various synthetic modulators with altered expression of gluconeogenic genes in liver cells, and to reduce function have been developed. GSK4112 EC Rev-erba 0.40 mM; SR9009 50 glucose output in primary hepatocytes (Grant et al., 2010). IC50 Rev-erba 0.67 mM, Rev-erbb 0.80 mM; SR9011 IC50 Rev-erba 0.79 mM, Rev-erbb 0.56 mM; SR8278 EC50 Rev-erba 0.47 mM. GSK4112 was also reported to induce adipogenesis in 3T3-L1 cells, leading to increased lipid accumulation and increased synthesis through regulation of cholesterol 7a-hydroxylase expression of adipogenic genes including aP2, NR1C3, and (Cyp7A1), the rate-limiting enzyme in the classic pathway of AdipoQ (Kumar et al., 2010c). Interestingly, GSK4112 was also bile acid synthesis from cholesterol in the liver (Duez et al., shown to synergize with rosiglitazone in promoting lipid accumu- 2008). Mice lacking Rev-erba have asynchronous circadian lation (Kumar et al., 2010c). rhythms, with a predisposition to diet-induced obesity, impaired More recently, the Rev-erb agonists SR9009 and SR9011 glucose, and lipid utilization leading to increased susceptibility to were described, representing chemical tools with improved diabetes (Delezie et al., 2012; Preitner et al., 2002). Depletion of pharmacokinetics over GSK4112 that were sufficient to enable both Rev-erb a/b isoforms in mice results in elevated hepatic in vivo studies (Solt et al., 2012b). Treatment of obese mice triglyceride levels triggering hepatosteatosis (Bugge et al., with SR9009 resulted in weight loss, improved dyslipidemia, 2012). Rev-erbs have been shown to regulate hepatic glucose and improved hyperglycemia. SR9009 and SR9011 were re- production, with increased heme binding demonstrated to ported to alter gene expression in several tissues including liver, repress the gluconeogenic gene PepCK in HepG2 cells (Yin skeletal muscle, and WAT. In skeletal muscle the Rev-erb et al., 2007). Synthetic modulators of Rev-erbs have been shown agonists induced expression of genes involved in fatty acid to alter expression of G6Pase, the enzyme controlling the final oxidation and glycolysis such as Cpt1b, Ucp3, Ppargc1b, step of gluconeogenesis (Grant et al., 2010; Kojetin et al., Pkm2, and Hk1. These Rev-erb agonists also decreased expres- 2011). These findings support the role of Rev-erbs in both lipid sion of the lipogenic enzymes Fasn and Scd1 and the cholester- and glucose homeostasis, making these receptors attractive tar- ologenic regulatory proteins Hmgcr and Srebf2, consistent with gets of therapeutic intervention for metabolic disorders. the decreased triglyceride and cholesterol synthesis observed in liver and WAT from treated animals. Taken together, these Rev-Erb Ligands observations suggest Rev-erb agonists increase whole-body Rev-erba and Rev-erbb were identified as orphan nuclear recep- energy expenditure, offering an approach for treatment of meta- tors based on their conserved NR domain structure and homol- bolic syndrome. ogy with other NRs (Bonnelye et al., 1994; Dumas et al., 1994; Recently, the first synthetic Rev-erba antagonist (SR8278) Lazar et al., 1989). The identification of the porphorin heme as was described to oppose the action of heme and drive

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