1、Nuclear receptors are generally classified as ligand- regulated transcription factors, as many members of the nuclear receptor superfamily serve as receptors for phys- iological ligands, including steroid hormones, lipids and fatty acids. The nuclear receptor superfamily is one of the primary classe

2、s of therapeutic drug targets for human disease. Among the drugs that target nuclear receptors are the anti-inflammatory glucocorticoids, steroidal con- traceptives and hormone replacement therapies, as well as the fibrate class of lipid-lowering agents. Members of the nuclear receptor family have a

3、 conserved modular domain structure (FIG. 1a). The binding of ligands to a region called the ligand-binding domain (LBD) causes a conformational change in this domain, which results in a cascade of downstream events. For some nuclear receptors, such as the glucocorticoid receptor and other steroid r

4、eceptors, these events include dissociation from heat shock proteins and translocation of the receptor from the cytoplasm to the nucleus. Subsequent to ligand binding, the conformational change in the receptor facil- itates the recruitment of transcriptional co-regulatory proteins to receptor-specif

5、ic gene promoter complexes to activate or repress transcription. However, many other nuclear receptors, such as thyroid hormone receptors and peroxisome proliferator-activated receptors (PPARs), are localized in the nucleus regardless of whether or not they are bound to a ligand and constitutively i

6、nteract with DNA response elements13. When various hormones such as thyroid hormones and the steroid hormones (oestrogens, progestins, glu- cocorticoids, androgens and mineralocorticoids) were identified, it was not known that they targeted members of the nuclear receptor superfamily; indeed, they w

7、ere identified before the existence of the superfamily was even known. Even today, the physiological ligands are known for only half of the nuclear receptor superfamily (of which there are 48 members in humans). The devel- opment of drugs that target ligand-regulated nuclear receptors led to the des

8、ign of many therapeutic com- pounds, which prompted substantial interest in the identification of either natural or synthetic ligands for the orphan members of the superfamily that could be used as chemical tools to probe receptor function and to understand the potential therapeutic value of these r

9、eceptors. In many cases, these efforts have led to the development of synthetic ligands with pharmacokinetic and pharmacodynamic profiles that are appropriate for their testing as therapeutic modulators in several animal models of disease. This chemical biology strategy has been successful in charac

10、terizing several orphan recep- tors as potential drug targets, including PPAR, liver X receptor (LXR), retinoid X receptor and the farnesoid X receptor (FXR; also known as bile acid receptor)46. The chemical biology strategy has recently been applied to additional orphan receptors, including REV-ERB

11、s and retinoic acid receptor-related orphan receptors (RORs). These two classes of nuclear receptors share many of the same target genes and thus have substantial overlap in functions that are known to include regulation of the circadian rhythm, metabolism and immune function710. In this Review, we

12、discuss the physiological and path- ological roles of these two classes of nuclear receptors 1Department of Molecular Therapeutics, The Scripps Research Institute, 130 Scripps Way, Jupiter, Florida 33458, USA. 2Department of Pharmacological consisting of ROR, ROR and ROR) are involved in many physio

13、logical processes, including regulation of metabolism, development and immunity as well as the circadian rhythm. The recent characterization of endogenous ligands for these former orphan nuclear receptors has stimulated the development of synthetic ligands and opened up the possibility of targeting

14、these receptors to treat several diseases, including diabetes, atherosclerosis, autoimmunity and cancer. This Review focuses on the latest developments in ROR and REV-ERB pharmacology indicating that these nuclear receptors are druggable targets and that ligands targeting these receptors may be usef

15、ul in the treatment of several disorders. REVIEWS NATURE REVIEWS | DRUG DISCOVERY VOLUME 13 | MARCH 2014 | 197 2014 Macmillan Publishers Limited. All rights reserved DBD A/BCDE Helix 12 130 112169579 4688418 4597610 5169731 188614 F 96%48% 91%63% 96%58% LBD REV-ERB REV-ERB ROR ROR ROR a b REV-ERB c

16、ROR and the discovery of their natural ligands, as well as the development of synthetic ligands targeting these recep- tors and their use in cellular and in vivo models of dis- ease. Finally, we highlight potential future directions for therapeutics targeting REV-ERB and ROR for the treat- ment of a

17、utoimmune diseases, central nervous system (CNS) disorders, diabetes and obesity. Roles of REV-ERB and ROR REV-ERBs. The REV-ERBs acquired their unusual name owing to the unique genomic organization of NR1D1, which encodes REV-ERB. REV-ERB is encoded by the opposite DNA strand of the ERBA (also know

18、n as THRA) oncogene1113, and hence its name is derived from reverse strand of ERBA. ERBA encodes the thyroid hormone receptor- and thus REV-ERB is encoded by sequences of DNA on the opposite strand of the gene that encodes thyroid hormone receptor-. Both REV-ERB and the closely related REV-ERB (enco

19、ded by NR1D2), which was identified a few years after REV-ERB, have an atypical LBD that lacks the carboxy-terminal activation function 2 (AF2) region1416 (FIG. 1b). Because the AF2 region recog- nizes co-activators that are necessary for transcriptional activation, REV-ERB and REV-ERB are generally

20、 char- acterized as being unable to activate transcription. Indeed, the REV-ERBs are constitutive repressors of transcription owing to their constant binding of co-repressors such as the nuclear receptor co-repressor 1 (NOR1)17. The recruitment of co-repressors to the target gene by a nuclear recept

21、or (via the DNA response element) leads to repression of the target gene owing to histone deacetylation and condensation of chromatin17,18. Unlike many other nuclear receptors that function as obligate heterodimers (either as homodimers or as heterodimers with retinoid X receptor) and recognize two

22、copies of a core sequence of nucleotides that are organized in either a palindromic or a repeated manner (termed the half site), REV-ERBs typically function as monomers and recognize a half site that consists of a single 5 extended AGGTCA sequence7. However, REV-ERB homodimers have been reported to

23、occur under some conditions18,19. REV-ERBs have overlapping patterns of temporal and spatial expression, which is consistent with our cur- rent understanding of their substantial overlapping functions. Both REV-ERB and REV-ERB are widely expressed throughout the body and, interestingly, both recepto

24、rs have a circadian pattern of expression that is essential for their role in the circadian regulation of transcription2023. RORs. The three members of the ROR subfamily ROR, ROR and ROR have sequence similarities to the retinoic acid receptor2427 and each receptor can constitutively activate transc

25、ription through the ligand- independent recruitment of transcriptional co-activators (FIG. 1b). ROR is widely expressed in many tissues, including cerebellar Purkinje cells, the liver, thymus, skeletal muscle, skin, lung, adipose tissue and kidney28,29. ROR has a similar broad pattern of expression

26、but is observed at very high levels within the thymus. ROR has a more restricted pattern of expression relative to the other RORs, and is found in regions of the CNS that are involved in the processing of sensory information, the retina and the pineal gland30. There is considerable overlap in the DN

27、A response elements that are recognized by REV-ERBs and RORs, and both receptors are often co-expressed in the same tissues31. Because RORs constitutively activate trans- cription, whereas REV-ERBs repress transcription, the balance of ROR and REV-ERB activity is crucial for the dynamic regulation o

28、f target genes containing the DNA response elements that are responsive to both classes of receptors (FIG. 2). Owing to the substantial overlap in expression patterns as well as the target genes that are regulated by these receptors, REV-ERBs and RORs are often involved in the regulation of similar

29、physiological processes, as outlined below. Regulation of the circadian rhythm Circadian rhythms have an essential role in the sleep wake cycle, feeding behaviour and metabolism, as well as in the control of body temperature, blood pressure and renal function32. The circadian rhythm is generated by

30、feedback loops in the expression patterns of genes encoding proteins that make up the so-called molecular clock (FIG. 3). Heterodimers of two transcription factors, brain and muscle ARNT-like 1 (BMAL1; also known as ARNTL) and circadian locomotor output cycles protein kaput (CLOCK), induce the expre

31、ssion of the Figure 1 | Structure of the RORs and REV-ERBs. a | The general organizational structure of members of the nuclear receptor superfamily. b | Structure of the REV-ERBs. c | Structure of the retinoic acid receptor-related orphan receptors (RORs). Numbers above each receptor represent the a

32、mino acid position. Percentages indicate amino acid identity within a particular domain relative to either REV-ERB or ROR. A/B, C, D, E and F refer to classically defined regions in the nuclear receptor domain structure. DBD, DNA-binding domain; LBD, ligand-binding domain. REVIEWS 198 | MARCH 2014 |

33、 VOLUME 13 2014 Macmillan Publishers Limited. All rights reserved Target genes ROR response element DNA Activation of transcription Repression of transcription Binding Binding Sterols Haem Nucleus LBD DBDAF1 Transcriptional co-activator LBD DBDAF1 REV-ERB ROR Transcriptional repressor E box A partic

34、ular DNA response element that is recognized by transcription factors belonging to the basic helixloophelix domain-containing family, such as circadian locomotor output cycles protein kaput (CLOCK) and brain and muscle ARNT-like 1 (BMAL1). Period () The time that elapses for one complete oscillation

35、 or cycle of a particular activity (for example, locomotor activity). Typically, the period for a circadian rhythm is almost 24 hours. In the absence of any extrinsic stimuli that act to entrain the circadian rhythm (such as light), the period may differ; for example, mice typically have a period of

36、 slightly less than 24 hours in the absence of entrainment. cryptochrome genes (CRY1 and CRY2) and the period circadian clock genes (PER1, PER2 and PER3) genes. As CRY and PER proteins reach crucial levels, they repress the stimulatory effect of the CLOCKBMAL1 dimer on the expression of their respec

37、tive genes. The dynamic interplay between the opposing circadian patterns of expression and the opposing transcriptional activity of RORs and REV-ERBs, resulting in the positive and nega- tive regulation of gene transcription, is readily apparent in this feedback loop as both classes of receptors ha

38、ve been shown to regulate BMAL1 expression7. As well as RORs and REV-ERBs, various other nuclear receptors have been implicated in the modula- tion and/or regulation of the circadian rhythm. Over half of the nuclear receptor superfamily members are expressed in a circadian manner33, and given their

39、role as transcription factors this probably leads to rhythmic expression of their target genes. Other direct links between nuclear receptor activity and circadian clock function have been identified. These include the direct interaction of the glucocorticoid receptor with CRY1 and CRY2, which mediat

40、es the rhythmic repression of glucocorticoid receptor transcriptional activity; this effect is essential for normal glucocorticoid signalling, which follows a clear circadian pattern34. Additionally, PER2 has been shown to interact with PPAR and REV- ERB at promoter sites to regulate their transcrip

41、tional activity35. REV-ERB represses the transcription of BMAL1 (REFS 22,36) through its actions on two DNA response elements that are located in the BMAL1 promoter. The circadian feedback loop shows additional complexity given that REV-ERB expression is itself regulated by BMAL1CLOCK heterodimers v

42、ia E box DNA response elements found within the Nr1d1 promoter37,38. Nr1d1/ mice have aberrant expression of Bmal1 and alterations in the period and phase of their circadian locomotor behaviour36. Nr1d2/ mice have a much more subtle circadian phenotype, but the Nr1d1/Nr1d2/ double knockout mice are

43、arrhythmic39 and have a similar phenotype to Bmal1/ mice40, Cry1/Cry2/ mice41 and Per1/Per2/ mice42. Indeed, the expression of genes encoding the REV-ERBs is driven by E-box DNA response elements in their promoter elements, which are similar to those that drive the circadian expression of the CRY an

44、d PER genes. These data suggest that the genes that encode REV-ERBs should be considered as core clock genes per se rather than components of an accessory loop that merely modulates the pattern of expression of the core clock genes. In contrast to the REV-ERBs, RORs stimulate BMAL1 expression43. Mic

45、e with a loss-of-function mutation in ROR (Rorasg/sg mice; also known as staggerer mice) have alterations in the circadian oscillator, indicating an essen- tial role for this receptor in normal circadian function43. REV-ERB (the repressor) and ROR (the activator) are expressed in an oscillatory fash

46、ion 12 hours out of phase with each other, leading to alternating activation and repression of BMAL1 expression36,43. ROR-null mice also have a circadian deficit with a longer period () than wild-type mice44,45, and ROR has also been impli- cated in the regulation of the circadian rhythm46. Given th

47、at RORs and REV-ERBs are regulated by ligands, syn- thetic ligands that act at these nuclear receptors could be used to modulate the circadian rhythm as well as to treat diseases that are associated with disrupted circa- dian rhythms, such as sleep disorders, metabolic disease and behavioural disord

48、ers. The initial studies that have aimed to test this hypothesis are addressed below. Regulation of metabolism Circadian rhythms are intricately linked to the regulation of metabolism, and genetic perturbations of core clock genes lead to a range of abnormal metabolic phenotypes in mice, including o

49、besity, dyslipidaemia and glucose intolerance4751. In humans, circadian disruption caused by shift work5254 or manipulated under controlled con- ditions causes metabolic disturbances55,56. The role of RORs and REV-ERBs in the regulation of metabolic pathways is well characterized. Both receptors are

50、 crucial components of the clock that link the core circadian oscillator to the regulation of clock-controlled genes, which in turn regulate metabolic pathways. Figure 2 | Molecular mechanism of action of the RORs and REV-ERBs. Retinoic acid receptor-related orphan receptors (RORs) and REV-ERBs are

51、involved in transcriptional regulation and are regulated by ligands. Haem functions as a ligand for REV-ERBs, whereas sterols (cholesterol, cholesterol sulphate and various oxysterols) function as ligands for RORs. Both classes of receptors recognize a similar DNA response element, typically denoted

52、 as a ROR response element. ROR activates transcription (via recruitment of transcriptional co-activators), whereas REV-ERB silences transcription (via recruitment of transcriptional co-repressors). REV-ERB functions as a ligand-dependent transcriptional repressor (haem binding is required for the r

53、ecruitment of the co-repressor and transcriptional repression), whereas ROR typically functions as a constitutive activator of transcription, and the binding of oxysterol ligands results in decreased activity. AF1, activation function 1; DBD, DNA-binding domain; LBD, ligand-binding domain. REVIEWS N

54、ATURE REVIEWS | DRUG DISCOVERY VOLUME 13 | MARCH 2014 | 199 2014 Macmillan Publishers Limited. All rights reserved Feedback loop BMAL1 CLOCK ROR REV-ERB CRY PER BMAL1 CLOCK ROR REV-ERB Positive arm Negative arm PER CRY ROR element ROR element E box E box a b Expression 01224 BMAL1/ CLOCK PER/ CRY Ex

55、pression 01224 ROR REV-ERB Time (hours)Time (hours) ROR response element A particular DNA response element that is recognized by retinoic acid receptor-related orphan receptors (RORs) and REV-ERBs. Loss-of-function studies both in vitro and in vivo clearly demonstrate that REV-ERBs have a crucial ro

56、le in lipid metabolism. REV-ERB-null mice have dyslipi- daemia with elevated levels of very-low-density lipo- protein (VLDL) triglyceride and increased serum levels of apolipoprotein C3 (APOC3)57,58. Rorasg/sg mice have the opposite phenotype with reduced APOC3 expression and lowered triglyceride le

57、vels58. APOA1, a component of high-density lipoprotein (HDL), is also regulated by both REV-ERB and ROR59. The expression of several genes involved in lipid metabolism was suppressed in a myocyte cell line expressing a dominant negative form of REV-ERB60; these genes included fatty acid translocase

58、(FAT; also known as CD36), fatty acid binding protein 3 (FABP3; also known as MDGL), FABP4 (also known as ALBP), mitochondrial uncoupling protein 3 (UCP3; also known as SLC25A9), sterol regulatory element-binding transcription factor 1 (SREBF1) and stearoyl-CoA desat- urase (SCD). Although it is unc

59、lear whether these are direct target genes of REV-ERB, this study clearly dem- onstrates that REV-ERB is involved in the regulation of genes that are involved in fatty acid and lipid absorption, energy expenditure and lipogenesis in muscle. Hepatic glucose metabolism is regulated by REV- ERB, which directly regulates the expression of the genes encoding the gluconeogenic enzymes phospho- enolpyruvate carboxykinase (PCK) and glucose-6-phos- phatase (G6PC)61. Mic