REV-ERB and ROR nuclear receptors as drug targets

Abstract

The nuclear receptors REV-ERB (consisting of REV-ERBα and REV-ERBβ) and retinoic acid receptor-related orphan receptors (RORs; consisting of RORα, RORβ and RORγ) are involved in many physiological 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 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 useful in the treatment of several disorders.

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 amino acid position. Percentages indicate amino acid identity within a particular domain relative to either REV-ERBα or RORα. 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.

Figure 2. Molecular mechanism of action of the RORs and REV-ERBs

Retinoic acid receptor-related orphan receptors (RORs) and REV-ERBs are 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 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 recruitment 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.

Figure 3. Role of RORs and REV-ERBs in regulation of the mammalian clock

a | The core mammalian clock is composed of a heterodimer of the transcription factors circadian locomotor output cycles protein kaput (CLOCK) and brain and muscle ARNT-like 1 (BMAL1) (known as the positive arm), which activate the transcription of period circadian clock (PER) and cryptochrome (CRY) genes via E box sequences within their promoters. PER and CRY proteins (known as the negative arm) form dimers and directly interact with the CLOCK–BMAL1 heterodimers, thus suppressing their activity. This feedback loop follows a 24-hour rhythm where peak expression of the CLOCK–BMAL1 complex is 12 hours out of phase with peak PER and CRY expression. A retinoic acid receptor-related orphan receptor (ROR) response element within the BMAL1 promoter is responsive to both ROR and REV-ERB (encoded by the genes NR1D1 and NR1D2); ROR activates the transcription of BMAL1, whereas REV-ERB suppresses its transcription. The expression of ROR and REV-ERB also oscillates in a circadian manner (12 hours out of phase with one another), reinforcing the core circadian oscillator. The REV-ERB promoter also contains an E box, allowing direct regulation of NR1D1 and NR1D2 transcription by BMAL1–CLOCK. PER2 has also been demonstrated to directly interact with REV-ERB at REV-ERB-responsive promoters and to regulate its activity. b | The expression of PER and CRY as well as BMAL1 and CLOCK oscillates over the course of 24 hours. REV-ERB and ROR expression also undergoes circadian oscillations.

Figure 4. Structures of REV-ERB and ROR demonstrate their capacity to bind to natural ligands

The apo structure of REV-ERBβ (part a) indicated that the putative ligand-binding pocket was filled with large hydrophobic residues and thus devoid of the space that would be necessary for a ligand to bind. However, the haem-bound REV-ERBβ structure (parts b and c) shows that its ligand-binding pocket can profoundly change its shape to accommodate haem, a large porphyrin natural ligand. Intriguingly, although studies indicate that the binding of haem to REV-ERB increases its interaction with the nuclear receptor co-repressor (NCOR)^,, the structure of apo REV-ERB bound to an NCOR peptide (part d) indicates that the binding of haem may not be an absolute requirement for mediating the REV-ERB–NCOR interaction. The co-crystal structure of the ligand-binding domain of retinoic acid receptor-related orphan receptor-α (RORα) bound to cholesterol (parts e and f) sets the stage for other studies indicating that various cholesterol derivatives, such as 7-oxygenated sterols, may act as physiological ligands to influence ROR activity. Structures are shown as space-filling models (parts a, c, d and e), with and without transparency to allow visualization of ligands bound to the internal ligand-binding pocket (haem-bound REV-ERBβ and cholesterol-bound RORα). A snapshot of the residues mediating the interaction of haem with REV-ERB (part b) illustrates that the repositioned hydrophobic residues that were originally thought to block the ligand-binding pocket in fact cooperate in binding to the large hydrophobic porphyrin haem scaffold. Protein Data Bank (PDB) codes: apo REV-ERB, 2V0V; haem-bound REV-ERB, 3CQV; apo REV-ERB with an NCOR fragment (co-repressor nuclear receptor (CoRNR) box motif peptide, 3N00; cholesterol-bound ROR, 1N83.

Figure 5. Development of selective ROR ligands

Following a screen of known nuclear receptor ligands against the entire nuclear receptor superfamily, the liver X receptor (LXR) agonist T0901317 was identified as a retinoic acid receptor-related orphan receptor (ROR) ligand. T0901317 has substantial promiscuity against other nuclear receptors. Various alterations in the structure led to the discovery of an agonist of RORα and RORγ (SR1078), an inverse agonist of RORα and RORγ (SR1001), and a RORγ-selective inverse agonist (SR2211). FXR, farnesoid X receptor; PXR, pregnane X receptor.

Figure 6. ROR inverse agonists alter T_H cell development

T helper 1 (T_H1), T_H2, T_H17 and regulatory T (T_Reg) cells develop from naive CD4⁺ T_H cells. The differentiation of naive CD4⁺ T_H cells into these effector CD4⁺ T cells is initiated via an interaction of dendritic cells with naive CD4⁺ T_H cells. Effector cell types are defined by their production of specific cytokines, function, modulation of distinct signalling pathways and the expression of distinct transcription factors. Retinoic acid receptor-related orphan receptor (ROR) inverse agonists suppress T_H17 cell differentiation and function. The RORγ inverse agonist SR1555 promotes T_Reg cell differentiation as well. ATRA, all-trans retinoic acid; FOXO3, forkhead box protein O3; GATA3, GATA-binding protein 3; IFNγ, interferon-γ; IL, interleukin; STAT, signal transducer and activator of transcription; TBX21, T-box protein 21 (T-bet); TGFβ1, transforming growth factor-β1.

Figure 7. ROR and REV-ERB in the regulation of physiological processes

Physiological processes are shown in pink boxes, and the potential therapeutic indications of synthetic ligands that target retinoic acid receptor-related orphan receptor (ROR) and the nuclear receptor REV-ERB are indicated in grey boxes.