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Modulators of the Nuclear Receptor Retinoic Acid Receptor-Related Orphan Receptor-γ (RORγ or RORc) Benjamin P. Fauber* and Steven Magnuson Discovery Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States ABSTRACT: As the biology surrounding the nuclear receptor retinoic acid receptor-related orphan receptor-gamma (RORγ or RORc) continues to evolve, significant effort has been invested in discovering modulators of this potentially important target for the treatment of metabolic and immunological diseases. Several major pharmaceutical and biotechnology companies have disclosed RORc inhibitors or partnered with other players in the field. In this perspective, we discuss both the biology and the underlying structural biology of RORc, and summarize the RORc modulators disclosed in the scientific and patent literature.
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(Figure 1).17 The tissue expression levels and functional effects of the two isoforms are quite varied. Messenger ribonucleic acid
INTRODUCTION Nuclear receptors (NR) are a large family of transcription factors involved in the regulation of key physiological functions such as cell differentiation, embryonic development, and organ physiology.1 Nuclear receptors have also been identified as important pathological regulators in diseases such as cancer, diabetes, and autoimmune disorders. As a result of their prevalence in human biology, this class of targets has given rise to some of the most notable pharmaceutical agents of the past century, including progesterone (contraceptive), cortisone (anti-inflammatory), tamoxifen (hormone and cancer therapy), and more recently, rosiglitazone (diabetes treatment).2 Modulators of NRs have proven to be a rich source of pharmaceuticals, and it was estimated in 2009 that this class of compounds accounted for roughly 13% of all pharmaceutical sales in the United States and an appreciable portion of global sales.3 The NR retinoic acid receptor-related orphan receptorgamma (RORγ or RORc, also known as NR1F3)4 was the third and most recent ROR group member to be discovered, following the prior disclosures of RORα (RORa or NR1F1)5 and RORβ (RORb or NR1F2).6 The ROR name was ascribed to these proteins given their sequence homology to the NR retinoic acid receptor (RAR). Although the RORs and RARs share sequence homology, there is now evidence that members of the ROR group may preferentially bind to and be modulated by oxysterol derivatives7−11 and not retinoic acid, which is the agreed upon endogenous ligand for RARs.12 It should also be noted that although there have been several attempts to identify a physiologically active ligand of RORc, it is still referred to as an orphan NR because there is no broad agreement on the identity of the ligand.13 RORc was initially discovered in Jetten’s lab from human pancreas poly(A)+ RNA.4 Murine RORγ and the human form (RORc) share an 88% sequence homology.14 Murine RORγ exists in two distinct isoforms, RORγ (RORγ1)15 and RORγt (RORγ2),16 which only differ in their N-terminal sequences © 2014 American Chemical Society
Figure 1. Structures and domains of the murine RORγ isoforms: RORγ and RORγt. Amino-terminal domain (A/B or AF1), DNAbinding domain (DBD), hinge region, ligand-binding domain (LBD), and the activation function helix-2 (AF2).
(mRNA) for RORγ is expressed in many tissues and is predominantly expressed in the kidneys, liver, and skeletal muscle.4,17 Expression of the RORγt isoform is restricted to lymphoid organs, such as the thymus. This observation suggests that RORγt may play a role in the development and regulation of the immune system. Studies with RORγ−/− mice noted that colonies of these animals were viable but that they lacked Peyer’s patches and peripheral lymph nodes.18 Thymic tissue in RORγ−/− mice contained 75% (±9%) fewer thymocytes than that typically found in wild-type animals.19 This reduction in thymocyte numbers was due to the massive apoptosis of double negative thymocytes. Furthermore, the expression of the antiapoptotic factor Bcl-xL was abolished in the thymus of the RORγ−/− mice. This apoptotic phenotype was rescued by crossing the RORγ−/− mice to Bcl-xL transgenic mice. It is not clear if RORγ directly regulates the expression of Bcl-xL, but initial data suggests that the ROR DNA response element (RORE) may not be required for such a process to occur.20 In addition to the reduction in thymocytes, there was a high incidence of thymic lymphomas in RORγ−/− mice older than Received: December 11, 2013 Published: February 6, 2014 5871
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four months.21 The original RORγ−/− litter mates in the survival study were derived from mixed parental backgrounds, and the heterozygous and wild-type cohorts had no notable thymic lymphomas. Breeding of the RORγ−/− mice to a single genetic background also had no notable impact on survival rates in the study. Interestingly, there were no other detectable tumor types in any of the RORγ−/− animals, even in the animals that survived for more than one year. These results suggested that RORγ signaling may be required for thymic function and homeostasis in younger animals, but such a concern would be minimized in human adults since the thymus involutes after puberty.22 An appropriate study to explore the murine homeostasis hypothesis would be the use of inducible RORγ knockout adult mice, but to our knowledge, such a study has yet to be reported in the literature. In 2006, Professor Littman’s lab at NYU disclosed the direct linkage of RORγt signaling to the differentiation of proinflammatory interleukin (IL)-17A+ (also referred to as IL-17 in the literature) T helper cells (TH17 cells).23 In a comparison of wild-type and RORγt null mice, it was found that the development of TH17 cells was critically dependent on the induction of RORγt during TH17 differentiation. De novo differentiation of purified naı̈ve CD4+ T cells to the TH17 phenotype required the presence of IL-6 and transforming growth factor (TGF)-β. RORγt mRNA and IL-17A transcription were induced from naı̈ve T cells upon T cell receptor activation only in the presence of both IL-6 and TGF-β. Under these conditions, the mRNA levels of RORγt peaked at ∼16 h post-stimulation, whereas the IL-17 mRNA levels peaked at 48 h post-stimulation. It was also noted that the induction of IL17A from naı̈ve T cells collected from RORγt null animals was approximately 50-fold lower than that observed in T cells from wild-type animals. IL-23 is another major cytokine that is indispensable for the maintenance of TH17 cells and IL-17A production.24 IL-23 can further enhance the expression of RORγt and IL-17A in TH17 cells. It should be noted that the regulation of human TH17 development is consistent with results from mouse T cells,25 and RORc is also essential for IL17A production from human TH17 cells.26 In addition to IL-17A, TH17 cells also produce IL-17F, another IL-17 family cytokine.27 The IL-17 family contains six members, IL-17A, IL-17B, IL-17C, IL-17D, IL-17E (also called IL-25), and IL-17F. Of these family members, IL-17A and IL17F share the highest sequence homology (50%). IL-17A and IL-17F can form either homodimers (IL-17AA and IL-17FF) or heterodimers (IL-17AF). Structurally, the homodimers of IL17A and IL-17F form a “cystine-knot” structure using four conserved cysteine residues, similar to those found in TGF-β and other growth factors.28,29 All forms of IL-17A and IL-17F ligands signal through the same heterodimeric receptor complexes, namely, IL-17RA and IL-17RC, and induce similar downstream biological effects from target cells.27 IL-17 targets tissue epithelial cells and other stromal cells, but not leukocytes, to induce the production of proinflammatory cytokines, chemokines, and antimicrobial peptides. IL-17A and IL-17F produced by TH17 cells exert essential roles during hose defense against various bacterial and fungal infections.30 Elevated levels of IL-17A and IL-17F have been detected in several human autoimmune diseases, including rheumatoid arthritis (RA),31 multiple sclerosis (MS),32 and inflammatory bowel disease (IBD).33,34 Higher quantities of TH17 cells are consistently found in the tissues and peripheral blood samples from patients with autoimmunity. Therefore, IL-17A and IL-
17F may share redundant biological effects and contribute to the pathogenesis of autoimmune diseases.35 Studies in preclinical autoimmune models provide additional support for the role of IL-17 in inflammation. Exploration of the collagen-induced arthritis (CIA) mouse model for RA in IL17(A)−/− mice noted a marked suppression of the disease histology relative to wild-type control animals.36 Treatment of wild-type mice with a polyclonal rabbit antimurine IL-17(A) antibody, after the onset of CIA, significantly reduced the severity of the disease as measured by clinical scoring and histology.37 In an effort to understand the impact of IL-17 signaling in the experimental autoimmune encephalomyelitis (EAE) mouse model for MS, purified T helper 1 (TH1) and TH17 cells were injected into wild-type mice.38 The mice that received the TH17 cells showed severe clinical signs of EAE, whereas the mice that received the TH1 cells showed relatively mild EAE. Additionally, the EAE disease severity demonstrated a dose-dependent correlation with the number of TH17 cells injected into the mice. Administration of an anti-IL-17(A) antibody to mice that received the TH17 cells was able to attenuate the disease as measured by EAE clinical scores and histology. Given the critical role of IL-17 signaling in inflammatory disease, several humanized anti-IL-17 antibodies have been developed and recently entered human clinical trials.39 Secukinumab (AIN457), an anti-IL-17A monoclonal antibody under development by Novartis, is the most advanced molecule in the clinic.40 Novartis recently announced the results of its phase III clinical trial in psoriasis patients in which secukinumab demonstrated head-to-head superiority over the TNFα-blocker etanercept, which is the current standard of care for psoriasis.41 These positive clinical results underscore the importance of the IL-17 signaling pathway in inflammatory disease, and they also illustrate the potential opportunity to treat inflammatory diseases by impacting the IL-17 signaling pathway via RORc inhibitors. Inhibition of RORc signaling may have additional effects beyond the regulation of IL-17A expression. In addition to the expression of IL-17A and IL-17F, TH17 cells also preferentially express proinflammatory cytokines such as IL-21,30 IL-22,42 and granulocyte-macrophage colony stimulating factor (GMCSF).43 IL-22 has been identified as an important cytokine in the pathogenesis of skin inflammation44 and gut homeostasis, as well as other proinflammatory pathways.45 GM-CSF plays a central role in maintaining chronic inflammation.46 GMCSF−/− mice are resistant to EAE, and they also display decreased antigen-specific proliferation of splenocytes.43,47 RORγt is also critical for the development and function of innate lymphoid cells (ILC).48 RORγt is preferentially expressed in Group 3 ILCs, which are composed of lymphoid tissue inducer (LTi) cells and natural killer cell receptor (NCR)+ ILCs.49 LTi cells are required for the development of many lymphoid organs in mice such as Peyer’s patch, lymph nodes, and isolated lymphoid follicles. In RORγt−/− mice, the development of LTi cells was compromised,48 which provided a cellular explanation for the lack of various lymphoid organs in these mice. In humans, Group 3 ILCs produce proinflammatory cytokines similar to TH17 cells such as IL-17A and IL-22;50 however, the pathogenic role of Group 3 ILCs and their cytokines in autoimmune diseases remains unclear. It is also unknown how the long-term inhibition of RORc will impact Group 3 ILCs. 5872
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impact of RORc inhibitors may have an enhanced benefit over that already identified with anti-IL-17 antibodies and could provide additional benefit for patients suffering from inflammatory diseases.59,60 Additionally, there is emerging evidence that RORγ signaling may play a role in the regulation of metabolic processes and could serve as a molecular target for the treatment of obesity-related insulin resistance.
RORγ and RORc signaling have also been implicated in the regulation of circadian rhythms in mice and humans, respectively.51 A RORE competition was noted across the ROR family, as all ROR family members share a conserved RORE binding motif.52 It initially was thought that modulation of the ROREs by any member of the ROR family (e.g., RORc) could impact similar downstream biology. For example, ROREs have been implicated in the regulation of circadian transcription via the aryl hydrocarbon receptor nuclear translocator-like transcription factor (a.k.a. BMAL1).53 However, recent studies in mice suggest that RORα, not RORγ, is the predominant ROR family member involved in the regulation of intracellular circadian rhythms,54 although it may not be a primary driver of the key regulatory mechanisms.55 RORγ also may play a role in the regulation of adipogenesis and insulin sensitivity. RORγ null mice showed decreased adipocyte size and also were protected from hyperglycemia and insulin resistance when fed a high-fat diet.56 Importantly, neither glucose uptake nor lipolysis were altered by varying RORγ expression levels in the mice. The same study also explored the gene transcription profiles of the visceral adipose stromal vascular fraction from lean and obese humans. This analysis revealed that RORc was the highest upregulated genetic transcription factor in the obese human patients. Deletion of peroxisome proliferator-activated receptorgamma (PPARγ) in the adipose tissue of mice led to protection against high fat diet-induced obesity and insulin resistance.57 Furthermore, studies with murine 3T3-L1 preadipocyte cells evaluated the interplay of PPARγ and RORγ signaling.56 These studies led to the conclusion that RORγ acted upstream of PPARγ. Transcriptional activity from the RORγ promoters was not affected by a PPARγ-activating ligand (i.e., rosiglitazone), suggesting that PPARγ does not regulate RORγ expression. Taken collectively, these results suggested that inhibition of RORc expression in humans might offer a novel target for the treatment of obesity-related insulin resistance with a distinct safety profile compared to that observed with PPARγ-activating ligands.58 In summary, the modulation of RORc can impact the IL-17, IL-22, and GM-CSF signaling pathways (Figure 2). The broad
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RORC RECEPTOR STRUCTURE AND FUNCTION Nuclear receptors act in the nucleus where they regulate gene transcription.1 In order to achieve this regulatory function, a typical NR contains a variable N-terminal domain (A/B or AF1, also known as activation function helix 1), a DNA-binding domain (DBD), a flexible hinge region, and a C-terminal ligand-binding domain (LBD) (Figure 1).61 The A/B region is believed to play a role in the specificity of DBD binding to DNA, but the A/B region does not directly interact with DNA.62 The DBD binds directly to DNA via two zinc-finger motifs (the P-Box) that interact with the corresponding NR response elements encoded on the proximal promoter region of the DNA. In the case of the ROR family, the DBD RORE is identical across all family members (nucleotide sequence: WWCWAGGTCA where W = A or T).18,20 Within the DBD, there is a high sequence homology in humans across the family, with RORb being 91% homologous to RORa, and RORc being 89% homologous to RORa.63 A typical NR LBD adopts a three-layered fold of 12 α-helices, with two or three β-strands forming a shorter sheet structure.61 A ligand-binding pocket resides within the core of the LBD and is typically hydrophobic. There is some LBD sequence homology within the human ROR family, with RORc sharing 48% and 46% sequence identity with RORa and RORb, respectively.11 Since murine RORγ and RORγt only differ in their N-terminal A/B domains (see above discussion), it is our opinion that modulators of their respective LDBs should have equivalent effects on each isoform and any downstream targets and that it would be challenging to selectively modulate only one isoform with a small molecule ligand. To our knowledge, the potential differences or similarities in the downstream signaling effects of these murine RORγ isoforms have not been studied. The RORc-LBD comprises the 12 canonical α-helices (H1− H12) along with two additional helices (H2′ and H11′).63 The α-helix nomenclature is shared by all ROR family members and was initially established with the X-ray structure of rodent RORβ.64 While several bound structures have been disclosed, an unliganded (apo) X-ray crystal structure of RORc has not been reported. In general, a NR LBD can interact with coactivator and corepressor complexes to regulate gene transcription. Activation by a coactivator complex is thought to involve the movement of helix 12 (activation function helix 2 or AF2) into a hydrophobic groove on the surface of the LBD near helices 3−5.61 This cumulative interaction region is referred to as the NR Box.65 The interaction of the LBD with coactivators is generally mediated by the LXXLL sequence motif (where X is any amino acid) on the AF2 helix, along with the neighboring helices that form the NR Box.62 There are multiple pathways for modulating the gene transcription levels of a NR. Recruitment of a coactivator complex generally enhances gene transcription, and ligands that promote such interactions are termed receptor agonists (Figure 3).66 Recruitment of a corepressor complex generally decreases
Figure 2. Proposed signaling pathway of RORc and downstream cytokines. Naı̈ve precursor T cells and lymphoid cells can generate T helper 17 (TH17) through multiple pathways, including stimulation by interleukin-6 (IL-6) and transforming growth factor (TGF)-β or interleukin-23 (IL-23). The NR retinoic acid receptor-related orphan receptor-gamma (RORγ or RORc) resides within the nuclei of TH17 cells. To induce gene transcription, RORc associates with a coactivator complex and then binds to the retinoic acid receptor-related orphan receptor response element (RORE) embedded within the DNA of the TH17 cell. The gene transcription induced by RORc leads to the expression of several pro-inflammatory cytokines including interleukin17 (IL-17), interleukin-22 (IL-22), and granulocyte-macrophage colony stimulating factor (GM-CSF). 5873
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structure illustrates the primary helical structural features described earlier in this section (Figure 5a) [PDB: 3L0L; the residue numbering scheme is based on full-length human RORc]. Compound 1 was bound in the hydrophobic core of the ligand-binding pocket, and the costructure also had a steroid receptor coactivator-2 (SRC2) peptide bound. The AF2 helix was stabilized via a hydrogen bond network71 between His479 and Tyr502 (3.0 Å), along with Gln487 and Ser507 (2.8 Å). His479 also engaged Phe506 in an edge-to-face π−π stacking interaction72 (3.2−3.7 Å), and Tyr502 and neighboring Phe506 interacted in an edge-to-face π−π stacking interaction (3.6−3.8 Å) (Figure 5b). Compound 1 made no direct RORc ligand-binding pocket hydrogen-bonding interactions with the residues on helix 3 or helices 11−12. The 25hydroxyl group of 1 made one water-mediated hydrogen bond (2.6 Å) to Tyr502 (Figure 5c). At the other end of the ligandbinding pocket, near the C-terminus of helix 3 and helices 5 and 7, polar residues were presented toward the binding pocket along with a network of water molecules. In this region of the pocket, the 3β-hydroxyl group of 1 made a direct hydrogenbonding interaction with Gln286 (3.1 Å) and also made a water-mediated hydrogen bond (2.8 Å) to Arg367 (Figure 5d). In an effort to identify modulators of RORγt, a set of 700 small molecules was screened against murine lymphoma EL-4 cells to identify inhibitors of IL-17 production. The screen identified digoxin (2) as an inverse agonist of RORγt (Figure 6).73 Analysis of a 2.2 Å resolution cocrystal structure of 2 and the human RORc-LBD revealed the structural basis of inverse agonism [PDB: 3B0W; the residue numbering scheme is based on full-length human RORc]. Compound 2 resided in the hydrophobic ligand-binding pocket of RORc. The digitoxose Y and Z glycosides pushed toward the helices 11−12 region of the LBD resulting in the disorder of the C-terminus of helix 11 and all of helices 11′−12 (Figure 7a). The AF2 residues were present but randomly oriented and poorly defined in the electron density. Compound 2 made several hydrogen bonds throughout the ligand-binding pocket. The 3Xα-OH group of digitoxose X formed a direct hydrogen bond with His479 (3.1 Å), and the 12β-OH group on the C-ring engaged the backbone carbonyl of Phe377 (3.0 Å) (Figure 7b). The carbonyl group of the D-ring α,β-unsaturated lactone formed a direct hydrogen bond with Arg367 (3.0 Å), and Arg367 was, in turn, stabilized by a salt bridge interaction with Glu414 (2.7− 2.8 Å). The 14β-OH group also formed a water-mediated hydrogen bond to the backbone carbonyl of Val361 (2.5 Å). An analysis of the RORc-LBD and compound 1 cocrystal structure [PDB: 3L0L] superimposed with the RORc-LBD and compound 2 inverse agonist costructure [PDB: 3B0W] revealed that the digitoxose Y and Z glycosides of 2 effectively occupied the region where helix 11 would otherwise reside.73 This steric clash between the ligand and receptor presumably led to the disordering of helices 11−12 and the inhibition of RORc-mediated IL-17 expression observed with compound 2 cell culture studies. Related structural effects have been associated with the inverse agonist mechanism of action in other members of the NR superfamily.74 Thus, a molecular basis for the agonist and inverse agonist mechanisms of the RORc-LBD have been defined, and this creates a framework for the investigation of synthetic modulators of RORc.
Figure 3. Graphical depiction of agonist, antagonist, and inverse agonist effects on NR co-activator recruitment and downstream transcription. A NR agonist ligand would increase the response, and an inverse agonist ligand would decrease the response. Partial agonists and inverse agonists would achieve a response less than ±100%.
gene transcription, and ligands that promote such processes are termed receptor inverse agonists. Ligands may also disfavor coactivator recruitment by inducing a LBD conformational change by which the AF2 region is disordered and therefore cannot interact with either a coactivator or corepressor complex. This type of ligand is considered an inverse agonist if the NR has a constitutive level of transcriptional activity. The degree of perturbation of coregulator complex recruitment can vary. Thus, there can be partial and full agonists and inverse agonists. A third method of NR modulation is receptor antagonism. Antagonist ligands intrinsically do not affect transcriptional activity; instead, they block or reduce the response induced by a receptor agonist. Although the topic is still debated within the field of ROR biology, it is most likely that RORc does utilize some sort of physiological ligand to modulate transcriptional activity.67 For example, it has been demonstrated that a range of exogenously supplemented oxysterols engendered murine RORγ transcriptional activity in an otherwise inactive insect cell system.68 It was demonstrated that 7-hydroxycholesterol regulated the expression of RORγ target genes glucose-6-phosphatase (G6 Pase) and phosphoenolpyruvate carboxykinase.9 A number of references now confirm that physiologically relevant ligands can modulate the activity of RORγ in cellular systems. These ligands will be summarized in a following section in greater detail. Early efforts to identify ligands of the ROR family focused on RORa.7,69,70 One of these ligands, 25-hydroxycholesterol (25HC, 1, Figure 4), was recently cocrystallized with the human RORc-LBD.11 An analysis of this 1.75-Å resolution cocrystal
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DISCOVERY OF RORC AGONISTS Sterols have long been known as natural ligands for a number of NRs.75 Thus, it is not surprising that some of the first
Figure 4. Structure of 1. 5874
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Figure 5. (a) Cocrystal structure (1.75 Å) of 1 (yellow) in complex with human RORc-LBD (gray) [PDB: 3L0L]. The helix 12 (AF2 helix) of RORc is highlighted (red). The costructure also contains the SRC2 coactivator peptide (blue) bound in the AF2 helix region. Side chains and water molecules have been omitted for clarity. (b) His479 (gray) formed a hydrogen bond (3.0 Å) with Tyr502 (red) and engaged the neighboring Phe506 (red) in an edge-to-face π−π stacking interaction (3.2−3.7 Å). Tyr502 and Phe506 also interacted in an edge-to-face π−π stacking interaction (3.6−3.8 Å). The hydrogen bond (2.8 Å) between Gln487 (gray) and Ser507 (red) further stabilized helix 12. The AF2 helix of RORc is highlighted (red), and 1 (yellow) is shown. Hydrogen bonds are depicted as dashed lines (black). The SRC2 coactivator peptide, side chains, and water molecules have been omitted for clarity. (c) Compound 1 (yellow) formed a water-mediated hydrogen bond (2.6 Å) to Tyr502 (red). His479 is shown (gray), and the AF2 helix of RORc is highlighted (red). Hydrogen bonds are depicted as dashed lines (black), and water molecules are shown as spheres (red). The side chains and SRC2 coactivator peptide have been omitted for clarity. (d) Compound 1 (yellow) formed a direct hydrogen bond (3.1 Å) to Gln286 (gray) and a water-mediated hydrogen bond (2.8 Å) to Arg367 (gray). The AF2 helix of RORc is highlighted (red). Hydrogen bonds are depicted as dashed lines (black), and water molecules are shown as spheres (red). The side chains and the SRC2 coactivator peptide have been omitted for clarity.
discovered RORc ligands were sterols. An AlphaScreen biochemical assay, using the RORc-LBD, revealed that RORc could promote steroid receptor coactivator-1 (SRC1) recruitment in the presence of the following sterol ligands: cholesterol (3), 20α-hydroxycholesterol (4), 22(R)-hydroxycholesterol (5) (Figure 8), and 1.11 These same oxysterol ligands also enhanced the affinity of RORc for other coactivator peptides containing the conserved LXXLL motifs including: steroid receptor coactivator 1−2 (SRC1−2), steroid receptor coactivator 1−4 (SRC1−4), CREB-binding protein (CBP), and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). At the same time, the oxysterol ligands were ineffective at recruiting the corepressor peptides NcoR-1 and NcoR-2. Mutagenesis studies designed to either disrupt or enhance binding of these hydroxycholesterols to RORc resulted in the expected decrease or increase, respectively, in RORc transcriptional activity. Collectively, these results, along with
Figure 6. Structure of 2, a RORc inverse agonist. 5875
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Figure 8. Structures of 3 and related sterol ligands that bind to RORc.
The team at Scripps Florida was the first to report a small molecule ligand of RORc that functioned as an agonist in the context of full-length receptors. The compound, SR1078 (7),76 was identified through modification of the heavily studied and promiscuous NR modulator T0901317 (8) (Figure 9).77−79
Figure 7. (a) Cocrystal structure (2.2 Å) of 2 (yellow) in complex with human RORc-LBD (gray) [PDB: 3B0W]. Side chains and water molecules have been omitted for clarity. The residues at the Cterminus of helix 11 and helices 11′−12 were present but randomly oriented and poorly defined in the X-ray density. (b) Compound 2 (yellow) formed direct hydrogen bonds to His479 (gray) (3.1 Å), Arg367 (gray) (3.0 Å), and the backbone carbonyl of Phe377 (residue not shown) (2.6 Å). Arg367 also engaged Glu414 (gray) in a salt bridge interaction (2.7−2.8 Å). The ligand formed a water-mediated hydrogen bond to the backbone carbonyl of Val361 (residue not shown) (2.5 Å). Hydrogen bonds are depicted as dashed lines (black), and water molecules are shown as spheres (red). Side chains have been omitted for clarity.
Figure 9. Structures of RORc agonist 7 and inverse agonist 8.
Compound 7 contained the same hexafluoroisopropanol carboxylic acid isostere as 8 but made use of a secondary amide, rather than a tertiary sulfonamide, to link the two phenyl rings. While 8 modulated the activity of RORa, RORc, liver X receptors (LXR)-α and -β, and farnesoid X receptor (FXR), compound 7 was more selective, modulating the activity of ROR receptors only. Using an AlphaScreen assay format, 7 dose-dependently decreased the ability of the RORc-LBD to interact with the LXXLL domain peptide derived from the thyroid hormone receptor-associated protein-22 (TRAP22) coactivator protein. This inverse agonist-like activity was also observed in a cell-based GAL4 cotransfection assay that included the RORc-LBD. Interestingly, when the compound was tested in cotransfection cell-based assays using full length RORc and luciferase reporter genes associated with ROR target genes, RORc agonist activity was observed. With two different reporter gene promoters, fibroblast growth factor-21 (FGF21) promoter and G6 Pase promoter, RORc-directed transcription demonstrated statistically significant stimulation starting at concentrations of 2 μM and 5 μM of compound 7, respectively. Agonist activity was also observed in a cell line expressing endogenous levels of RORc. In HepG2 cells, using a 10 μM
the analysis of the crystal structure discussed in the previous section, suggest that sterol ligands can act as modulators of RORc. Scripps Florida. Burris and co-workers at Scripps Florida also discussed the binding of 3 and cholesterol sulfate (6) (Figure 8) to RORc.9 Using a radioligand binding assay with tritiated 1, they determined that 3 barely displaced the radioligand. Furthermore, they found that 3 did not have an effect on transcriptional activity in a cell-based GAL4-RORc transfection assay at concentrations up to 500 μM. The Scripps Florida result may be explained by solubility limitations of the steroids in different assay formats, or there may be a biological disconnect between biochemical assays using the ligand binding domain and cellular assays with full-length RORc. In any event, this result highlights the need for synthetic small molecule modulators to better assess RORc cellular activity. 5876
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number of business partnerships over the past four years directed toward discovering RORc inhibitors.81 Because of the number of these partnerships and the complexity of compound ownership that may result from these deals, we will categorize and discuss the disclosed RORc modulators in chronological order grouped by the organization or partnership from which they arose. Scripps Florida. One of the first RORc inverse agonists discussed in the literature was the benzenesulfonamide 8 (Figure 9).82 This compound was first identified by Tularik as a potent LXR agonist77 and later noted as a ligand to other NRs.78,79 Given the compound’s binding affinity for multiple NRs, it is not entirely surprising that the Griffin and Burris laboratories at Scripps Florida found 8 to be a ligand for RORc as well. Compound 8 inhibited coactivator recruitment in GAL4-nuclear receptor LBD assays of both RORc and RORa with estimated IC50 values of 1.7 and 2.0 μM, respectively.81 Interestingly, 8 did not show similar inverse agonist activity in the RORb assay. Compound 8 was tested in radioligand receptor binding assays to confirm that the RORc and RORa results were due to direct interaction with the receptor, and these assays revealed Ki values of 51 nM for RORc and 132 nM for RORa. Additionally, 8 was found to block activation of an IL-17 promoter containing a ROR response element in HEK293 cells. The effect was attributed to RORc and/or RORa but not LXR activity, providing some evidence for the role of ROR in IL-17-related biology. With these results and a potent, albeit nonselective, RORc/a inverse agonist in hand, the team at Scripps Florida embarked on a SAR investigation in search of selective RORc ligands. Three of these compounds, SR1001 (12),83 SR2211 (13),84 and SR1555 (14)85 (Figure 11) highlight the team’s progress toward this goal. Compound 12, like 8, has a hexafluoroisopropanol benzene group with an N-linked sulfonamide at the para-position. The major structural differences between the two molecules are (1) a more elaborate substituted thiazole attached to the sulfonamide sulfur in 12, rather than a phenyl ring in 8; and (2) the trifluoroethyl group on the sulfonamide nitrogen in 8 was simplified to an N-H in 12. In radioligand binding assays, 12 bound to the LBDs of RORc and RORa with Ki values comparable to that of 8 (111 and 172 nM, respectively). Although these two compounds had similar RORc Ki values, they differed significantly in coactivator recruitment assays. Compound 12 repressed coactivator recruitment in RORc and RORa GAL4 assays but had no activity in an LXRα cell assay or any of the other 45 NRs tested, including RORb. Thus, 12 was a RORc/a selective compound and an improved tool to probe ROR pharmacology.
concentration of 7, both G6 Pase mRNA and FGF21 mRNA levels were increased. Compound 7 was also dosed intraperitoneally (i.p.) in mice at 10 mg/kg, and similar effects on mRNA levels of G6 Pase and FGF21 also were observed after mRNA purification of harvested mice livers (2 h post-dosing). GlaxoSmithKline. A team from GlaxoSmithKline (GSK) reported additional modulators of RORc. Initially, compounds 9, 10, and 11 (Figure 10) were found to inhibit recruitment of a
Figure 10. Structures of RORc agonists from GSK.
peptide fragment of the SRC1 coactivator in a TR-Fret assay using the RORc-LBD.80 Conversely, these compounds were found to enhance anti-CD3 stimulated IL-17 reporter activity in a Jurkat cell reporter assay. If a compound is a RORc agonist and activates the IL-17 reporter, it should be able to do so in the absence of anti-CD3 treatment. Indeed, activation of the IL17 reporter was also observed for compounds 9−11 in the absence of anti-CD3 (EC 50 ∼0.1 μM). Furthermore, compound 10 was assayed in TH17 differentiation experiments, and it was observed that production of IL-17 could be dosedependently increased up to 220% of basal activity with a 3 μM concentration of compound 10.
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DISCOVERY OF RORC INVERSE AGONISTS In the previous section discussing RORc agonists, there were multiple examples of ligands that were initially identified as RORc inverse agonists using coactivator recruitment assays with the RORc-LBD, but then the same compounds had agonist activity when tested in cell assays using full-length RORc. Because of the compelling preclinical biology surrounding RORc, there was a strong interest in identifying RORc inverse agonists. Fortunately, many RORc inverse agonists hits identified using the LBD maintained an inverse agonist profile when tested in confirmatory cellular assays. The potential to optimize inhibitors through the use of structural biology and medicinal chemistry has led to the formation of a
Figure 11. Optimization of RORc inverse agonists by Scripps Florida. 5877
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17 protein expression, both when compound treatment was initiated at the same time as TH17 polarizing conditions and when compound treatment occurred well after TH17 cell differentiation. Compound 14 affected gene expression (elevated Foxp3, decreased RORγt) when splenocytes were cultured under T regulatory (Treg) cell-polarizing conditions and also led to an increase in the proportion of Treg cells after 5day treatment. This latter result was surprising, given that previously tested RORc ligands, including 13, had no effect on Treg cell populations. It is not clear why 14 had a different effect on Treg cells than 13, but a ligand that can target both TH17 and Treg cells would be of interest. In addition to the detailed disclosure of 13, the team at Scripps Florida also disclosed SAR for a selected set of related analogues (Figure 12).87 Methylation at various positions on
Elegant hydrogen−deuterium exchange (HDX) mass spectrometry studies were performed on the RORc-LBD using 12.83 The results indicated that 12 made many contacts with the ligand-binding pocket that were similar to those in the cocrystal structure of 1 with the RORc-LBD. Although compounds 1 and 12 have different functional activities in coactivator peptide recruitment assays, the HDX studies provide insights into how 12 can block coactivator recruitment and thereby repress the transcriptional output of target genes. Compound 12 displayed inverse agonist activity in an IL-17 promoter-driven luciferase reporter assay in HEK293 cells and similarly reduced endogenous IL-17A gene expression in an EL4 murine lymphoma cell line. Using splenocytes cultured under TH 17 polarizing conditions, 12 blunted mRNA expression of a number of cytokines, including IL-17A and IL-17F, relative to control cells. Compound 12 was dosed i.p. in mice at 25 mg/kg b.i.d. in an established multiple-sclerosisrelated murine model of TH17 cell-mediated autoimmune disease (EAE). Significant decreases in mean clinical scores, as well as reduced mRNA expression of IL-17A and IL-22, relative to vehicle controls, all indicated that 12 was acting in vivo to suppress TH17 cell function. This body of work speaks to the positive effects that can be obtained through inverse agonism of RORc and RORa, but the next advance in understanding the role of RORc came with the identification of 13. Compound 13 (Figure 11) maintained the hexafluoroisopropanol group of the previously discussed compounds and added a novel para-substituted biphenyl motif with an extended tail containing a piperazine.84 It also had a 3-fluoro group on the phenyl ring containing the hexafluoroisopropanol attachment. Compound 13 bound to RORc with a Ki value of 105 nM and blocked transcriptional activity in a GAL4 RORc-LBD cell based reporter assay with an EC50 value of 320 nM. The compound did not show activity in the corresponding LXRα or FXR reporter assays, and most importantly, it also was inactive in the RORa assay. In 293T cells, this RORc selective inverse agonist suppressed transcriptional activity driven by an IL-17 promoter cotransfected with full length RORc, but it did not have activity when the cotransfection was done with RORa. In an EL-4 murine lymphoma cell line, 13 inhibited endogenous IL-17 gene expression, IL-23 receptor expression, and IL-17 production. Recently disclosed mouse CIA model results revealed that i.p. administration of 13 b.i.d. for 15 days resulted in a statistically significant reduction in joint inflammation versus the vehicle control arm.86 The latest compound disclosed by Burris and co-workers (14, Figure 11) was similar in structure to 13, but it lacked the 3-fluoro group on the hexafluoroisopropanol-containing phenyl ring and had its piperazine tail capped with an acetyl group.85 This compound bound to the RORc-LBD with an IC50 value of 1 μM and functioned as a RORc inverse agonist in a GAL4RORc cotransfection assay (EC50 = 1.5 μM). Compound 14 did not have significant activity when tested in RORa, LXR, or FXR cotransfection assays. In HEK293 cells, 14 suppressed IL17 promoter-driven activity when cotransfected with RORc but not RORa. Compound 14 also led to reduced IL-17 gene expression in EL4 cells, all of this indicating a pharmacological profile similar to that of 13. The effects of 14 on TH17 cell differentiation were investigated in murine splenocytes cultured under TH17 polarizing conditions. Compound 14 inhibited mRNA expression of IL-17A but not IL-17F, thus differentiating the effects of this compound from the RORc/a inverse agonist (13) discussed above. Compound 14 also reduced IL-
Figure 12. Structures for the methylated analogues of 13.
the scaffold provided a 2-to-3-fold improvement in binding potency as assessed by a [3H]T0901317 scintillation proximity binding assay (SPA) with the RORc-LBD. Compound 13 was fairly potent in the SPA assay (IC50 = 150 nM), whereas an analogue that was methylated at one of the benzylic carbons (15) was less potent (IC50 = 430 nM). Methylation on the ortho-carbon of the central biaryl ring (16) resulted in improved potency (IC50 = 70 nM), and methylation at the meta-carbon of the terminal biaryl ring (17) provided a similar improvement in potency (IC50 = 90 nM). New York University. As discussed previously, 2 (Figure 6) was identified as a RORγt inverse agonist.73 The Littman group also performed a focused screen and found that 2 inhibited RORγt transcriptional activity in a luciferase-based cotransfection assay (IC50 = 1.98 μM).68 The compound was not active in the corresponding RORα assay. In a competitive binding assay with RORγ-LBD, 2 displaced fluorescein-labeled 25-HC with an IC50 value of 4.1 μM. Compound 2 also caused a significant reduction in expression of IL-17A when naı̈ve mouse CD4+ T cells were subjected to TH17 polarizing conditions. In wild-type cells treated with 2, a reduction of TH17 cell differentiation was observed with and without IL-23 induction. Similar to 13, but unlike 14, compound 2 had no effect on Treg cell differentiation. Daily dosing of 2, via i.p. administration, for 20 days in a mouse EAE model resulted in delayed onset and reduced severity of encephalomyelitis. In this multiple-sclerosis-related model, 2 demonstrated a 50% reduction in IL-17-producing T cells entering the spinal column compared to vehicle control. There was no change in the number of IFNγ-producing TH1 cells. Because of its inhibitory activity against sodium/potassium-ATPase, compound 2 is toxic to human cells at relatively low concentrations (>300 nM),88 partially explaining why so much of the original work described in this perspective focused on murine cell lines and the use of RORγ instead of RORc in human cell lines. Two 5878
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in a cell-based gene reporter assay (EC50 = 3.3 μM) but had no activity in a corresponding RORa assay.90 Compound 22 also inhibited mouse TH17 cell differentiation but had no effect on TH1 cell differentiation. Maintaining the core 3,3-diphenylpropanamide motif, SAR was explored on three separate regions of the molecule leading to eventual identification of 23.90 Over 40 amine variations were explored with cis-3,5-dimethylpiperidine giving a 3-fold improvement in potency over the parent compound. Exploration of the other regions yielded no significant improvements in potency. Chiral separation of the enantiomers led to the isolation of the (−)-enantiomer 24 that had an EC50 value of 0.5 μM in the cell-based RORc gene reporter assay, while the (+)-enantiomer had an EC50 value of 10.5 μM.90 Compound 24 was inactive against RORa and also was largely inactive against a panel of 20 other NRs with measurable EC50 values obtained for only ERRα (14 μM), LXRα (10 μM), thyroid receptor (TR)-α (4.5 μM), and TRβ (13 μM). The racemate (23) inhibited murine TH17 cell differentiation, while it had no differential effects on naı̈ve CD4+ T cells. This compound also suppressed RORc-directed IL-17A expression in human T cells, while it had no effect on the corresponding RORa-directed assay. Further lead optimization by this group is reported to be ongoing. In another publication,91 a team at Scripps Florida used compound 24 as a lead identified from the patent literature.92 This publication expanded the number of compounds made to explore this scaffold. The SAR presented in the Scripps Florida paper was consistent with the areas explored in the prior Litmann paper. No significantly improved analogues were identified relative to the Litmann group’s work. Phenex. Phenex described the interaction of various retinoids with RORc as assessed by a RORc-LBD SRC1 coactivator peptide recruitment assay (Figure 15). Retinoid antagonist LE540 (25)94 had modest activity as a RORc inverse agonist (EC50 = 4 μM).93 Other retinoids95 such as Ro13− 7410 (26) (RORc EC50 = 9 μM), 9-cis-retinoic acid (27) (RORc EC50 = 14 μM), and all-trans-retinoic acid (ATRA, 28) (RORc EC50 = 18 μM) also displayed some RORc inverse agonist activity.93 In a separate disclosure, Phenex described a series of tetrahydrobenzoxazocine analogues as inverse agonists of RORc as assessed in the RORc-LBD SRC1 coactivator peptide recruitment assay (Figure 16). Compounds 29 and 30 from the Phenex patent exhibited RORc inverse agonist EC50 values below 500 nM.96 Compound 29 was progressed into a human peripheral blood mononuclear cell (PBMC) cytokine release assay, and it displayed favorable inhibition of IL-17A (EC50 = 208 nM) with minimal impact on the IL-4 and IL-10 cytokines. Compound 30 also was administered at 10 mg/kg via i.p. administration approximately every 12 h for 17 days in a mouse
analogues, Dig(dhd) (18) and Dig(sal) (19) (Figure 13), were made in an effort to achieve selectivity over the ATPase.
Figure 13. Structures of some analogues of 2.
Complete reduction of the butenolide functionality in 2 afforded the diol moiety in 18, while aldol condensation of the butenolide found in 2 with salicylaldehyde produced the conjugated phenol in 19. Compound 18 was not cytotoxic to human cells up to concentrations of 40 μM but did retain some RORγ binding (IC50 = 12 μM). Compound 19 was also found to bind to RORγ and both compounds inhibited RORc activities. Compound 19 also reduced RORc-mediated IL-17A induction in human CD4+ T cells. In addition to 2 and the digoxin analogues described above, Littman’s group also disclosed some additional SAR data on related analogues in a separate publication. Reduction of the exo-olefin on the α,β-unsaturated lactone found in 19 resulted in an analogue (20, Figure 13) with an EC50 value
