RORγ Inverse Agonists Research Articles

Discovery and pharmacological characterization of 1,2,3,4-tetrahydroquinoline derivatives as RORγ inverse agonists against prostate cancer.

The retinoic acid receptor-related orphan receptor γ (RORγ) is regarded as an attractive therapeutic target for the treatment of prostate cancer. Herein, we report the identification, optimization, and evaluation of 1,2,3,4-tetrahydroquinoline derivatives as novel RORγ inverse agonists, starting from high throughput screening using a thermal stability shift assay (TSA). The representative compounds 13e (designated as XY039) and 14a (designated as XY077) effectively inhibited the RORγ transcriptional activity and exhibited excellent selectivity against other nuclear receptor subtypes. The structural basis for their inhibitory potency was elucidated through the crystallographic study of RORγ LBD complex with 13e. Both 13e and 14a demonstrated reasonable antiproliferative activity, potently inhibited colony formation and the expression of AR, AR regulated genes, and other oncogene in AR positive prostate cancer cell lines. Moreover, 13e and 14a effectively suppressed tumor growth in a 22Rv1 xenograft tumor model in mice. This work provides new and valuable lead compounds for further development of drugs against prostate cancer.

Tumor mitochondrial oxidative phosphorylation stimulated by the nuclear receptor RORγ represents an effective therapeutic opportunity in osteosarcoma

Osteosarcoma (OS) is the most common malignant bone tumor with a poor prognosis. Here, we show that the nuclear receptor RORγ may serve as a potential therapeutic target in OS. OS exhibits a hyperactivated oxidative phosphorylation (OXPHOS) program, which fuels the carbon source to promote tumor progression. We found that RORγ is overexpressed in OS tumors and is linked to hyperactivated OXPHOS. RORγ induces the expression of PGC-1β and physically interacts with it to activate the OXPHOS program by upregulating the expression of respiratory chain component genes. Inhibition of RORγ strongly inhibits OXPHOS activation, downregulates mitochondrial functions, and increases ROS production, which results in OS cell apoptosis and ferroptosis. RORγ inverse agonists strongly suppressed OS tumor growth and progression and sensitized OS tumors to chemotherapy. Taken together, our results indicate that RORγ is a critical regulator of the OXPHOS program in OS and provides an effective therapeutic strategy for this deadly disease.

Inhibition of Nuclear Receptor Related Orphan Receptor γ Ameliorates Mechanical Hypersensitivity Through the Suppression of Spinal Microglial Activation

Microglia are crucial in induction of central sensitization under a chronic pain state. Therefore, control of microglial activity is important to ameliorate nociceptive hypersensitivity. The nuclear receptor retinoic acid related orphan receptor γ (RORγ) contributes to the regulation of inflammation-related gene transcription in some immune cells, including T cells and macrophages. Their role and function in regulation of microglial activity and nociceptive transduction have yet to be elaborated. Treatment of cultured microglia with specific RORγ inverse agonists, SR2211 or GSK2981278, significantly suppressed lipopolysaccharide (LPS)-induced mRNA expression of pronociceptive molecules interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor (TNF). Intrathecal treatment of naïve male mice with LPS markedly induced mechanical hypersensitivity and upregulation of ionized calcium-biding adaptor molecule (Iba1) in the spinal dorsal horn, indicating microglial activation. In addition, intrathecal treatment with LPS significantly induced mRNA upregulation of IL-1β and IL-6 in the spinal dorsal horn. These responses were prevented by intrathecal pretreatment with SR2211. In addition, intrathecal administration of SR2211 significantly ameliorated established mechanical hypersensitivity and upregulation of Iba1 immunoreactivity in the spinal dorsal horn of male mice following peripheral sciatic nerve injury. The current findings demonstrate that blockade of RORγ in spinal microglia exerts anti-inflammatory effects, and that RORγ may be an appropriate target for the treatment of chronic pain.

Ginseng-derived panaxadiol ameliorates STZ-induced type 1 diabetes through inhibiting RORγ/IL-17A axis.

Retinoic-acid-receptor-related orphan receptor γ (RORγ) is a major transcription factor for proinflammatory IL-17A production. Here, we revealed that the RORγ deficiency protects mice from STZ-induced Type 1 diabetes (T1D) through inhibiting IL-17A production, leading to improved pancreatic islet β cell function, thereby uncovering a potential novel therapeutic target for treating T1D. We further identified a novel RORγ inverse agonist, ginseng-derived panaxadiol, which selectively inhibits RORγ transcriptional activity with a distinct cofactor recruitment profile from known RORγ ligands. Structural and functional studies of receptor-ligand interactions reveal the molecular basis for a unique binding mode for panaxadiol in the RORγ ligand-binding pocket. Despite its inverse agonist activity, panaxadiol induced the C-terminal AF-2 helix of RORγ to adopt a canonical active conformation. Interestingly, panaxadiol ameliorates mice from STZ-induced T1D through inhibiting IL-17A production in a RORγ-dependent manner. This study demonstrates a novel regulatory function of RORγ with linkage of the IL-17A pathway in pancreatic β cells, and provides a valuable molecule for further investigating RORγ functions in treating T1D.

1,5-Disubstituted Acylated 2-Amino-4,5-dihydroimidazoles as a New Class of Retinoic Acid Receptor-Related Orphan Receptor (ROR) Inhibitors.

A growing body of evidence suggests a pathogenic role for pro-inflammatory T helper 17 cells (Th17) in several autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, type I diabetes, and psoriasis—diseases for which no curative treatment is currently available. The nuclear retinoic acid receptor–related orphan receptors alpha and gamma (RORα/γ), in particular the truncated isoform RORγt that is specifically expressed in the thymus, play a critical role in the activation of a pro-inflammatory Th17 response, and RORγ inverse agonists have shown promise as negative regulators of Th17 for the treatment of autoimmune diseases. Our study underscores the screening of a large combinatorial library of 1,5-disubstituted acylated 2-amino-4,5-dihydroimidazoles using a demonstrated synthetic and screening approach and the utility of the positional scanning libraries strategy for the rapid identification of a novel class of ROR inhibitors. We identified compound 1295-273 with the highest activity against RORγ (3.3 µM IC50) in this series, and almost a two-fold selectivity towards this receptor isoform, with 5.3 and 5.8 µM IC50 against RORα and RORβ cells, respectively.

In silico design of RORγ inverse agonists based on 3D-QSAR and molecular docking

Computationally designing novel RORγ inverse agonists with higher activity using a systematic modeling study.

Discovery and Characterization of Benzimidazole Derivative XY123 as a Potent, Selective, and Orally Available RORγ Inverse Agonist.

Receptor-related orphan receptor γ (RORγ) has emerged as an attractive therapeutic target for the treatment of cancer and inflammatory diseases. Herein, we report our effort on the discovery, optimization, and evaluation of benzothiazole and benzimidazole derivatives as novel inverse agonists of RORγ. The representative compound 27h (designated as XY123) potently inhibited the RORγ transcription activity with a half-maximal inhibitory concentration (IC50) value of 64 nM and showed excellent selectivity against other nuclear receptors. 27h also potently suppressed cell proliferation, colony formation, and the expression of androgen receptor (AR)-regulated genes in AR-positive prostate cancer cell lines. In addition, 27h demonstrated good metabolic stability and a pharmacokinetic property with reasonable oral bioavailability (32.41%) and moderate half-life (t1/2 = 4.98 h). Significantly, oral administration of compound 27h achieved complete and long-lasting tumor regression in the 22Rv1 xenograft tumor model in mice. Compound 27h may serve as a new valuable lead compound for further development of drugs for the treatment of prostate cancer.

Computer‐Aided Discovery of Novel Human RORγ Inverse Agonists

Overexpression of pro‐inflammatory cytokine IL‐17 can lead to various autoimmune disorders, including psoriasis, Crohn’s disease, and Ankylosing Spondylitis. However, when an inverse agonist binds to the ligand‐binding domain (LBD) of human retinoic acid receptor‐related orphan receptor γ (RORγ), the transcriptional function of RORγ is down‐regulated, resulting in diminished IL‐17 expression. It makes RORγ an ideal drug target for treating autoimmune diseases. This has been studied for over ten years, but only several RORγinverse agonists are under clinical trials. New methods may be needed to develop new drugs efficiently. In this project, we aim to discover potential RORγ inverse agonists by developing a structure‐based virtual screening method that effectively screens millions of compounds in a shorter time scale. We here applied the in silico program iDock to screen a ligand‐like compound library by docking every compound into the binding pocket of RORγ LBD. iDock evaluated the performance of each compound in the pocket and outputted the top 1000 hits. Through excluding the hits structurally similar to the known RORγ inverse agonists, we focused on the ones with unique chemical scaffolds and tested their bioactivity via luciferase reporter assay. A family of compounds sharing the same backbone was found. Two members in this family, RG14 and RG14‐2, inhibited the RORγ transcriptional function with IC50 values at 3.2 μM and 1.5 μM, respectively. To further study the interactions between the compounds and the protein, binding affinities of RG14 and RG14‐2 to the LBD are under investigation by thermal shift assay. RORγ LBD has been successfully co‐crystallized with RG14 and RG14‐2. With solving crystal structures of the protein‐ligand complexes, we will obtain more information to interpret the interactions structurally. A lead compound of a novel class of RORγ inverse agonists is expected to develop, basing on the chemical structures of RG14 and RG14‐2 and their binding actions to RORγ LBD.Support or Funding InformationNSF 1833181

(Inverse) Agonists of Retinoic Acid-Related Orphan Receptor γ: Regulation of Immune Responses, Inflammation, and Autoimmune Disease.

Retinoic acid-related orphan receptor γt (RORγt) functions as a ligand-dependent transcription factor that regulates multiple proinflammatory genes and plays a critical role in several inflammatory and autoimmune diseases. Various endogenous and synthetic RORγ (inverse) agonists have been identified that regulate RORγ transcriptional activity, including many cholesterol intermediates and oxysterols. Changes in cholesterol biosynthesis and metabolism can therefore have a significant impact on the generation of oxysterol RORγ ligands and, consequently, can control RORγt activity and inflammation. These observations contribute to a growing literature that connects cholesterol metabolism to the regulation of immune responses and autoimmune disease. Loss of RORγ function in knockout mice and in mice treated with RORγ inverse agonists results in reduced production of proinflammatory cytokines, such as IL-17A/F, and increased resistance to autoimmune disease in several experimental rodent models. Thus, RORγt inverse agonists might provide an attractive therapeutic approach to treat a variety of autoimmune diseases.

Nucleophilic Aromatic Substitutions of 2-Halo-5-(sulfamoyl)benzoic Acids and N,O-Bis-alkylation via Phase Transfer Catalysis: Synthesis of RoRγ Inverse Agonist GSK2981278A

GSK2981278A (1) is a RORγ inverse agonist used as a potential topical nonsteroidal therapy for psoriasis. New synthesis of 1 was developed based on a SNAr reaction of (tetrahydro-2H-pyran-4-yl)methanol with an aryl halide intermediate, prepared from 2-halobenzoic acids. The dianion underwent in situ N,O-bis-isobutylation, followed by a reduction to provide 1. The new route eliminated a genotoxic tosylate of (tetrahydro-2H-pyran-4-yl)methanol and a difficult reductive amination from the original synthesis starting from methyl salicylate. A primary version of the route has been scaled up to deliver 125 kg of 1. However, the heating of a strong base in DMSO for an extended period during the bis-alkylation was found to be a safety concern for manufacturing. A safer and greener process was then developed utilizing a facile N,O-bis-alkylation, which was conducted under phase transfer conditions with mild bases such as potassium carbonate and in green solvents such as water. The concise four stage sequence from ...

Discovery of novel quinoline sulphonamide derivatives as potent, selective and orally active RORγ inverse agonists.

A high-throughput screen against Inventiva's compound library using a Gal4/RORγ-LBD luciferase reporter gene assay led to the discovery of a new series of quinoline sulphonamides as RORγ inhibitors, eventually giving rise to a lead compound having an interesting in vivo profile after oral administration. This lead was evaluated in a target engagement model in mouse, where it reduced IL-17 cytokine production after immune challenge. It also proved to be active in a multiple sclerosis model (EAE) where it reduced the disease score. The synthesis, structure activity relationship (SAR) and biological activity of these derivatives is described herein.

Discovery and Characterization of XY101, a Potent, Selective, and Orally Bioavailable RORγ Inverse Agonist for Treatment of Castration-Resistant Prostate Cancer.

We report the design, optimization, and biological evaluation of nuclear receptor RORγ inverse agonists as therapeutic agents for prostate cancer treatment. The most potent compound 27 (designated as XY101) exhibited cellular activity with an IC50 value of 30 nM in a cell-based reporter gene assay with good selectivity against other nuclear receptor subtypes. The cocrystal structure of 27 in complex with the RORγ ligand binding domain provided a solid structural basis for its antagonistic mechanism. 27 potently inhibited cell growth, colony formation, and the expression of AR, AR-V7, and PSA. 27 also exhibited good metabolic stability and a pharmacokinetic profile with oral bioavailability of 59% and a half-life of 7.3 h. Notably, 27 demonstrated promising therapeutic effects with significant tumor growth inhibition in a prostate cancer xenograft model in mice. The potent, selective, metabolically stable, and orally available RORγ inverse agonists represent a new class of compounds as potential therapeutics against prostate cancer.

Unique Polypharmacology Nuclear Receptor Modulator Blocks Inflammatory Signaling Pathways.

Obesity and rheumatic disease are mechanistically linked via chronic inflammation. The orphan receptor TREM-1 (triggering receptor expressed on myeloid cells-1) is a potent amplifier of proinflammatory and noninfectious immune responses. Here, we show that the pan modulator SR1903 effectively blocks TREM-1 activation. SR1903 emerged from a chemical series of potent RORγ inverse agonists, although unlike close structural analogues, it has modest agonist activity on LXR and weak repressive activity (inverse agonism) of PPARγ, three receptors that play essential roles in inflammation and metabolism. The anti-inflammatory and antidiabetic efficacy of this unique modulator in collagen-induced arthritis and diet-induced obesity mouse models is demonstrated. Interestingly, in the context of obesity, SR1903 aided in the maintenance of the thymic homeostasis unlike selective RORγ inverse agonists. SR1903 was well-tolerated following chronic administration, and combined, these data suggest that it may represent a viable strategy for treatment of both metabolic and inflammatory disease. More importantly, the ability of SR1903 to block LPS signaling suggests the potential utility of this unique polypharmacological modulator for treatment of innate immune response disorders.

RORγ regulates the NLRP3 inflammasome

RAR-related orphan receptor γ (RORγ) is a nuclear receptor that plays an essential role in the development of T helper 17 (Th17) cells of the adaptive immune system. The NLRP3 inflammasome is a component of the innate immune system that processes interleukin (IL)-1β into a mature cytokine. Elevated activity of the NLRP3 inflammasome contributes to the progression of an array of inflammatory diseases. Bone marrow-derived macrophages (BMDMs) isolated from RORγ-null mice displayed reduced capacity to secrete IL-1β, and they also displayed a reduction in Nlrp3 and Il1b gene expression. Examination of the promoters of the Il1b and Nlrp3 genes revealed multiple putative ROR response elements (ROREs) that were occupied by RORγ. RORγ inverse agonists were effective inhibitors of the inflammasome. RORγ inverse agonists suppressed lipopolysaccharide (LPS)/ATP-stimulated IL-1β secretion and expression of Il1b and Nlrp3 in BMDMs. Additionally, the ability of the RORγ inverse agonists to suppress IL-1β secretion was lost in Nlrp3-null macrophages. The potential for targeting the NLRP3 inflammasome in vivo using RORγ inverse agonists was examined in two models: LPS-induced sepsis and fulminant hepatitis. Pharmacological inhibition of RORγ activity reduced plasma IL-1β as well as IL-1β production by peritoneal macrophages in a model of LPS-induced sepsis. Additionally, RORγ inverse agonists reduced mortality in an LPS/d-galactosamine-induced fulminant hepatitis mouse model. These results illustrate a major role for RORγ in regulation of innate immunity via modulation of NLRP3 inflammasome activity. Furthermore, these data suggest that inhibiting the NLRP3 inflammasome with RORγ inverse agonists may be an effective method to treat NLRP3-associated diseases.

LB1578 Identification and characterization of highly optimized RORγ inverse agonists for the topical treatment of psoriasis

LB1578 Identification and characterization of highly optimized RORγ inverse agonists for the topical treatment of psoriasis

Sulfoximines as potent RORγ inverse agonists

Progress in the identification of suitable RORγ inverse agonists as clinical candidates has been hampered by the high lipophilicity that seems required for high potency on this nuclear receptor. In this context, we decided to focus on the replacement of the hydroxymethyl group found on known modulators to determine if more polarity could be tolerated in this position. SAR of the replacement of this moiety is presented in this article leading to the identification of sulfoximine derivatives as potent modulators with pharmacological activity in the in vivo mouse Imiquimod psoriasis model.

Identification of an aminothiazole series of RORβ modulators

Crystallography has identified stearic acid, ALRT 1550 and ATRA as ligands that bind RORβ, however, none of these molecules represent good starting points to develop optimized small molecule modulators. Recently, Compound 1 was identified as a potent dual RORβ and RORγ inverse agonist with no activity towards RORα (Fig. 1). To our knowledge, this is one of only two small molecule RORβ inverse agonists identified in the primary literature from a tractable chemical series and represents an ideal starting point from which to design RORβ-selective modulators. Herein we describe our SAR optimization efforts that led to a series of potent neutral antagonists of RORβ.

Discovery and Characterization of CD12681, a Potent RORγ Inverse Agonist, Preclinical Candidate for the Topical Treatment of Psoriasis.

With possible implications in multiple autoimmune diseases, the retinoic acid receptor-related orphan receptor RORγ has become a sought-after target in the pharmaceutical industry. Herein are described the efforts to identify a potent RORγ inverse agonist compatible with topical application for the treatment of skin diseases. These efforts culminated in the discovery of N-(2,4-dimethylphenyl)-N-isobutyl-2-oxo-1-[(tetrahydro-2H-pyran-4-yl)methyl]-2,3-dihydro-1H-benzo[d]imidazole-5-sulfonamide (CD12681), a potent inverse agonist with in vivo activity in an IL-23-induced mouse skin inflammation model.

Discovery of phenoxyindazoles and phenylthioindazoles as RORγ inverse agonists

Targeting the IL17 pathway and more specifically the nuclear receptor RORγ is thought to be beneficial in multiple skin disorders. The Letter describes the discovery of phenoxyindazoles and thiophenoxy indazoles as potent RORγ inverse agonists. Optimization of the potency and efforts to mitigate the phototoxic liability of the series are presented. Finally, crystallization of the lead compound revealed that the series bound to an allosteric site of the nuclear receptor. Such compounds could be useful as tool compounds for understanding the impact of topical treatment on skin disease models.

Discovery of selective RORγ inverse agonists and demonstration of efficacy in inflammatory disease models

Abstract The nuclear hormone receptor RORγ controls the differentiation of Th17 cells that plays a key pro-inflammatory role in a variety of autoimmune diseases. RORγ inverse agonists from multiple structural classes were discovered and screened in a RORγ radio-ligand binding assay as well as in a cell based reporter assay and functional effect was evaluated using primary mouse/human CD4+ve T-cells differentiation to Th17 cells. Pharmacokinetic profile of potent compounds was determined to support evaluation of efficacy as well as safety profile in rodents. Lead compounds have demonstrated good activity (< 100 nM) in reporter assay. Crystal structure of RORγ in complex with known inverse agonists as well as novel compounds, were solved to support lead optimization. Selected compounds demonstrated >100 fold selectivity against RORα as well as other nuclear receptors with significant inhibition of IL-17 release in Th17 differentiation assay. Lead compounds have shown optimal physicochemical profile including good solubility and high free fraction, leading to excellent pharmacokinetic profile in mice and rats. Efficacy of lead compounds has been demonstrated in multiple disease models including asthma, psoriasis, Experimental auto immune encephalomyelitis and collagen induced arthritis. Potential lead candidates have been identified with excellent in-vitro safety profile including CEREP-44 safety panel, Ames test and cytotoxicity. One of the lead candidates has also demonstrated good therapeutic window as determined by a 7 day rat toxicity study. Conclusions We have identified structurally diverse small molecule inverse agonists of RORγ and have demonstrated efficacy in relevant disease models as well as good therapeutic window.