Retinoic Acid Receptor-related Orphan Research Articles

Coproporphyrin-I as a Selective OATP1B Biomarker Can Be Used to Delineate the Mechanisms of Complex Drug-Drug Interactions: Cedirogant Case Study.

Cedirogant is an inverse agonist of retinoic acid-related orphan receptor gamma thymus developed for the treatment of chronic plaque psoriasis. Cedirogant induces cytochrome P450 (CYP) 3A4 while inhibiting P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), organic anion transporting polypeptide (OATP) 1B1, and OATP1B3 in vitro. Static drug-drug interactions (DDIs) predictions suggested possible clinical induction of CYP3A4, and inhibition of P-gp, BCRP, and OATP1B1, leading to challenges in interpreting DDI studies between cedirogant and substrates of CYP3A, P-gp, BCRP, and OATP1B1/3. Here the effects of cedirogant on the pharmacokinetics of two statin drugs were investigated in healthy participants. Coproporphyrin-I (CP-I), a selective endogenous OATP1B biomarker, was used to assess the impact of cedirogant on OATP1B. Cedirogant (375 mg once daily) increased rosuvastatin maximum plasma concentration (Cmax) and area under the plasma concentration curve (AUCtau) by 141% and 55%, respectively when co-administered, whereas atorvastatin Cmax increased by 40% with no effect on its AUCtau compared with administration of rosuvastatin/atorvastatin alone. Cedirogant did not increase CP-I exposures, indicating no clinical OATP1B inhibition. The increased rosuvastatin exposure and minimal change in atorvastatin exposure with co-administration of cedirogant is attributed to BCRP inhibition and interplay between P-gp/BCRP inhibition and CYP3A induction, respectively. Correlation analysis with data from two investigational drugs (glecaprevir and flubentylosin) demonstrated that OATP1B1 R-value of > 1.5 and [Cmax,u]/[OATP1B1 IC50] of > 0.1 are associated with > 1.25-fold increase in CP-I Cmax ratio. This demonstrates the utility of CP-I in disentangling mechanisms underlying a complex DDI involving multiple transporters and enzymes and proposes refined criteria for static OATP1B inhibition predictions.

Anti-Inflammatory and Immunoregulatory Effects of Xiaoqinglong Decoction on a Murine Model of Combined Allergic Rhinitis and Asthma Syndrome

Background Xiaoqinglong Decoction (XQLD) is a traditional Chinese medicine formula used for the treatment of allergic rhinitis and asthma, including combined allergic rhinitis and asthma syndrome (CARAS), due to its anti-inflammatory and anti-seizure properties. However, the pharmacological activities and the underlying molecular mechanisms of XQLD remain to be elucidated . Hence, we investigated the effects of XQLD against inflammation in an ovalbumin (OVA)-induced CARAS model in BALB/c mice. Methods BALB/c mice were sensitized by intraperitoneal injection and aerosol inhalation of OVA. XQLD or dexamethasone was administered by oral gavage prior to OVA challenge for 7 consecutive days. Specific airway resistance (sRAW) was evaluated 24 h after the final challenge with OVA. Enzyme-linked immunosorbent assay was used to measure the levels of serum inflammatory factors. The nasal mucosa and lung tissue were examined for general morphology and goblet hyperplasia using hematoxylin and eosin or periodic acid Schiff staining. Flow cytometry was employed to analyze the frequencies of helper T lymphocytes in peripheral blood. Immunohistochemistry was performed to determine the expression of transcription factors in helper T lymphocytes and γδ TCR. Results XQLD significantly ameliorated the symptoms of CARAS model mice, suppressed serum levels of interferon-γ, interleukin (IL)-2, IL-4, IL-5, IL-17, while increasing levels of IL-10, transforming growth factor-β. Epithelium impairment, cilia loss, eosinophil infiltration, and goblet cell metaplasia were significantly reduced in the nasal mucosa and lung tissue sections. XQLD restored the balance of Th1/Th2 and Th17/Treg cell frequency in peripheral blood. Furthermore, XQLD down-modulated the expression of GATA-binding protein 3 (GATA3), retinoic acid receptor-related orphan receptor γt, and γδ T cell receptor (TCR) in the nasal mucosa, along with GATA3 and γδ TCR in the lung tissue. Conclusions XQLD alleviated OVA-induced CARAS in BALB/c mice through its anti-allergic effects and modulation of the immune system.

Renal IL-23-Dependent Type 3 Innate Lymphoid Cells Link Crystal-induced Intrarenal Inflammasome Activation with Kidney Fibrosis.

Chronic inflammasome activation in mononuclear phagocytes (MNPs) promotes fibrosis in various tissues, including the kidney. The cellular and molecular links between the inflammasome and fibrosis are unclear. To address this question, we fed mice lacking various immunological mediators an adenine-enriched diet, which causes crystal precipitation in renal tubules, crystal-induced inflammasome activation, and renal fibrosis. We found that kidney fibrosis depended on an intrarenal inflammasome-dependent type 3 immune response driven by its signature transcription factor Rorc (retinoic acid receptor-related orphan receptor C gene), which was partially carried out by type 3 innate lymphoid cells (ILC3s). The role of ILCs in the kidney is less well known than in other organs, especially that of ILC3. In this article, we describe that depletion of ILCs or genetic deficiency for Rorc attenuated kidney inflammation and fibrosis. Among the inflammasome-derived cytokines, only IL-1β expanded ILC3 and promoted fibrosis, whereas IL-18 caused differentiation of NKp46+ ILC3. Deficiency of the type 3 maintenance cytokine, IL-23, was more protective than IL-1β inhibition, which may be explained by the downregulation of the IL-1R, but not of the IL-23R, by ILC3 early in the disease, allowing persistent sensing of IL-23. Mechanistically, ILC3s colocalized with renal MNPs invivo as shown by multiepitope-ligand cartography. Cell culture experiments indicated that renal ILC3s caused renal MNPs to increase TGF-β production that stimulated fibroblasts to produce collagen. We conclude that ILC3s link inflammasome activation with kidney inflammation and fibrosis and are regulated by IL-1β and IL-23.

Retinoic acid receptor-related orphan receptor alpha and synthetic RORα agonist against invasion and metastasis in tongue squamous cell carcinoma

Retinoic acid receptor-related orphan receptor alpha (RORα), an essential tumor suppressor in a range of human malignancies, is classified as a member of the orphan nuclear receptor family. The most prevalent form of oral cancer, tongue squamous cell carcinoma (TSCC) is characterized by its severe malignancy and unfavorable prognosis. However, the extent to which its tumorigenesis mechanisms are associated with RORα expression levels is still not fully understood. The objective of this study was to examine the molecular mechanisms by which RORα is involved in TSCC. Through the use of immunohistochemistry (IHC), it was discovered that the expression level of RORα was significantly downregulated in TSCC tissues when compared to adjacent normal tissues in this study. To further investigate the role of RORα in TSCC, we activated the expression of RORα in human TSCC cell line (SCC9 cells) by transfecting RORα cDNA and using the selective RORα agonist SR1078. The results show that RORα can significantly inhibit the invasion, migration, proliferation, and adhesion of TSCC cells and induce cell apoptosis. In addition, xenograft models confirmed the conclusion that stable activation or treatment with SR1078 to increase RORα content significantly inhibited tumor growth and development. Taken together, this study provides solid evidence for the inhibitory role of RORα in the progression of TSCC. In addition, the preliminary application results of SR1078 in TSCC show that SR1078 is expected to be a potential therapeutic medication for TSCC. These findings provide innovative perspectives on the development of potential biomarkers and agents for TSCC therapy. The objective is to introduce novel strategy and alternatives for the prevention and treatment of TSCC.

Effects of dietary Chitosan oligosaccharides supplementation on Th17/Treg balance and gut microbiota of early weaned pigeon squabs

Our previous study found that early weaning is associated with decreased growth performance, intestinal barrier impairment, and an imbalance in Th17/Treg in pigeon squabs. Chitosan oligosaccharides (COS) has been substantiated to regulate gut microbiota and restore Th17/Treg equilibrium in mammals, thereby ameliorating growth performance. However, the potential effects of COS in altricial birds remain unclear. Three hundred healthy 7-day-old American king pigeon squabs were selected with similar body weights and randomly divided into 5 groups. The 5 treatment groups were as follows: the control group (CON), fed with artificial pigeon milk; 4 supplementation groups, fed with artificial pigeon milk +100 (COS1), 150 (COS2), 200 (COS3), and 250 (COS4) mg/kg COS, respectively. Results showed that dietary supplementation of COS significantly enhanced the growth performance of weaned squabs. Compared to the CON group, the COS groups exhibited increased villus length and villus area in the jejunum and ileum, accompanied by improvements in morphological structure and mucosal permeability. COS was found to reduce the levels of Th17-associated cytokines and increase the levels of Treg-associated cytokines. COS downregulated the expression of retinoic acid receptor-related orphan receptor C (RORC), a key transcription factor of Th17 cells, while upregulated the expression of Forkhead box protein P3 (FOXP3), a key transcription factor of Treg cells. Dietary COS supplementation increased gut bacterial diversity, altered the relative abundance of several bacteria taxa and enhanced the concentration of short-chain fatty acids (SCFA). Correlation analysis demonstrated a close association between gut microbiota, SCFAs, and indicators related to the Th17/Treg balance. Moreover, we found that SCFAs correlated more strongly with Th17/Treg-related indexes than gut microbiota. These results demonstrated that COS could relieve early weaning stress in pigeon squabs and the optimal dosage of dietary COS supplementation was suggested to be 200 mg/kg. In addition, COS had a protective effect on maintaining intestinal immune balance by modulating microbiota and Th17/Treg related signaling pathways, in which SCFAs might play a crucial role as messengers.

Impairment of Gal-9 and Tim-3 crosstalk between Tregs and Th17 cells drives tobacco smoke-induced airway inflammation.

Overexpression of T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) on T cells has been observed in smokers. However, whether and how galectin-9 (Gal-9)/TIM-3 signal between T-regulatory cells (Tregs) and type 17 helper (Th17) cells contributes to tobacco smoke-induced airway inflammation remains unclear. Here, we aimed to explore the role of the Gal-9/TIM-3 signal between Tregs and Th17 cells during chronic tobacco smoke exposure. Tregs phenotype and the expression of TIM-3 on CD4+ T cells were detected in a mouse model of experimental emphysema. The role of TIM-3 in CD4+ T cells was explored in a HAVCR2-/- mouse model and in mice that received recombinant anti-TIM3. The crosstalk between Gal-9 and Tim-3 was evaluated by coculture Tregs with effector CD4+ T cells. We also invested the expression of Gal-9 in Tregs in patients with COPD. Our study revealed that chronic tobacco smoke exposure significantly reduces the frequency of Tregs in the lungs of mice and remarkably shapes the heterogeneity of Tregs by downregulating the expression of Gal-9. We observed a pro-inflammatory but restrained phenotypic transition of CD4+ T cells after tobacco smoke exposure, which was maintained by TIM-3. The restrained phenotype of CD4+ T cells was perturbed when TIM-3 was deleted or neutralised. Tregs from the lungs of mice with emphysema displayed a blunt ability to inhibit the differentiation and proliferation of Th17 cells. The inhibitory function of Tregs was partially restored by using recombinant Gal-9. The interaction between Gal-9 and TIM-3 inhibits the differentiation of Th17 cells and promotes apoptosis of CD4+ T cells, possibly by interfering with the expression of retinoic acid receptor-related orphan receptor gamma t. The expression of Gal-9 in Tregs was reduced in patients with COPD, which was associated with Th17 response and lung function. These findings present a new paradigm that impairment of Gal-9/Tim-3 crosstalk between Tregs and Th17 cells during chronic tobacco smoke exposure promotes tobacco smoke-induced airway/lung inflammation.

The Role of Notch Signaling Pathway in Adult Patients with Epstein-Barr Virus-induced Infectious Mononucleosis

To investigate the changes of Notch signaling molecules and Th22 cells in adult patients with infectious mononucleosis (IM), and assess the regulatory function of Notch signaling inhibition to Th22 cells. Forty-two IM patients and twenty-one healthy controls were enrolled in this study. Their peripheral blood was collected, from which plasma and peripheral blood mononuclear cells (PBMCs) were isolated. Plasma interleukin (IL)-17 and IL-22 were measured by enzyme-linked immunosorbent assay. The percentages of CD3+ CD4+ IL-17+ Th17 cells and CD3+ CD4+ IL-22+ Th22 cells were investigated by flow cytometry. The mRNA relative levels corresponding to Th17 transcription factor retinoic acid related orphan receptor γt (RORγt), Th22 transcription factor aryl hydrocarbon receptor (AhR), and Notch signaling pathway molecules (including Notch receptors, Notch ligands, Notch downstream molecules) were semi-quantified by real-time PCR. CD4+ T cells were purified and stimulated with γ-secretase inhibitor (GSI). Cellular proliferation, Th17 and Th22 percentage, IL-17 and IL-22 secretion, transcription factor mRNA were measured in response to GSI stimulation. The relative expression levels of Notch1 and Notch2 mRNA in PBMCs of IM group were 13.58±3.18 and 4.73±1.16, respectively, which were significantly higher than 1.09±0.12 and 1.07±0.15 in PBMCs of control group (both P < 0.001). However, there were no significant differences in relative expression levels of Notch3 and Notch4 mRNA between IM group and control group (P >0.05). The relative expression levels of Notch ligands (including DLL1 and Jagged1 ) mRNA and Notch downstream molecules (including Hes1, Hes5, and Hey1 ) were increased in IM group compared with control group (all P < 0.001). In IM group, the Th17 and Th22 percentage were 5.03%±1.15% and 4.48%±1.29%, respectively, which were both higher than 4.36%±0.82% and 3.83%±0.55% in control group (both P < 0.05). In IM group, the IL-17 and IL-22 level were (301.1±53.82) and (101.2±16.45) pg/ml, respectively, which were both higher than (237.2±72.18) and (84.75±11.83) pg/ml in control group (both P < 0.001). In IM group, the relative expression levels of RORγt and AhR mRNA were 1.25±0.22 and 1.21±0.12, respectively, which were both higher than 0.99±0.15 and 1.04±0.11 in control group (both P < 0.001). There were no remarkable differences in CD4+ T cell proliferation, Th17 percentage, IL-17 secretion, and relative expression level of RORγt mRNA between cells with GSI stimulation and without GSI stimulation (P >0.05). GSI stimulation reduced Th22 percentage, IL-22 secretion, and relative expression level of AhR mRNA compared with non-stimulation (all P < 0.05). Notch signaling pathway regulates IL-22 secretion by CD4+ T cells via AhR in IM patients. Notch-AhR-Th22 pathway may take part in the pathogenesis of IM.

Correlation of Professional Antigen-Presenting Tbet+CD11c+ B Cells With Bone Destruction in Untreated Rheumatoid Arthritis.

Subsets of CD21-/low memory B cells (MBCs), including double-negative (DN, CD27-IgD-) and Tbet+CD11c+ cells, are expanded in chronic inflammatory diseases. In rheumatoid arthritis (RA), CD21-/low MBCs correlate with joint destruction. However, whether this is due to the Tbet+CD11c+ subset, its function and pathogenic contribution to RA are unknown. This study aims to investigate the association between CD21-/lowTbet+CD11c+ MBCs and joint destruction as well as other clinical parameters and to elucidate their functional properties in patients with untreated RA (uRA). Clinical observations were combined with flow cytometry (n = 36) and single-cell RNA sequencing (scRNA-seq) and V(D)J sequencing (n = 4) of peripheral blood (PB) MBCs from patients with uRA. The transcriptome of circulating Tbet+CD11c+ MBCs was compared with scRNA-seq data of synovial B cells. In vitro coculture of Tbet+CD11c+ B cells with T cells was used to assess costimulatory capacity. CD21-/lowTbet+CD11c+ MBCs in PB correlated with bone destruction but no other clinical parameters analyzed. The Tbet+CD11c+ MBCs have undergone clonal expansion and express somatically mutated V genes. Gene expression analysis of these cells identified a unique signature of more than 150 up-regulated genes associated with antigen presentation functions, including B cell receptor activation and clathrin-mediated antigen internalization; regulation of actin filaments, endosomes, and lysosomes; antigen processing, loading, presentation, and costimulation; a transcriptome mirrored in their synovial tissue counterparts. In vitro, Tbet+CD11c+ B cells induced retinoic acid receptor-related orphan nuclear receptor γT expression in CD4+ T cells, thereby polarizing to Th17 cells, a T cell subset critical for osteoclastogenesis and associated with bone destruction. This study suggests that Tbet+CD11c+ MBCs contribute to the pathogenesis of RA by promoting bone destruction through antigen presentation, T cell activation, and Th17 polarization.

Regulation of RORα Stability through PRMT5-Dependent Symmetric Dimethylation.

Retinoic acid receptor-related orphan receptor alpha (RORα), a candidate tumor suppressor, is prevalently downregulated or lost in malignant breast cancer cells. However, the mechanisms of how RORα expression is regulated in breast epithelial cells remain incompletely understood. Protein arginine N-methyltransferase 5 (PRMT5), a type II methyltransferase catalyzing the symmetric methylation of the amino acid arginine in target proteins, was reported to regulate protein stability. To study whether and how PRMT5 regulates RORα, we examined the direct interaction between RORα and PRMT5 by immunoprecipitation and GST pull-down assays. The results showed that PRMT5 directly bound to RORα, and PRMT5 mainly symmetrically dimethylated the DNA-binding domain (DBD) but not the ligand-binding domain (LBD) of RORα. To investigate whether RORα protein stability is regulated by PRMT5, we transfected HEK293FT cells with RORα and PRMT5-expressing or PRMT5-silencing (shPRMT5) vectors and then examined RORα protein stability by a cycloheximide chase assay. The results showed that PRMT5 increased RORα protein stability, while silencing PRMT5 accelerated RORα protein degradation. In PRMT5-silenced mammary epithelial cells, RORα protein expression was decreased, accompanied by an enhanced epithelial-mesenchymal transition morphology and cell invasion and migration abilities. In PRMT5-overexpressed mammary epithelial cells, RORα protein was accumulated, and cell invasion was suppressed. These findings revealed a novel mechanism by which PRMT5 regulates RORα protein stability.

Retinoic acid related orphan receptor α is a genetic modifier that rescues retinal degeneration in a mouse model of Stargardt disease and Dry AMD

Degeneration of the macula is associated with several overlapping diseases including age-related macular degeneration (AMD) and Stargardt Disease (STGD). Mutations in ATP Binding Cassette Subfamily A Member 4 (ABCA4) are associated with late-onset dry AMD and early-onset STGD. Additionally, both forms of macular degeneration exhibit deposition of subretinal material and photoreceptor degeneration. Retinoic acid related orphan receptor α (RORA) regulates the AMD inflammation pathway that includes ABCA4, CD59, C3 and C5. In this translational study, we examined the efficacy of RORA at attenuating retinal degeneration and improving the inflammatory response in Abca4 knockout (Abca4−/−) mice. AAV5-hRORA-treated mice showed reduced deposits, restored CD59 expression and attenuated amyloid precursor protein (APP) expression compared with untreated eyes. This molecular rescue correlated with statistically significant improvement in photoreceptor function. This is the first study evaluating the impact of RORA modifier gene therapy on rescuing retinal degeneration. Our studies demonstrate efficacy of RORA in improving STGD and dry AMD-like disease.

Nobiletin Stimulates Adrenal Hormones and Modulates the Circadian Clock in Mice.

Polymethoxyflavonoids, such as nobiletin (abundant in Citrus depressa), have been reported to have antioxidant, anti-inflammatory, anticancer, and anti-dementia effects, and are also a circadian clock modulator through retinoic acid receptor-related orphan receptor (ROR) α/γ. However, the optimal timing of nobiletin intake has not yet been determined. Here, we explored the time-dependent treatment effects of nobiletin and a possible novel mechanistic idea for nobiletin-induced circadian clock regulation in mice. In vivo imaging showed that the PER2::LUC rhythm in the peripheral organs was altered in accordance with the timing of nobiletin administration (100 mg/kg). Administration at ZT4 (middle of the light period) caused an advance in the peripheral clock, whereas administration at ZT16 (middle of the dark period) caused an increase in amplitude. In addition, the intraperitoneal injection of nobiletin significantly and potently stimulated corticosterone and adrenaline secretion and caused an increase in Per1 expression in the peripheral tissues. Nobiletin inhibited phosphodiesterase (PDE) 4A1A, 4B1, and 10A2. Nobiletin or rolipram (PDE4 inhibitor) injection, but not SR1078 (RORα/γ agonist), caused acute Per1 expression in the peripheral tissues. Thus, the present study demonstrated a novel function of nobiletin and the regulation of the peripheral circadian clock.

The Circadian Clock Component RORA Increases Immunosurveillance in Melanoma by Inhibiting PD-L1 Expression.

Circadian clock perturbation frequently occurs in cancer and facilitates tumor progression by regulating malignant growth and shaping the immune microenvironment. Emerging evidence has indicated that clock genes are disrupted in melanoma and linked to immune escape. Herein, we found that the expression of retinoic acid receptor-related orphan receptor-α (RORA) is downregulated in melanoma patients and that patients with higher RORA expression have a better prognosis after immunotherapy. Additionally, RORA was significantly positively correlated with T-cell infiltration and recruitment. Overexpression or activation of RORA stimulated cytotoxic T-cell-mediated antitumor responses. RORA bound to the CD274 promoter and formed an inhibitory complex with HDAC3 to suppress PD-L1 expression. In contrast, the DEAD-box helicase family member DDX3X competed with HDAC3 for binding to RORA, and DDX3X overexpression promoted RORA release from the suppressive complex and thereby increased PD-L1 expression to generate an inhibitory immune environment. The combination of a RORA agonist with an anti-CTLA4 antibody synergistically increased T-cell antitumor immunity in vivo. A score based on the combined expression of HDAC3, DDX3X, and RORA correlated with immunotherapy response in melanoma patients. Together, this study elucidates a mechanism of clock component-regulated antitumor immunity, which will help inform the use of immunotherapy and lead to improved outcomes for melanoma patients receiving combined therapeutic treatments. Significance: RORA forms a corepressor complex to inhibit PD-L1 expression and activate antitumor T-cell responses, indicating that RORA is a potential target and predictive biomarker to improve immunotherapy response in melanoma patients.

RAR-related orphan receptor alpha and the staggerer mice: a fine molecular story.

The retinoic acid-related orphan receptor alpha (RORα) protein first came into the limelight due to a set of staggerer mice, discovered at the Jackson Laboratories in the United States of America by Sidman, Lane, and Dickie (1962) and genetically deciphered by Hamilton etal. in 1996. These staggerer mice exhibited cerebellar defects, an ataxic gait, a stagger along with several other developmental abnormalities, compensatory mechanisms, and, most importantly, a deletion of 160 kilobases (kb), encompassing the RORα ligand binding domain (LBD). The discovery of the staggerer mice and the subsequent discovery of a loss of the LBD within the RORα gene of these mice at the genetic level clearly indicated that RORα's LBD played a crucial role in patterning during embryogenesis. Moreover, a chance study by Roffler-Tarlov and Sidman (1978) noted reduced concentrations of glutamic acid levels in the staggerer mice, indicating a possible role for the essence of a nutritionally balanced diet. The sequential organisation of the building blocks of intact genes, requires the nucleotide bases of deoxyribonucleic acid (DNA): purines and pyrimidines, both of which are synthesized, upon a constant supply of glutamine, an amino acid fortified in a balanced diet and a byproduct of the carbohydrate and lipid metabolic pathways. A nutritionally balanced diet, along with a metabolic "enzymatic machinery" devoid of mutations/aberrations, was essential in the uninterrupted transcription of RORα during embryogenesis. In addition to the above, following translation, a ligand-responsive RORα acts as a "molecular circadian regulator" during embryogenesis and not only is expressed selectively and differentially, but also promotes differential activity depending on the anatomical and pathological site of its expression. RORα is highly expressed in the central nervous system (CNS) and the endocrine organs. Additionally, RORα and the clock genes are core components of the circadian rhythmicity, with the expression of RORα fluctuating in a night-day-night sigmoidal pattern and undoubtedly serves as an endocrine-like, albeit "molecular-circadian regulator". Melatonin, a circadian hormone, along with tri-iodothyronine and some steroid hormones are known to regulate RORα-mediated molecular activity, with each of these hormones themselves being regulated rhythmically by the hypothalamic-pituitary axis (HPA). The HPA regulates the circadian rhythm and cyclical release of hormones, in a self-regulatory feedback loop. Irregular sleep-wake patterns affect circadian rhythmicity and the ability of the immune system to withstand infections. The staggerer mice with their thinner bones, an altered skeletal musculature, an aberrant metabolic profile, the ataxic gait and an underdeveloped cerebellar cortex; exhibited compensatory mechanisms, that not only allowed the survival of the staggerer mice, but also enhanced protection from microbial invasions and resistance to high-fat-diet induced obesity. This review has been compiled in its present form, more than 14years later after a chromatin immunoprecipitation (ChIP) cloning and sequencing methodology helped me identify signal transducer and activator oftranscription 5 (STAT5) target sequences, one of which was mapped to the firstintron of the RORα gene. The 599-base-long sequence containing one consensus TTCNNNGAA (TTCN3GAA) gamma-activated sequence (GAS) and five other non-consensus TTN5AA sequences had been identified from the clones isolated from the STAT5 target sites (fragments) in human phytohemagglutinin-activated CD8+ T lymphocytes, during my doctoral studies between 2006 and 2009. Most importantly, preliminary studies noted a unique RORα expression profile, during a time-course study on the ribonucleic acid (RNA), extracted from human phytohemagglutinin (PHA) activated CD8+ T lymphocytes stimulated with interleukin-2 (IL-2). This review mainly focuses on the "staggerer mice" with one of its first roles materialising during embryogenesis, a molecular-endocrine mediated circadian-like regulatory process.

Cedirogant in adults with psoriasis: a phase 2, randomized, placebo-controlled clinical trial.

Dysregulated interleukin (IL)-17/IL-23 signaling contributes to psoriasis pathogenesis. Cedirogant is an inverse agonist of retinoic acid-related orphan receptor gamma thymus (RORγt), a key transcription factor responsible for IL-17 synthesis and a regulator of the T helper 17 cell lineage program. To evaluate the efficacy and safety of cedirogant to treat moderate-to-severe psoriasis. In this phase 2b, multicenter, double-blind, 16-week study (NCT05044234), adults aged 18-65 years were randomized 1:1:1:1 to once-daily oral cedirogant 75 mg, 150 mg, 375 mg, or placebo. Assessments included ≥50%/75%/90%/100% improvement from baseline in Psoriasis Area and Severity Index (PASI 50/75/90/100), static Physician Global Assessment 0/1, Psoriasis Symptoms Scale 0, and improvements in itch, adverse events (AEs), pharmacokinetics, and IL-17A/F levels. Efficacy results based on observed cases were summarized descriptively. Of 156 enrolled patients, most were male (70.5%); 39 patients were randomized to each treatment. Only 47 patients completed the study; the study was terminated early due to preclinical findings. At week 16, PASI 75 achievement rates (primary endpoint) were 28.6%, 7.7%, and 41.7% in the cedirogant 75 mg, 150 mg, and 375 mg groups, respectively, and 0% in the placebo group. AE rates were similar in the cedirogant 75 mg, 150 mg, and placebo groups and higher in the cedirogant 375-mg group; most AEs were mild or moderate. Patients with psoriasis who received cedirogant showed PASI improvement and cedirogant was generally well tolerated. Results should be interpreted in the context of early study termination. Cedirogant development has been discontinued.

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.

RORγt inverse agonist TF-S14 inhibits Th17 cytokines and prolongs skin allograft survival in sensitized mice

Chronic antibody mediated rejection (AMR) is the major cause of solid organ graft rejection. Th17 contributes to AMR through the secretion of IL17A, IL21 and IL22. These cytokines promote neutrophilic infiltration, B cell proliferation and donor specific antibodies (DSAs) production. In the current study we investigated the role of Th17 in transplant sensitization. Additionally, we investigated the therapeutic potential of novel inverse agonists of the retinoic acid receptor-related orphan receptor gamma t (RORγt) in the treatment of skin allograft rejection in sensitized mice. Our results show that RORγt inverse agonists reduce cytokine production in human Th17 cells in vitro. In mice, we demonstrate that the RORγt inverse agonist TF-S14 reduces Th17 signature cytokines in vitro and in vivo and leads to blocking neutrophilic infiltration to skin allografts, inhibition of the B-cell differentiation, and the reduction of de novo IgG3 DSAs production. Finally, we show that TF-S14 prolongs the survival of a total mismatch grafts in sensitized mice. In conclusion, RORγt inverse agonists offer a therapeutic intervention through a novel mechanism to treat rejection in highly sensitized patients.

Screening, Deconvolution and Parallel Synthesis of Trisubstituted Piperazine and Trisubstituted 2,3-diketopierazine Libraries for the Rapid Identification of Antagonists of the Nuclear Retinoic Acid Receptor-related Orphan Receptor Gamma (RORγ)

Background: Genetic studies support a key role for RORγ and RORα in the differentiation of proinflammatory Th17 cells, and a growing body of evidence suggests a pathogenic role for Th17 in several autoimmune diseases, including MS, rheumatoid arthritis, inflammatory bowel disease, type I diabetes, and psoriasis. RORγ antagonists have been shown to suppress Th17 differentiation and delay the onset of disease in an experimental autoimmune encephalomyelitis mouse model of MS. Objective: Given the high therapeutic interest of RORγ antagonists and the promising activity of currently known ligands, small molecules with higher potency and receptor selectivity (in particular within the ROR family) are highly desirable. We used our small molecule compound library to discover, characterize, and optimize novel RORγ antagonists for the treatment of autoimmune diseases from Mixture-based Combinatorial Chemical Libraries. Methods: We screened the FIU collection of small molecule libraries (&gt;30 million compounds) composed of 75 molecular scaffolds systematically arranged in positional scanning and scaffold ranking formats. We identified scaffolds that selectively inhibit the binding of RORγ, RORγ, and RORβ but not RORα, and others that function as antagonists of all three receptors. Results: The deconvolution of selected PS-SCL mixtures led to the identification of novel chemical entities, trisubstituted piperazine and diketopiperazine that function as RORγ antagonists. Conclusion: The screening of a large complex library led to the rapid identification of novel trisubstituted piperazine and diketopiperazine antagonists of the nuclear retinoic acid receptor-related orphan receptor gamma (RORγ).

Thiazole Derivatives as RORγt Inhibitors: Synthesis, Biological Evaluation, and Docking Analysis

Background: Cancer is a serious concern in developing countries. Due to various worldwide impacts, it has surpassed coronary disorders becoming the leading cause of mortality. In cancer progression, the involvement of RORs (retinoic acid receptor-related orphan receptors) has gained attention in the search for versatile agents. Thiazole has the potential to be used as a scaffold in the development of anticancer drugs. Objective: This work’s major goal was to design and synthesize promising anticancer candidates. Methods: Docking investigations concerning the RORγt receptor were utilized in the design of the compounds. A series of novel thiazole derivatives was synthesized employing Hantsch synthesis. Melting points, TLC development, and spectral analyses were considered for the synthesized compounds' characterization. Corresponding spectra were analyzed and determined to be consistent with the assigned structure. On 60 human cancer cell lines, selected compounds were investigated in vitro by NCI (National Cancer Institute), USA, for anticancer action. Results: Varying ratios of anticancer potential were presented by the compounds in in vitro studies. Compound 3c was determined to be the most active compound in the series, with a -15.03 mean growth percent. In silico study revealed that the synthesized compounds could have anticancer possibilities. In the targeted pdb id (6q7a) binding site, compound 3c demonstrated excellent docking scores in molecular docking analyses, supporting its ability to be utilized as a lead compound in rational drug design. Conclusion: Compounds with satisfactory docking scores within the binding pocket of chosen pdb id (6q7a) validated their suitability for usage as lead compounds in rational drug design. Outcomes of in silico investigations were in agreement with the findings of the cytotoxicity experiments. Leukemia, renal, breast, and lung cancer cell lines were most sensitive to the substance-treated tumor cells. Electronwithdrawing groups were essential for antiproliferative action, according to the findings.

Clostridium butyricum inhibits the inflammation in children with primary nephrotic syndrome by regulating Th17/Tregs balance via gut-kidney axis

BackgroundPrimary nephrotic syndrome (PNS) is a common glomerular disease in children. Clostridium butyricum (C. butyricum), a probiotic producing butyric acid, exerts effective in regulating inflammation. This study was designed to elucidate the effect of C. butyricum on PNS inflammation through the gut-kidney axis.MethodBALB/c mice were randomly divided into 4 groups: normal control group (CON), C. butyricum control group (CON+C. butyricum), PNS model group (PNS), and PNS with C. butyricum group (PNS+C. butyricum). The PNS model was established by a single injection of doxorubicin hydrochloride (DOX) through the tail vein. After 1 week of modeling, the mice were treated with C. butyricum for 6 weeks. At the end of the experiment, the mice were euthanized and associated indications were investigated.ResultsSince the successful modeling of the PNS, the 24 h urine protein, blood urea nitrogen (BUN), serum creatinine (SCr), urine urea nitrogen (UUN), urine creatinine (UCr), lipopolysaccharides (LPS), pro-inflammatory interleukin (IL)-6, IL-17A were increased, the kidney pathological damage was aggravated, while a reduction of body weights of the mice and the anti-inflammatory IL-10 significantly reduced. However, these abnormalities could be dramatically reversed by C. butyricum treatment. The crucial Th17/Tregs axis in PNS inflammation also was proved to be effectively regulated by C. butyricum treatment. This probiotic intervention notably affected the expression levels of signal transducer and activator of transcription 3 (STAT3), Heme oxygenase-1 (HO-1) protein, and retinoic acid-related orphan receptor gamma t (RORγt). 16S rRNA sequencing showed that C. butyricum could regulate the composition of the intestinal microbial community and found Proteobacteria was more abundant in urine microorganisms in mice with PNS. Short-chain fatty acids (SCFAs) were measured and showed that C. butyricum treatment increased the contents of acetic acid, propionic acid, butyric acid in feces, acetic acid, and valeric acid in urine. Correlation analysis showed that there was a closely complicated correlation among inflammatory indicators, metabolic indicators, microbiota, and associated metabolic SCFAs in the gut-kidney axis.ConclusionC. butyricum regulates Th17/Tregs balance via the gut-kidney axis to suppress the immune inflammatory response in mice with PNS, which may potentially contribute to a safe and inexpensive therapeutic agent for PNS.

Screening of Chelidonium majus isoquinoline alkaloids reveals berberine and chelidonine as selective ligands for the nuclear receptors RORβ and HNF4α, respectively.

The nuclear receptors hepatocyte nuclear factor 4α (HNF4α) and retinoic acid receptor-related orphan receptor-β (RORβ) are ligand-regulated transcription factors and potential drug targets for metabolic disorders. However, there is a lack of small molecular, selective ligands to explore the therapeutic potential in further detail. Here, we report the discovery of greater celandine (Chelidonium majus) isoquinoline alkaloids as nuclear receptor modulators: Berberine is a selective RORβ inverse agonist and modulated target genes involved in the circadian clock, photoreceptor cell development, and neuronal function. The structurally related chelidonine was identified as a ligand for the constitutively active HNF4α receptor, with nanomolar potency in a cellular reporter gene assay. In human liver cancer cells naturally expressing high levels of HNF4α, chelidonine acted as an inverse agonist and downregulated genes associated with gluconeogenesis and drug metabolism. Both berberine and chelidonine are promising tool compounds to further investigate their target nuclear receptors and for drug discovery.