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

The nuclear receptors hepatocyte nuclear factor 4α (HNF4α) and retinoic acid receptor-related orphan receptor-ß (RORß) 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 RORß 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.

Discovery of Biaryl Amide Derivatives as Potent, Selective, and Orally Bioavailable RORγt Agonists for Cancer Immunotherapy.

The master transcription factor receptor retinoic acid receptor-related orphan receptor γt (RORγt) regulates the differentiation of T-helper 17 (Th17) cells and the production of interleukin-17 (IL-17). Activation of RORγt+ T cells in the tumor microenvironment promotes immune infiltration to more effectively inhibit tumor growth. Therefore, RORγt agonists provide a reachable approach to cancer immunotherapy. Herein, a series of biaryl amide derivatives as novel RORγt agonists were designed, synthesized, and evaluated. Starting from the reported RORγt inverse agonist GSK805 (1), "functionality switching" and structure-based drug optimization led to the discovery of a promising RORγt agonist lead compound 14, which displayed potent and selective RORγt agonist activity and significantly improved metabolic stability. With excellent in vivo pharmacokinetic profiles, compound 14 demonstrated robust efficacy in preclinical tumor models of mouse B16F10 melanoma and LLC lung adenocarcinoma. Taken together, current studies indicate that 14 deserves further investigation as a potential lead RORγt agonist for cancer immunotherapy.

Development and therapeutic potential of allosteric retinoic acid receptor-related orphan receptor γt (RORγt) inverse agonists for autoimmune diseases.

The transcription factor retinoic acid receptor-related orphan receptor γt (RORγt) is an attractive drug target for some autoimmune diseases owing to its roles in the differentiation of human T helper 17 (Th17) cells which produce pro-inflammatory cytokine interleukin (IL)-17. RORγt agonists and inverse agonists are classically targeted to the hydrophobic and highly conserved orthosteric binding pocket of RORγt ligand binding domain (LBD). Although successful, this approach also brings some challenges, including off-target effects due to lack of selectivity over other nuclear receptors (NRs). Allosteric regulation of RORγt by synthetic small molecules has recently emerged as novel research interests for its interesting modes of action (MOA), satisfying bioactivity profile and improved selectivity. In this review, we delineated the discovery and identification of the allosteric pocket of RORγt. Subsequently, we focused on examples of small molecules that allosterically inhibit RORγt, with a central attention on structural-activity-relationship (SAR) information, biological activity, pharmacokinetic (PK) property, and the ligand binding mode of these compounds. We also discussed the potential role of RORγt allosteric inverse agonists as small molecule therapeutics for autoimmune diseases.

Discovery of novel triazine derivatives as potent retinoic acid receptor-related orphan receptor γt (RORγt) inverse agonists.

Retinoic Acid Receptor-Related Orphan Receptor γt (RORγt) has been exploited as a promising target for the new small molecule therapeutics to treat inflammatory and autoimmune diseases via modulating the interleukin-17 (IL-17) production by T helper 17 (Th17) cells. Herein, we reported a series of triazine-based derivatives as novel RORγt inverse agonists. By screening of our in-house compound library, the hit compound 1 was identified with weak RORγt inhibitory activity. Subsequently, we engineered detailed structural modifications to explore the structure-activity relationships (SARs) of triazines derivatives, which led to discovery of a number of potent RORγt inverse agonists with IC50 values in the range of 7 nM-50 nM in RORγt dual FRET assay. Among them, compound 14g displayed potent RORγt inverse agonistic activity with an IC50 value of 22.9 nM in dual FRET assay. In a cell-based reporter gene assay, compound 14g showed an IC50 value of 0.428 µM and maximum inhibition rate of 108.9%. Compound 14g also exhibited good metabolic stability and a decent pharmacokinetic profile with a low clearance (CL = 0.229 L/h/kg) and a reasonable oral exposure (AUC0-Last = 5058 ng/mL*h). Most importantly, 14g alleviated the severity of imiquimod-induced psoriasis in mice. Taken together, triazine-based derivatives represent a new chemical class of RORγt inverse agonists as potential therapeutic agents against autoimmune diseases.

Discovery, Synthesis, and In Vitro Characterization of 2,3 Derivatives of 4,5,6,7-Tetrahydro-Benzothiophene as Potent Modulators of Retinoic Acid Receptor-Related Orphan Receptor γt.

Retinoic acid receptor-related orphan receptor γt (RORγt) is a nuclear receptor that is expressed in a variety of tissues and is a potential drug target for the treatment of inflammatory and auto-immune diseases, metabolic diseases, and resistant cancer types. We herein report the discovery of 2,3 derivatives of 4,5,6,7-tetrahydro-benzothiophene modulators of RORγt. We also report the solubility in acidic/neutral pH, mouse/human/dog/rat microsomal stability, Caco-2, and MDR1-MDCKII permeabilities of a set of these derivatives. For this group of modulators, inverse agonism by steric clashes and push-pull mechanisms induce greater instability to protein conformation compared to agonist lock hydration. Independent of the two mechanisms, we observed a basal modulatory activity of the tested 2,3 derivatives of 4,5,6,7-tetrahydro-benzothiophene toward RORγt due to the interactions with the Cys320-Glu326 and Arg364-Phe377 hydrophilic regions. The drug discovery approach reported in the current study can be employed to discover modulators of nuclear receptors and other globular protein targets.

Optimization of carbazole carboxamide RORγt agonists: Challenges in improving the metabolic stability and maintaining the agonistic activity.

Based on two previously discovered carbazole carboxamide retinoic acid receptor-related orphan receptor-γt (RORγt) agonists 6 and 7 (t1/2 = 8.7 min and 16.4 min in mouse liver microsome, respectively), new carbazole carboxamides were designed and synthesized according to the molecular mechanism of action (MOA) and metabolic site analysis with the aim of identifying novel RORγt agonists with optimal pharmacological and metabolic profiles. By modifying the "agonist lock" touching substitutions on carbazole ring, introducing heteroatoms into different parts of the molecule and attaching a side chain to the sulfonyl benzyl moiety, several potent RORγt agonists were identified with greatly improved metabolic stability. Best overall properties were achieved in compound (R)-10f with high agonistic activities in RORγt dual FRET (EC50 = 15.6 nM) and Gal4 reporter gene (EC50 = 141 nM) assays and greatly improved metabolic stability (t1/2 > 145 min) in mouse liver microsome. Besides, the binding modes of (R)-10f and (S)-10f in RORγt ligand binding domain (LBD) were also studied. Altogether, the optimization of carbazole carboxamides led to the discovery of (R)-10f as a potential small molecule therapeutics for cancer immunotherapy.

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 ß 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 ß cells, and provides a valuable molecule for further investigating RORγ functions in treating T1D.

Modes of action insights from the crystallographic structures of retinoic acid receptor-related orphan receptor-γt (RORγt).

RORγt plays an important role in mediating IL-17 production and some tumor cells. It has four functional domains, of which the ligand-binding domain (LBD) is responsible for binding agonists to recruit co-activators or inverse agonists to prevent co-activator recruiting the agonists. Thus, potent ligands targeting the LBD of this protein could provide novel treatments for cancer and autoimmune diseases. In this perspective, we summarized and discussed various modes of action (MOA) of RORγt-ligand binding structures. The ligands can bind with RORγt at either orthosteric site or the allosteric site, and the binding modes at these two sites are different for agonists and inverse agonist. At the orthosteric site, the binding of agonist is to stabilize the H479-Y502-F506 triplet interaction network of RORγt. The binding of inverse agonist features as these four apparent ways: (1) blocking the entrance of the agonist pocket in RORγt; (2) directly breaking the H479-Y502 pair interactions; (3) destabilizing the triplet H479-Y502-F506 interaction network through perturbing the conformation of the side chain in M358 at the bottom of the binding pocket; (4) and destabilizing the triplet H479-Y502-F506 through changing the conformation of the side chain of residue W317 side chain. At the allosteric site of RORγt, the binding of inverse agonist was found recently to inhibit the activation of protein by interacting directly with H12, which results in unfolding of helix 11' and orientation of H12 to directly block cofactor peptide binding. This overview of recent advances in the RORγt structures is expected to provide a guidance of designing more potent drugs to treat RORγt-related diseases.

RORγt agonist enhances anti-PD-1 therapy by promoting monocyte-derived dendritic cells through CXCL10 in cancers.

BACKGROUND: The overall response rate to checkpoint blockade remains unsatisfactory, partially due to the immune-suppressive tumor microenvironment. A retinoic acid-related orphan receptor γt (RORγt) agonist (LYC-55716) is currently used in clinical trials combined with anti-PD-1, but how the Th17 cell transcription factor RORγt enhances antitumor immunity of PD-1 in the tumor microenvironment remains elusive. METHODS: The expression of mRNA was analyzed using qPCR assays. Flow cytometry was used to sort and profile cells. Cell migration was analyzed using Transwell assays. Biacore was used to determine the binding affinity to the RORγt protein. The RORγt GAL4 cell-based reporter gene assay was used to measure activity in the RORγt driven luciferase reporter gene expression. RESULTS: We designed a potent and selective small-molecule RORγt agonist (8-074) that shows robust antitumor efficacy in syngeneic tumor models and improves the efficacy of anti­PD­1 in a murine lung cancer model. RORγt agonist treatment increased intratumoral CD8+ T cells, which were correlated with CXCL10 and monocyte-derived dendritic cells (MoDCs). In addition, the RORγt agonist promoted Type 17 T cell migration by upregulating CCL20 and CCR6 expression, and Type 17 T cell tumor infiltration. CCL20 induces MoDCs migration, and CXCL10 derived from MoDCs promotes CD8+ T cell migration. CONCLUSION: Our results revealed that the RORγt agonist improved the efficacy of anti-PD-1. The RORγt agonist increased the migration of MoDCs, which increased the local levels of CXCL10, thus promoting CD8+ T cell tumor infiltration. Our findings provide the mechanistic insights implicating the RORγt agonist in immunotherapy and offer a strategy for targeting the RORγt agonist to improve PD-1 antibody efficacy in cancers.

Identification of Corosolic and Oleanolic Acids as Molecules Antagonizing the Human RORγT Nuclear Receptor Using the Calculated Fingerprints of the Molecular Similarity.

RORγT is a protein product of the RORC gene belonging to the nuclear receptor subfamily of retinoic-acid-receptor-related orphan receptors (RORs). RORγT is preferentially expressed in Th17 lymphocytes and drives their differentiation from naive CD4+ cells and is involved in the regulation of the expression of numerous Th17-specific cytokines, such as IL-17. Because Th17 cells are implicated in the pathology of autoimmune diseases (e.g., psoriasis, inflammatory bowel disease, multiple sclerosis), RORγT, whose activity is regulated by ligands, has been recognized as a drug target in potential therapies against these diseases. The identification of such ligands is time-consuming and usually requires the screening of chemical libraries. Herein, using a Tanimoto similarity search, we found corosolic acid and other pentacyclic tritepenes in the library we previously screened as compounds highly similar to the RORγT inverse agonist ursolic acid. Furthermore, using gene reporter assays and Th17 lymphocytes, we distinguished compounds that exert stronger biological effects (ursolic, corosolic, and oleanolic acid) from those that are ineffective (asiatic and maslinic acids), providing evidence that such combinatorial methodology (in silico and experimental) might help wet screenings to achieve more accurate results, eliminating false negatives.

Crystallographic landscape provides molecular insights into the modes of action of diverse ROR-γt modulators.

ROR-γt, the master regulator of Th-17 cells, is activated by the binding of small molecules at its orthosteric site, followed by the recruitment of co-activators or co-repressors in the ligand binding domain (LBD). Th-17 cells provide immune-dependent protection against cancers and pathogens. Their dysregulation causes inflammation and is therefore implicated in various autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, and psoriasis. Consequently, there is enormous interest in the development of ROR-γt modulators, both agonist and inverse-agonists. Here, we review advances in the development of ROR-γt modulators that have been made over the past decade, focusing on the rich crystallography landscape for ROR-γt co-crystals that has delineated the relationship between the binding patterns of modulators and the resulting biological activities.

Discovery of N-(2-benzyl-4-oxochroman-7-yl)-2-(5-(ethylsulfonyl) pyridin-2-yl) acetamide (b12) as a potent, selective, and orally available novel retinoic acid receptor-related orphan receptor γt inverse agonist.

The nuclear receptor retinoic acid receptor-related orphan receptor γ (RORγ, NR1F3, or RORc) exists in two isoforms, with one isoform (RORγ or RORc1) widely expressed in a variety of tissues, and the expression of the second isoform (RORγt or RORc2) restricted to the thymus and cells of the immune system. RORγt is a key regulator of the development and functions of T-helper 17 (Th17) cells. Clinical proof-of-concept (PoC) with small molecule inverse agonists of RORγt has been achieved with VTP-43742 (Phase II) for the treatment of psoriasis, and pre-clinical PoC for this mechanism has also been established for the treatment of autoimmune diseases. A series of aryl sulfonyl derivatives as novel RORγt inverse agonists were designed and synthesized based on VTP-43742. We conducted structural modifications that improved the activity profile. In pharmacodynamic (PD) studies, oral administration of compound b12 showed robust and dose-dependent inhibition of IL-6 and IL-17A cytokine expression. The ability of compound b12 to reduce the levels of IL-6 and IL-17A in vivo after oral dosing in mice, and a corresponding reduction in skin inflammation further supports the potential of small molecule RORγt modulation as a therapeutic target for the treatment of inflammatory diseases.

Discovery of Chromane-6-Sulfonamide Derivative as a Potent, Selective, and Orally Available Novel Retinoic Acid Receptor-Related Orphan Receptor γt Inverse Agonist.

Interleukin-17 (IL-17) is a proinflammatory cytokine that plays a dominant role in inflammation, autoimmunity, and host defense. RORγt is a key transcription factor mediating T helper 17 (Th17) cell differentiation and IL-17 production, which is able to activate CD8+ T cells and elicit antitumor efficacy. A series of sulfonamide derivatives as novel RORγt inverse agonists were designed and synthesized. Using GSK2981278 (phase II) as a starting point, we engineered structural modifications that significantly improved the activity and pharmacokinetic profile. In animal studies, oral administration of compound d3 showed a robust and dose-dependent inhibition of the IL-17A cytokine expression in a mouse imiquimod-induced skin inflammation model. Docking analysis of the binding mode revealed that the compound d3 occupied the active pocket suitably. Thus, compound d3 was selected as a clinical compound for the treatment of Th17-driven autoimmune diseases.

Discovery of 2-(Ortho-Substituted Benzyl)-Indole Derivatives as Potent and Orally Bioavailable RORγ Agonists with Antitumor Activity.

RORγ is a dual-functional drug target, which involves not only induction of inflammation but also promotion of cancer immunity. The development of agonists of RORγ promoting Th17 cell differentiation could provide a novel mechanism of action (MOA) as an immune-activating anticancer agent. Herein, we describe new 2-(ortho-substituted benzyl)-indole derivatives as RORγ agonists by scaffold hopping based on clinical RORγ antagonist VTP-43742. Interestingly, subtle structural differences of the compounds led to the opposite biological MOA. After rational optimization for structure-activity relationship and pharmacokinetic profile, we identified a potent RORγ agonist compound 17 that was able to induce the production of IL-17 and IFNγ in tumor tissues and elicit antitumor efficacy in MC38 syngeneic mouse colorectal tumor model. This is the first comprehensive work to demonstrate the in vivo antitumor efficacy of an RORγ agonist.

Discovery of LYC-55716: A Potent, Selective, and Orally Bioavailable Retinoic Acid Receptor-Related Orphan Receptor-γ (RORγ) Agonist for Use in Treating Cancer.

Retinoic acid receptor-related orphan receptor γ (RORc, RORγ, or NR1F3) is the nuclear receptor master transcription factor that drives the function and development of IL-17-producing T helper cells (Th17), cytotoxic T cells (Tc17), and subsets of innate lymphoid cells. Activation of RORγ+ T cells in the tumor microenvironment is hypothesized to render immune infiltrates more effective at countering tumor growth. To test this hypothesis, a family of benzoxazines was optimized to provide LYC-55716 (37c), a potent, selective, and orally bioavailable small-molecule RORγ agonist. LYC-55716 decreases tumor growth and enhances survival in preclinical tumor models and was nominated as a clinical development candidate for evaluation in patients with solid tumors.

RORγt agonist synergizes with CTLA-4 antibody to inhibit tumor growth through inhibition of Treg cells via TGF-ß signaling in cancer.

To date, the overall response rate to checkpoint blockade remains unsatisfactory, partially due to the limited understanding of the tumor immune microenvironment. The retinoic acid-related orphan receptor γt (RORγt) is the key transcription factor of T helper cell 17 (Th17) cells and plays an essential role in tumor immunity. In this study, we used JG-1, a potent and selective small-molecule RORγt agonist to evaluate the therapeutic potential and mechanism of action of targeting RORγt in tumor immunity. JG-1 promotes Th17 cells differentiation and inhibition of regulatory T (Treg) cells differentiation. JG-1 demonstrates robust tumor growth inhibition in multiple syngeneic models and shows a synergic effect with the Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) antibody. In tumors, JG-1 not only promotes Th17 cells differentiation and increases C-C Motif Chemokine Receptor 6 (CCR6)- Chemokine (C-C motif) ligand 20 (CCL20) expression, but also inhibits both the expression of transforming growth factor-ß1 (TGF-ß1) and the differentiation and infiltration of Treg cells. In summary, JG-1 is a lead compound showing a potent activity in vitro and robust tumor growth inhibition in vivo with synergetic effects with anti-CTLA-4.

Agonist Lock Touched and Untouched Retinoic Acid Receptor-Related Orphan Receptor-γt (RORγt) Inverse Agonists: Classification Based on the Molecular Mechanisms of Action.

Retinoic acid receptor-related orphan receptor-gamma-t (RORγt) is a potential drug target for autoimmune diseases with a clear biological mechanism in the Th17/IL-17 pathway. The "agonist lock", which is formed by residues His479-Tyr502-Phe506 in RORγt, makes H12 tightly contact H11 in a suitable conformation for coactivator binding and, thus, is related to RORγt transcriptional activation. The inverse agonism of RORγt is complex because not all RORγt inverse agonists directly break the agonist lock to interfere with coactivator recruitment and the transcription of RORγt. Here, we analyze the complex structures, binding modes, and biological activities of various RORγt inverse agonists and classify them as "agonist lock touched" and "agonist lock untouched" RORγt inverse agonists according to whether they infringe on the agonist lock directly or not. We aim at providing a comprehensive review and insights into drug discovery of RORγt inverse agonists.

Discovery of tert-amine-based RORγt agonists.

The nuclear receptor retinoic acid receptor-related orphan receptor gamma-t (RORγt) is a transcription factor regulating Th17 cell differentiation and proliferation from naive CD4+ T cells. Since Th17 cells have demonstrated the antitumor efficacy by eliciting remarkable activation of CD8+ T cells, RORγt agonists could be applied as potential small molecule therapeutics for cancer immunotherapy. Based on the previously reported RORγt agonist 1 and its resolved co-crystal structure, a series of new tertiary amines were designed, synthesized and biologically evaluated, yielding optimal moieties with improved chemical properties and biological responses. The combination of these optimal moieties resulted in identification of novel RORγt agonists such as 8b with further elevated RORγt agonism responses at a target-based level as well as in cell-based assays, which provided some structural knowledge for further optimization of RORγt agonists as small molecule therapeutics for cancer immunotherapy.

Retinoic acid-related orphan receptor gamma t (RORγt) inverse agonists/antagonists for the treatment of inflammatory diseases - where are we presently?

Introduction: The transcription factor retinoic acid-related orphan receptor gamma t (RORγt) has been identified as the master regulator of TH17 cell differentiation and IL-17/22 production and is therefore an attractive target for the treatment of inflammatory diseases. Several orally or topically administered small molecule RORγt inverse agonists (RIAs) have progressed up to the end of clinical Phase 2.Areas covered: Based on publications and patent evaluations this review summarizes the evolution of the chemical matter for all 16 pharmaceutical companies, who develop(ed) a clinical-stage RIAs (until March 2021). Structure proposals for some clinical stage RIAs are presented and the outcome of the clinical trials is discussed.Expert opinion: So far, the clinical trials have been plagued with a high attrition rate. Main reasons were lack of efficacy (topical) or safety signals (oral) as well as, amongst other things, thymic lymphomas as seen with BMS-986251 in a preclinical study and liver enzyme elevations in humans with VTP-43742. Possibilities to mitigate these risks could be the use of RIAs with different chemical structures not interfering with thymocytes maturation and no livertox-inducing properties. With new frontrunners (e.g., ABBV-157 (cedirogant), BI 730357 or IMU-935) this is still an exciting time for this treatment approach.

Discovery of novel N-sulfonamide-tetrahydroisoquinolines as potent retinoic acid receptor-related orphan receptor γt agonists.

Cancer immunotherapy has become a research hotspot in recent years. A variety of targets were developed for small molecule immuno-oncology agents, including retinoic acid-related orphan receptor gamma t (RORγt), chemokine receptor, stimulator of interferon genes (Sting), indoleamine 2,3-dioxygenase (IDO), toll-like receptors (TLR), etc. Among them, the retinoic acid receptor-related orphan receptor γt (RORγt) has gradually attracted more attention in these years. In particular, LYC-55716 (cintirorgon), a small molecule RORγt agonist developed by Lycera, has entered the phase II clinical study. In this work, starting from compound 7, compound 28 was obtained after 4 rounds of compound design, synthesis and SAR studies, which had an EC50 of 0.021 ± 0.002 µM in dual Fluorescence Resonance Energy Transfer (dual-FRET) assay and an EC50 of 0.021 ± 0.002 µM in mouse Th17 cell differentiation assay. It indicated that compound 28 had excellent RORγt agonistic activity and was expected to be developed as a new type of small molecule drug for cancer immunotherapy. The molecular dynamic simulation revealed that the agonist 28 formed a strong HYF triplet intramolecular interaction to stabilize H12, which helped RORγt to form the protein-binding site and therefore made the receptor ready to recruit coactivator. When the inverse agonist s27 bound with RORγt, the steric hindrance between s27 and H479 caused the destruction of the HYF triplet, leading to the collapse of H12, thus the transcription function of RORγt was interrupted due to the failure of recruiting a coactivator molecule. The triplet HYF in RORγt and the rigidity of 28 and s27 were identified to be the structural determinants for the functional switch of RORγt.