Cryo-EM Structure of the Human Cannabinoid Receptor CB2-Gi Signaling Complex.

Drugs selectively targeting CB2 hold promise for treating neurodegenerative disorders, inflammation, and pain while avoiding psychotropic side effects mediated by CB1. The mechanisms underlying CB2 activation and signaling are poorly understood but critical for drug design. Here we report the cryo-EM structure of the human CB2-Gi signaling complex bound to the agonist WIN 55,212-2. The 3D structure reveals the binding mode of WIN 55,212-2 and structural determinants for distinguishing CB2 agonists from antagonists, which are supported by a pair of rationally designed agonist and antagonist. Further structural analyses with computational docking results uncover the differences between CB2 and CB1 in receptor activation, ligand recognition, and Gi coupling. These findings are expected to facilitate rational structure-based discovery of drugs targeting the cannabinoid system.

Pharmaceutical targeting of the cannabinoid type 1 receptor impacts the crosstalk between immune cells and islets to reduce insulitis in humans.

AIMS/HYPOTHESIS: Insulitis, a hallmark of inflammation preceding autoimmune type 1 diabetes, leads to the eventual loss of functional beta cells. However, functional beta cells can persist even in the face of continuous insulitis. Despite advances in immunosuppressive treatments, maintaining functional beta cells to prevent insulitis progression and hyperglycaemia remains a challenge. The cannabinoid type 1 receptor (CB1R), present in immune cells and beta cells, regulates inflammation and beta cell function. Here, we pioneer an ex vivo model mirroring human insulitis to investigate the role of CB1R in this process. METHODS: CD4+ T lymphocytes were isolated from peripheral blood mononuclear cells (PBMCs) from male and female individuals at the onset of type 1 diabetes and from non-diabetic individuals, RNA was extracted and mRNA expression was analysed by real-time PCR. Single beta cell expression from donors with type 1 diabetes was obtained from data mining. Patient-derived human islets from male and female cadaveric donors were 3D-cultured in solubilised extracellular matrix gel in co-culture with the same donor PBMCs, and incubated with cytokines (IL-1ß, TNF-α, IFN-γ) for 24-48 h in the presence of vehicle or increasing concentrations of the CB1R blocker JD-5037. Expression of CNR1 (encoding for CB1R) was ablated using CRISPR/Cas9 technology. Viability, intracellular stress and signalling were assayed by live-cell probing and real-time PCR. The islet function measured as glucose-stimulated insulin secretion was determined in a perifusion system. Infiltration of immune cells into the islets was monitored by microscopy. Non-obese diabetic mice aged 7 weeks were treated for 1 week with JD-5037, then euthanised. Profiling of immune cells infiltrated in the islets was performed by flow cytometry. RESULTS: CNR1 expression was upregulated in circulating CD4+ T cells from individuals at type 1 diabetes onset (6.9-fold higher vs healthy individuals) and in sorted islet beta cells from donors with type 1 diabetes (3.6-fold higher vs healthy counterparts). The peripherally restricted CB1R inverse agonist JD-5037 arrested the initiation of insulitis in humans and mice. Mechanistically, CB1R blockade prevented islet NO production and ameliorated the ATF6 arm of the unfolded protein response. Consequently, cyto/chemokine expression decreased in human islets, leading to sustained islet cell viability and function. CONCLUSIONS/INTERPRETATION: These results suggest that CB1R could be an interesting target for type 1 diabetes while highlighting the regulatory mechanisms of insulitis. Moreover, these findings may apply to type 2 diabetes where islet inflammation is also a pathophysiological factor. DATA AVAILABILITY: Transcriptomic analysis of sorted human beta cells are from Gene Expression Omnibus database, accession no. GSE121863, available at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM3448161 .

Effects of CB1R inverse agonist, INV-202, in patients with features of metabolic syndrome. A randomized, placebo-controlled, double-blind phase 1b study.

AIMS: To evaluate the clinical safety, tolerability, and pharmacokinetic and pharmacodynamic profile of the novel cannabinoid receptor-1 (CB1R) inverse agonist, INV-202, in adults with features of metabolic syndrome. MATERIALS AND METHODS: This was a multicentre, randomized, double-blind, placebo-controlled, 28-day repeat-dose (INV-202 [25 mg] or placebo, once-daily oral tablet), parallel-group study in 37 participants aged 18 to 65 years (46% female, mean age 55 years, glycated haemoglobin 5.7% [39 mmol/mol], body mass index [BMI] 38.1 kg/m2 ) with features of metabolic syndrome and glucose intolerance. An oral glucose tolerance test (OGTT) was performed at baseline and at the end of the study. Lipid profiles, weight, waist circumference and biomarkers were assessed weekly. Statistical comparisons were performed post hoc. RESULTS: INV-202 was well tolerated with no serious or severe treatment-emergent adverse events; the most common events related to known effects of CB1R blockade in the gastrointestinal tract. INV-202 produced a significant mean weight loss of 3.5 kg (3.3% compared with placebo participants who gained a mean 0.6 kg [0.5%]). INV-202 also exhibited significant reductions in waist circumference and BMI (P ≤ 0.03). There was no significant difference in OGTT 0- to 3-hour area under the curve for INV-202 versus placebo: least squares mean 29.38 versus 30.25 h*mmol/L, with an INV-202: placebo ratio of 97.1% (95% confidence interval 90.2, 105.6; P = 0.43). CONCLUSIONS: INV-202 was well tolerated, producing a signal for rapid weight loss with improvements in other metabolic syndrome markers in this population. These findings support further exploration and long-term assessment of cardiometabolic effects.

Cannabinoid receptor 2 (CB2) modulators: A patent review (2016-2024).

Cannabinoid receptors CB1 and CB2 play critical roles in regulating numerous central and peripheral physiological activities. While efforts have been made to develop ligands for both CB1 and CB2 receptors, CB1 receptor ligands often have restricted use due to undesirable psychotropic side effects. Consequently, recent cannabis research has increasingly focused on CB2-specific ligands. Pharmacological agonists of CB2 receptors have shown potential in managing pain, inflammation, arthritis, neuroprotection, cancer, and other disorders. Despite several CB2 receptor ligands entering clinical trials, none have achieved market approval except natural cannabinoids and their derivatives, primarily due to insufficient CB2/CB1 receptor selectivity. However, new-generation ligands developed in recent years have demonstrated improved selectivity. This review covers patent literature on CB2 modulators from 2016 to 2024, highlighting the major advances in the field. During this period, the majority of research has concentrated on using CB2 modulators to alleviate inflammation and pain. Additionally, patents have explored CB2 modulators for a range of specific diseases, including: psychiatric and neuropsychiatric disorders, schizophrenia, multiple myeloma and osteoporosis, ocular inflammation and neuropathic Pain, cancer anorexia and weight loss, antioxidant and anti-aging agents, lymphocytopenia, hearing loss, Alzheimer's disease, cancer and non-malignant tumors. Notably, recent years have seen increased interest in CB2 antagonists/inverse agonists, with few candidates advancing to clinical studies. Significant progress has been made in the synthesis and modulation of selective CB2 agonists and antagonists, paving the way for future developments in CB2 modulators. This review provides insights and prospects for the continued evolution of CB2-targeted therapies.

Liver X receptor inverse agonist SR9243 attenuates rheumatoid arthritis via modulating glycolytic metabolism of macrophages.

Liver X receptors (LXRs) which link lipid metabolism and inflammation, were overexpressed in experimental rheumatoid arthritis (RA) rats as observed in our previous studies, while suppression of LXRα by silybin ameliorates arthritis and abnormal lipid metabolism. However, the role of LXRs in RA remains undefined. In this study, we investigated the inhibition role of LXRs in the polarization and activation of M1 macrophage by using a special LXRs inverse agonist SR9243, which led to ameliorating the progression of adjuvant-induced arthritis (AIA) in rats. Mechanistically, SR9243 disrupted the LPS/IFN-γ-induced Warburg effect in M1 macrophages, while glycolysis inhibitor 2-DG attenuated the inhibition effect of SR9243 on M1 polarization and the cytokines expression of M1 macrophages including iNOS, TNF-α, and IL-6 in vitro. Furthermore, SR9243 downregulated key glycolytic enzymes, including LDH-A, HK2, G6PD, GLUT1, and HIF-1α in M1 macrophages, which is mediated by increased phosphorylation of AMPK (Thr172) and reduced downstream phosphorylation of mTOR (Ser2448). Importantly, gene silencing of LXRs compromises the inhibition effect of SR9243 on M1 macrophage polarization and activation. Collectively, for the first time, our findings suggest that the LXR inverse agonist SR9243 mitigates adjuvant-induced rheumatoid arthritis and protects against bone erosion by inhibiting M1 macrophage polarization and activation through modulation of glycolytic metabolism via the AMPK/mTOR/HIF-1α pathway.

Histaminergic System Activity in the Central Nervous System: The Role in Neurodevelopmental and Neurodegenerative Disorders.

Histamine (HA), a biogenic monoamine, exerts its pleiotropic effects through four H1R-H4R histamine receptors, which are also expressed in brain tissue. Together with the projections of HA-producing neurons located within the tuberomammillary nucleus (TMN), which innervate most areas of the brain, they constitute the histaminergic system. Thus, while remaining a mediator of the inflammatory reaction and immune system function, HA also acts as a neurotransmitter and a modulator of other neurotransmitter systems in the central nervous system (CNS). Although the detailed causes are still not fully understood, neuroinflammation seems to play a crucial role in the etiopathogenesis of both neurodevelopmental and neurodegenerative (neuropsychiatric) diseases, such as autism spectrum disorders (ASDs), attention-deficit/hyperactivity disorder (ADHD), Alzheimer's disease (AD) and Parkinson's disease (PD). Given the increasing prevalence/diagnosis of these disorders and their socioeconomic impact, the need to develop effective forms of therapy has focused researchers' attention on the brain's histaminergic activity and other related signaling pathways. This review presents the current state of knowledge concerning the involvement of HA and the histaminergic system within the CNS in the development of neurodevelopmental and neurodegenerative disorders. To this end, the roles of HA in neurotransmission, neuroinflammation, and neurodevelopment are also discussed.

Development of novel potent ligands for GPR85, an orphan G protein-coupled receptor expressed in the brain.

GPR85 is a member of the G protein-coupled receptor and is a super-conserved receptor expressed in the brain sub-family (Super Conserved Receptor Expressed in Brain; SREB) with GPR27 and GPR173. These three receptors are "orphan receptors"; however, their endogenous ligands have not been identified. SREB has garnered the interest of many scientists because it is expressed in the central nervous system and is evolutionarily conserved. In particular, brain mass is reported to be increased and learning and memory are improved in GPR85 knockout mice (Matsumoto et al. 2008). In this study, we characterized newly synthesized compounds using a GPR85-Gsα fusion protein and the [35 S]GTPγS binding assay and identified novel GPR85 inverse-agonists with IC50 values of approximately 1 µM. To analyze the neurochemical character of the compounds and investigate the physiological significance of GPR85, we used cerebellar Purkinje cells expressing GPR85 and an electrophysiological technique. Based on the results, the inverse-agonist compound for GPR85 modulated potassium channel opening. Together with the results of previous gene analysis of GPR85, we expect that the development of the GPR85 ligand will provide new insights into a few types of neurological disorders.

Discovery and characterization of orally bioavailable 4-chloro-6-fluoroisophthalamides as covalent PPARG inverse-agonists.

PPAR gamma (PPARG) is a ligand activated transcription factor that regulates genes involved in inflammation, bone biology, lipid homeostasis, as well as a master regulator of adipogenesis and a potential lineage driver of luminal bladder cancer. While PPARG agonists lead to transcriptional activation of canonical target genes, inverse agonists have the opposite effect through inducing a transcriptionally repressive complex leading to repression of canonical target gene expression. While many agonists have been described and tested clinically, inverse agonists offer an underexplored avenue to modulate PPARG biology in vivo. Current inverse agonists lack favorable in vivo properties; herein we describe the discovery and characterization of a series of orally bioavailable 4-chloro-6-fluoroisophthalamides as covalent PPARG inverse-agonists, BAY-5516, BAY-5094, and BAY-9683. Structural studies of this series revealed distinct pre- and post-covalent binding positions, which led to the hypothesis that interactions in the pre-covalent conformation are primarily responsible for driving affinity, while interactions in the post-covalent conformation are more responsible for cellular functional effects by enhancing PPARG interactions with its corepressors. The need to simultaneously optimize for two distinct states may partially explain the steep SAR observed. Exquisite selectivity was achieved over related nuclear receptors in the subfamily due in part to a covalent warhead with low reactivity through an SNAr mechanism in addition to the specificity gained through covalent binding to a reactive cysteine uniquely positioned within the PPARG LBD. BAY-5516, BAY-5094, and BAY-9683 lead to pharmacodynamic regulation of PPARG target gene expression in vivo comparable to known inverse agonist SR10221 and represent new tools for future in vivo studies to explore their potential utility for treatment of disorders of hyperactivated PPARG including luminal bladder cancer and other disorders.

The adenosine A2A receptor antagonist/inverse agonist, KW-6356 enhances the anti-parkinsonian activity of L-DOPA with a low risk of dyskinesia in MPTP-treated common marmosets.

The adenosine A2A receptor antagonist/inverse agonist, KW-6356 has been shown to be effective in Parkinson's disease (PD) patients as monotherapy and as an adjunct therapy to L-3,4-dihydroxyphenylalanine (L-DOPA)/decarboxylase inhibitor. However, the effects of KW-6356 combined with L-DOPA on anti-parkinsonian activity and established dyskinesia has not been investigated in preclinical experiments. We examined the effects of combination of KW-6356 with L-DOPA in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated common marmosets. Oral administration of KW-6356 (1 mg/kg) enhanced the anti-parkinsonian activities of various doses of L-DOPA (2.5-10 mg/kg). In MPTP-treated common marmosets primed with L-DOPA to show dyskinesia, KW-6356 (1 mg/kg) also enhanced the anti-parkinsonian activities of various doses of L-DOPA (1.25-10 mg/kg) but not dyskinesia. Chronic co-administration of KW-6356 (1 mg/kg) with a low dose of L-DOPA (2.5 mg/kg) for 21 days increased the degree of dyskinesia induced by the low dose of L-DOPA, but the amplitude of dyskinesia induced by combined administration of KW-6356 (1 mg/kg) with L-DOPA (2.5 mg/kg) was lower than that induced by an optimal dose of L-DOPA (10 mg/kg). These results suggest that KW-6356 can be used to potentiate the effects of a wide range of L-DOPA doses with a low risk of dyskinesia for the treatment of PD.

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.

Ligand-Free Signaling of G-Protein-Coupled Receptors: Physiology, Pharmacology, and Genetics.

G-protein-coupled receptors (GPCRs) are ubiquitous sensors and regulators of cellular functions. Each GPCR exists in complex aggregates with multiple resting and active conformations. Designed to detect weak stimuli, GPCRs can also activate spontaneously, resulting in basal ligand-free signaling. Agonists trigger a cascade of events leading to an activated agonist-receptor G-protein complex with high agonist affinity. However, the ensuing signaling process can further remodel the receptor complex to reduce agonist affinity, causing rapid ligand dissociation. The acutely activated ligand-free receptor can continue signaling, as proposed for rhodopsin and µ opioid receptors, resulting in robust receptor activation at low agonist occupancy with enhanced agonist potency. Continued receptor stimulation can further modify the receptor complex, regulating sustained ligand-free signaling-proposed to play a role in opioid dependence. Basal, acutely agonist-triggered, and sustained elevated ligand-free signaling could each have distinct functions, reflecting multi-state conformations of GPCRs. This review addresses basal and stimulus-activated ligand-free signaling, its regulation, genetic factors, and pharmacological implications, focusing on opioid and serotonin receptors, and the growth hormone secretagogue receptor (GHSR). The hypothesis is proposed that ligand-free signaling of 5-HT2A receptors mediate therapeutic effects of psychedelic drugs. Research avenues are suggested to close the gaps in our knowledge of ligand-free GPCR signaling.

G protein-coupled receptors can control the Hippo/YAP pathway through Gq signaling.

The Hippo pathway is an evolutionarily conserved kinase cascade involved in the control of tissue homeostasis, cellular differentiation, proliferation, and organ size, and is regulated by cell-cell contact, apical cell polarity, and mechanical signals. Miss-regulation of this pathway can lead to cancer. The Hippo pathway acts through the inhibition of the transcriptional coactivators YAP and TAZ through phosphorylation. Among the various signaling mechanisms controlling the hippo pathway, activation of G12/13 by G protein-coupled receptors (GPCR) recently emerged. Here we show that a GPCR, the ghrelin receptor, that activates several types of G proteins, including G12/13, Gi/o, and Gq, can activate YAP through Gq/11 exclusively, independently of G12/13. We revealed that a strong basal YAP activation results from the high constitutive activity of this receptor, which can be further increased upon agonist activation. Thus, acting on ghrelin receptor allowed to modulate up-and-down YAP activity, as activating the receptor increased YAP activity and blocking constitutive activity reduced YAP activity. Our results demonstrate that GPCRs can be used as molecular switches to finely up- or down-regulate YAP activity through a pure Gq pathway.

Thyrotropin receptor antagonists and inverse agonists, and their potential application to thyroid diseases.

The thyrotropin receptor (TSHR) plays critical roles in thyroid growth and function and in the pathogenesis of several thyroid diseases including Graves' hyperthyroidism and ophthalmopathy, non-autoimmune hyperthyroidism and thyroid cancer. Several low-molecular weight compounds (LMWCs) and anti-TSHR monoclonal antibodies (mAbs) with receptor antagonistic and inverse agonistic activities have been reported. The former binds to the pocket formed by the receptor transmembrane bundle, and the latter to the extracellular TSH binding site. Both are effective inhibitors of TSH/thyroid stimulating antibody-stimulated cAMP and/or hyaluronic acid production in TSHR-expressing cells. Anti-insulin-like growth factor 1 inhibitors are also found to inhibit TSHR signaling. Each agent has advantages and disadvantages; for example, mAbs have a higher affinity and longer half-life but are more costly than LMWCs. At present, mAbs appear most promising, yet the development of more efficacious LMWCs is desirable. These agents are anticipated to be efficacious not only for the above-mentioned diseases but also for resistance to thyroid hormone and have utility for thyroid cancer radionuclide scintigraphy/therapy as a new theranostic.

The discovery of a novel series of potential ERRα inverse agonists based on p-nitrobenzenesulfonamide template for triple-negative breast cancer in vivo.

Oestrogen related receptor α participated in the regulation of oxidative metabolism and mitochondrial biogenesis, and was overexpressed in many cancers including triple-negative breast cancer. A set of new ERRα inverse agonists based on p-nitrobenzenesulfonamide template were discovered and compound 11 with high potent activity (IC50 = 0.80 µM) could significantly inhibit the transcription of ERRα-regulated target genes. By regulating the downstream signalling pathway, compound 11 could suppress the migration and invasion of the ER-negative MDA-MB-231 cell line. Furthermore, compound 11 demonstrated a significant growth suppression of breast cancer xenograft tumours in vivo (inhibition rate 23.58%). The docking results showed that compound 11 could form hydrogen bonds with Glu331 and Arg372 in addition to its hydrophobic interaction with ligand-binding domain. Our data implied that compound 11 represented a novel and effective ERRα inverse agonist, which had broad application prospects in the treatment of triple-negative breast cancer.

An Inverse Agonist GSK5182 Increases Protein Stability of the Orphan Nuclear Receptor ERRγ via Inhibition of Ubiquitination.

The orphan nuclear receptor, estrogen-related receptor γ (ERRγ) is a constitutively active transcription factor involved in mitochondrial metabolism and energy homeostasis. GSK5182, a specific inverse agonist of ERRγ that inhibits transcriptional activity, induces a conformational change in ERRγ, resulting in a loss of coactivator binding. However, the molecular mechanism underlying the stabilization of the ERRγ protein by its inverse agonist remains largely unknown. In this study, we found that GSK5182 inhibited ubiquitination of ERRγ, thereby stabilizing the ERRγ protein, using cell-based assays and confocal image analysis. Y326 of ERRγ was essential for stabilization by GSK5182, as ligand-induced stabilization of ERRγ was not observed with the ERRγ-Y326A mutant. GSK5182 suppressed ubiquitination of ERRγ by the E3 ligase Parkin and subsequent degradation. The inhibitory activity of GSK5182 was strong even when the ERRγ protein level was elevated, as ERRγ bound to GSK5182 recruited a corepressor, small heterodimer partner-interacting leucine zipper (SMILE), through the activation function 2 (AF-2) domain, without alteration of the nuclear localization or DNA-binding ability of ERRγ. In addition, the AF-2 domain of ERRγ was critical for the regulation of protein stability. Mutants in the AF-2 domain were present at higher levels than the wild type in the absence of GSK5182. Furthermore, the ERRγ-L449A/L451A mutant was no longer susceptible to GSK5182. Thus, the AF-2 domain of ERRγ is responsible for the regulation of transcriptional activity and protein stability by GSK5182. These findings suggest that GSK5182 regulates ERRγ by a unique molecular mechanism, increasing the inactive form of ERRγ via inhibition of ubiquitination.

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.

Pharmacotherapy of Itch-Antihistamines and Histamine Receptors as G Protein-Coupled Receptors.

Itching can decrease quality of life and exacerbate skin symptoms due to scratching. Itching not only contributes to disease progression but also triggers complications such as skin infections and eye symptoms. Therefore, controlling itching is very important in therapeutic management. In addition to the well-known histamine, IL-31, IL-4 and IL-13 have recently been reported as factors that induce itching. Itching may also be caused by factors other than these histamines. However, we do not know the extent to which these factors are involved in each disease. In addition, the degree of involvement is likely to vary among individuals. To date, antihistamines have been widely used to treat itching and are often effective, suggesting that histamine is more or less involved in itchy diseases. This review discusses the ligand-receptor perspective and describes the dynamics of G protein-coupled receptors, their role as biased agonists, their role as inverse agonists, proactive antihistamine therapy, and drug selection with consideration of impaired performance and anti-PAF effects.

Delineation of the molecular determinants of the unique allosteric binding site of the orphan nuclear receptor RORγt.

Nuclear receptors (NRs) are high-interest targets in drug discovery because of their involvement in numerous biological processes and diseases. Classically, NRs are targeted via their hydrophobic, orthosteric pocket. Although successful, this approach comes with challenges, including off-target effects due to lack of selectivity. Allosteric modulation of NR activity constitutes a promising pharmacological strategy. The retinoic acid receptor-related orphan receptor-γt (RORγt) is a constitutively active NR that positively regulates the expression of interleukin-17 in T helper 17 cells. Inhibiting this process is an emerging strategy for managing autoimmune diseases. Recently, an allosteric binding pocket in the C-terminal region of the ligand-binding domain (LBD) of RORγt was discovered that is amenable to small-molecule drug discovery. Compounds binding this pocket induce a reorientation of helix 12, thereby preventing coactivator recruitment. Therefore, inverse agonists binding this site with high affinity are actively being pursued. To elucidate the pocket formation mechanism, verify the uniqueness of this pocket, and substantiate the relevance of targeting this site, here we identified the key characteristics of the RORγt allosteric region. We evaluated the effects of substitutions in the LBD on coactivator, orthosteric, and allosteric ligand binding. We found that two molecular elements unique to RORγt, the length of helix 11' and a Gln-487 residue, are crucial for the formation of the allosteric pocket. The unique combination of elements present in RORγt suggests a high potential for subtype-selective targeting of this NR to more effectively treat patients with autoimmune diseases.

Inverse agonism at the Na/K-ATPase receptor reverses EMT in prostate cancer cells.

The surface expression of Na/K-ATPase α1 (NKA) is significantly reduced in primary prostate tumors and further decreased in bone metastatic lesions. Here, we show that the loss of cell surface expression of NKA induces epithelial-mesenchymal transition (EMT) and promotes metastatic potential and tumor growth of prostate cancer (PCa) by decreasing the expression of E-cadherin and increasing c-Myc expression via the activation of Src/FAK pathways. Mechanistically, reduced surface expression of NKA in PCa is due to increased endocytosis through the activation of NKA/Src receptor complex. Using a high-throughput NKA ligand-screening platform, we have discovered MB5 as an inverse agonist of the NKA/Src receptor complex, capable of blocking the endocytosis of NKA. MB5 treatment increased NKA expression and E-cadherin in PCa cells, which reversed EMT and consequently decreased the invasion and growth of spheroid models and tumor xenografts. Thus, we have identified a hitherto unrecognized mechanism that regulates EMT and invasiveness of PCa and demonstrated for the first time the feasibility of identifying inverse agonists of receptor NKA/Src complex and their potential utility as anticancer drugs. We, therefore, conclude that cell surface expression of α1 NKA can be targeted for the development of new therapeutics against aggressive PCa and that MB5 may serve as a prototype for drug development against EMT in metastatic PCa.

Discovery of 6-Oxo-4-phenyl-hexanoic acid derivatives as RORγt inverse agonists showing favorable ADME profile.

The retinoic acid receptor-related orphan nuclear receptor gamma t (RORγt), which is a promising therapeutic target for immune diseases, is a major transcription factor of genes related to psoriasis pathogenesis, such as interleukin (IL)-17A, IL-22, and IL-23R. Inspired by the co-crystal structure of RORγt, a 6-oxo-4-phenyl-hexanoic acid derivative 6a was designed, synthesized, and identified as a ligand of RORγt. The structure-activity relationship (SAR) studies in 6a, which focus on the improvement of its membrane permeability profile by introducing chlorine atoms, led to finding 12a, which has a potent RORγt inhibitory activity and a favorable pharmacokinetic profile.