Endogenous Agonist Research Articles

2-Arachidonoylglycerol Synthesis: Facile and Handy Enzymatic Method That Allows to Avoid Isomerization.

A simple and practical synthesis of 2-arachidonoyl glycerol (2-AG), an endogenous agonist for cannabinoid receptors, based on a two-step enzymatic process and a chemical coupling, was achieved with a good yield and negligible amount of the isomerization product 1-AG. Commercial preparation of immobilized lipase from Mucor miehei (MML) was selected as the most suitable enzyme to catalyze the efficient protection of glycerol using vinyl benzoate as an acyl transfer reagent in tetrahydrofuran. The same enzyme was used to remove the protective groups in positions 1 and 3. Owing to the mild neutral conditions and easy suitability of the method, 2-AG was obtained without any isomerization to the more stable 1-AG and air oxidation of acid chain. The synthetic method proposed here allows us to easily obtain 2-AG from the protected precursor in a one-step reaction without purification requirement.

Cinnabarinic Acid Provides Hepatoprotection Against Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) is a chronic condition in which excess lipids accumulate in the liver and can lead to a range of progressive liver disorders including non-alcoholic steatohepatitis, liver cirrhosis, and hepatocellular carcinoma. While lifestyle and diet modifications have proven to be effective as NAFLD treatments, they are not sustainable in the long-term, and currently no pharmacological therapies are approved to treat NAFLD. Our previous studies demonstrated that cinnabarinic acid (CA), a novel endogenous Aryl hydrocarbon Receptor (AhR) agonist, activates the AhR target gene, Stanniocalcin 2, and confers cytoprotection against a plethora of ER/oxidative stressors. In this study, the hepatoprotective and anti-steatotic properties of CA were examined against free fatty-acid-induced in vitro and high-fat-diet fed in vivo NAFLD models. The results demonstrated that CA treatment significantly lowered weight gain and attenuated hepatic lipotoxicity both before and after the established fatty liver, thereby protecting against steatosis, inflammation, and liver injury. CA mitigated intracellular free fatty acid uptake concomitant with the downregulation of CD36/fatty acid translocase. Genes involved in fatty acid and triglyceride synthesis were also downregulated in response to CA treatment. Additionally, suppressing AhR and Stc2 expression using RNA interference in vitro verified that the hepatoprotective effects of CA were absolutely dependent on both AhR and its target, Stc2. Collectively, our results demonstrate that the endogenous AhR agonist, CA, confers hepatoprotection against NAFLD by regulating hepatic fatty acid uptake and lipogenesis. SIGNIFICANCE STATEMENT: In this study using in vitro and in vivo models, we demonstrate that cinnabarinic acid (CA), an endogenous AhR agonist, provides protection against non-alcoholic fatty liver disease. CA bestows cytoprotection against steatosis and liver injury by controlling expression of several key genes associated with lipid metabolism pathways, limiting the hepatic lipid uptake, and controlling liver inflammation. Moreover, CA-induced hepatoprotection is absolutely dependent on AhR and Stc2 expression.

Cryo-EM structure of human κ-opioid receptor-Gi complex bound to an endogenous agonist dynorphin A.

Cryo-EM structure of human κ-opioid receptor-Gi complex bound to an endogenous agonist dynorphin A.

Cannabinoids – a new therapeutic strategy in neurology

Cannabinoids' usefulness in the treatment of neurological disorders (epilepsy, and various neurodegenerative diseases, such as Multiple Sclerosis and Alzheimer's Disease) has been demonstrated in a growing number of studies. Of the 11 known general types of natural cannabinoids, the focus has been mainly directed at cannabidiol (CBD) due to its specificity in stimulating cannabinoid receptors and the low rate of side effects, as well as on Δ (9)-tetrahydrocannabinol (Δ9-THC). The natural and synthetic analogs of CBD have been described as a potential treatment in neurological diseases, as they showed their therapeutic benefits in reducing the seizures from epilepsy and their neuroprotectivity in neurodegenerative diseases. First and foremost, CBD's neuroprotective properties are due to its capacity to act as an endogenous cannabinoid receptor agonist. Second, CBD enhances neuroprotection by interacting with many signal transduction pathways mediated indirectly through cannabinoid receptors. CBD also reduces the hyperphosphorylation of glycogen synthetase kinase 3 (GSK-3) induced by the buildup of Amyloid β in the physiopathology of Alzheimer's disease.

D-aspartate oxidase gene duplication induces social recognition memory deficit in mice and intellectual disabilities in humans

The D-aspartate oxidase (DDO) gene encodes the enzyme responsible for the catabolism of D-aspartate, an atypical amino acid enriched in the mammalian brain and acting as an endogenous NMDA receptor agonist. Considering the key role of NMDA receptors in neurodevelopmental disorders, recent findings suggest a link between D-aspartate dysmetabolism and schizophrenia. To clarify the role of D-aspartate on brain development and functioning, we used a mouse model with constitutive Ddo overexpression and D-aspartate depletion. In these mice, we found reduced number of BrdU-positive dorsal pallium neurons during corticogenesis, and decreased cortical and striatal gray matter volume at adulthood. Brain abnormalities were associated with social recognition memory deficit at juvenile phase, suggesting that early D-aspartate occurrence influences neurodevelopmental related phenotypes. We corroborated this hypothesis by reporting the first clinical case of a young patient with severe intellectual disability, thought disorders and autism spectrum disorder symptomatology, harboring a duplication of a chromosome 6 region, including the entire DDO gene.

081 Modulation of the aryl hydrocarbon receptor by adipose tissue: Implications for skin carcinogenesis

The aryl hydrocarbon receptor(AhR) is a ligand-activated transcription factor that responds to chemical carcinogens. An endogenous AhR agonist, 6-Formylindolo[3,2-b]carbazole, is produced following ultraviolet light(UV) absorption by tryptophan. Epidemiological studies show a correlation between exposure to AhR agonists and non melanoma skin cancer(NMSC). As an example, AhR activation promotes metabolism of pro-carcinogens to carcinogens such as the metabolism of benzo(a)pyrene to benzo[a]pyrene diol epoxide(BPDE).

Olive Oil Phenolics and Platelets-From Molecular Mechanisms to Human Studies.

Chronically activated, dysfunctional platelets mediate the progression of the majority of non-communicable diseases in a pleiotropic fashion. Antiplatelet therapy remains an attractive therapeutic means which however hasn't reached the expected targets according to the promising preclinical studies. It is therefore obvious that the consumption of foods demonstrating antiplatelet activity may be a less drastic but on the other hand a more sustainable way of achieving daily antiplatelet therapy, either alone or in combination with antiplatelet drugs. Olive oil is probably the main cardioprotective component of the Mediterranean Diet according to the results of observational and dietary intervention studies. Among all phytochemicals of olive oil, its unique phenolics seems to be responsible for the majority of its cardioprotective properties. This review article aims to highlight the platelet modulating roles of olive oil polyphenols, trying to critically assess whether those properties could partially explain the cardioprotective role of olive oil. The cellular and animal studies clearly show that extra virging olive oil (EVOO) phenolics, mainly hydroxytyrosol, are able to inhibit the activation of platelets induced by several endogenous agonists and pathologies. However, the outcomes of the pre-clinical studies are difficult to be translated to humans mainly because the dosages and the chemical forms of the phenolics used to these studies are much higher and different to that found in human circulation. Despite the heterogeneity of the few human trials on the field so far, the results are promising showing that EVOO can exert antiplatelet actions in real, acute or long-term, trials and at least part of this antiplatelet action can be attributed to the phenolic content of EVOOs. Although we clearly need better, well-powered studies to give certain answers on this field the antiplatelet properties of olive oil phenolics is a promising, emerging mechanism which may explain some of the health properties of EVOO and the Mediterranean Diet.

The blockage of downstream P2Y2 receptor signaling inhibits the prostate cancer cell adhesion to endothelial cells

AimsProstate cancer is the second most frequently malignancy in men worldwide. Most deaths are caused by metastasis, and tumor cell dissemination involves the interaction with endothelial cells. However, the endothelial cell signaling involved in such interaction is not entirely understood. The tumor microenvironment contains extracellular ATP, an endogenous agonist of the purinergic P2Y2 receptor (P2Y2R). P2Y2R signaling changes endothelial cell phenotype, which may be relevant to cancer pathophysiology. Therefore, we hypothesized that P2Y2R activation could favor the metastatic prostate cancer cells adhesion to endothelial cells. Main methodsFor adhesion assays, confluent endothelial cells EA.hy926 were treated with P2Y2R agonists before adding and imaging stained DU-145 cells. Alternatively, fluorescent probes and antibodies were used to determine intracellular endothelial Ca2+, nitric oxide (NO), and flow cytometry assays. Key findingsEndothelial P2Y2R activation with ATP, UTP, or the selective agonist 2-thio-UTP increased DU-145 cell adhesion to EA.hy926 cells. This effect required endothelial cell Ca2+ mobilization and relied on the endothelial expression of VCAM-1 and ICAM-1. Conversely, inhibiting this proadhesive endothelial phenotype could impair DU-145 cell adhesion. To evaluate this, we chose atorvastatin based on its notable improvement of endothelial cell dysfunction. Atorvastatin blocked UTP-induced DU-145 cell adhesion to endothelial cell monolayer in a NO-dependent manner, unveiling a P2Y2R and NO signaling crosstalk. SignificanceEndothelial P2Y2R signaling contributes to the adhesion of metastatic prostate cancer cells suggesting that the downstream signaling blockade by statins could be a putative mechanism to reduce prostate cancer metastasis.

Application of a pharmacological transcriptome filter identifies a shortlist of mouse glucocorticoid receptor target genes associated with memory consolidation

Glucocorticoids regulate memory consolidation, facilitating long-term storage of relevant information to adequately respond to future stressors in similar conditions. This effect of glucocorticoids is well-established and is observed in multiple types of behaviour that depend on various brain regions. By and large, higher glucocorticoid levels strengthen event-related memory, while inhibition of glucocorticoid signalling impairs consolidation. The mechanism underlying this glucocorticoid effect remains unclear, but it likely involves the transcriptional effects of the glucocorticoid receptor (GR). We here used a powerful paradigm to investigate the transcriptional effects of GR in the dorsal hippocampus of mice after training in an auditory fear conditioning task, aiming to identify a shortlist of GR target genes associated to memory consolidation. Therefore, we utilized in an explorative study the properties of selective GR modulators (CORT108297 and CORT118335), alongside the endogenous agonist corticosterone and the classical GR antagonist RU486, to pinpoint GR-dependent transcriptional changes. First, we confirmed that glucocorticoids can modulate memory strength via GR activation. Subsequently, by assessing the specific effects of the available GR-ligands on memory strength, we established a pharmacological filter which we imposed on the hippocampal transcriptome data. This identified a manageable shortlist of eight genes by which glucocorticoids may modulate memory consolidation, warranting in-depth follow-up. Overall, we showcase the strength of the concept of pharmacological transcriptome filtering, which can be readily applied to other research topics with an established role of glucocorticoids.

Effects of Taurine Depletion on Body Weight and Mouse Behavior during Development.

Taurine (2-aminoethanesulfonic acid) plays an important role in various physiological functions and is abundant in the brain and skeletal muscle. Extracellular taurine is an endogenous agonist of gamma-aminobutyric acid type A and glycine receptors. Taurine actively accumulates in cells via the taurine transporter (TauT). Adult taurine-knockout (TauT−/−) mice exhibit lower body weights and exercise intolerance. To further examine the physiological role of taurine, we examined the effect of its depletion on mouse behavior, startle responses, muscular endurance, and body weight during development from postnatal day 0 (P0) until P60. In the elevated plus maze test, TauT−/− mice showed decreased anxiety-like behavior. In addition, TauT−/− mice did not show a startle response to startle stimuli, suggesting they have difficulty hearing. Wire-hang test revealed that muscular endurance was reduced in TauT−/− mice. Although a reduction of body weight was observed in TauT−/− mice during the developmental period, changes in body weight during 60% food restriction were similar to wild-type mice. Collectively, these results suggest that taurine has important roles in anxiety-like behavior, hearing, muscular endurance, and maintenance of body weight.

THE NEUROPROTECTION CAUSED BY PHYTOCANABINOIDS, THEIR EFFECTS AND THEIR APPLICABILITY IN BRAZIL

Introduction: The endocannabinoid system is composed of cannabinoid receptors type 1 (CB1) and type 2, their endogenous agonists, anandamide endocannabinoids and 2-aaquidonoilglycerol (2-AG). It is assumed that 2-AG is the binding receptor of cannabidioids to the Central Nervous System. CB1 inhibits the release of GABA and glutamate from pre-sinaptic terminals, being responsible for the ability to modulate neurotransmission as a mechanism of neuroprotection of CB1 against excitotoxicity. This action provides a good justification for such mechanisms to be investigated as therapeutic targets for severe neurodegenerative diseases, including Alzheimer’s disease, Huntington’s disease, and Parkinson’s disease. Development: Cannabidiol is more related to neuroprotective activity, tolerable at higher doses and less cardiovascular and psychological risks, THC to psychotropic effects, since there are medications that contain both substances, there should be a control of its prescription and care for self-medication. Final Considerations: Although we know that cannabidiol has an impact and medicinal value, more studies need to be done, and there should be greater rigor on composition and concentrations, due to the high power of interaction with other substances and their toxic power when used erroneously. Finally, it is known of its beneficial potential for various neurodegenerative diseases.

Michaelis-Menten Quantification of Ligand Signaling Bias Applied to the Promiscuous Vasopressin V2 Receptor.

Activation of G protein-coupled receptors by agonists may result in the activation of one or more G proteins and recruitment of arrestins. The extent of the activation of each of these pathways depends on the intrinsic efficacy of the ligand. Quantification of intrinsic efficacy relative to a reference compound is essential for the development of novel compounds. In the operational model, changes in efficacy can be compensated by changes in the "functional" affinity, resulting in poorly defined values. To separate the effects of ligand affinity from the intrinsic activity of the receptor, we developed a Michaelis-Menten based quantification of G protein activation bias that uses experimentally measured ligand affinities and provides a single measure of ligand efficacy. We used it to evaluate the signaling of a promiscuous model receptor, the Vasopressin V2 receptor (V2R). Using BRET-based biosensors, we show that the V2R engages many different G proteins across all G protein subfamilies in response to its primary endogenous agonist, arginine vasopressin, including Gs and members of the Gi/o and G12/13 families. These signaling pathways are also activated by the synthetic peptide desmopressin, oxytocin, and the nonmammalian hormone vasotocin. We compared bias quantification using the operational model with Michaelis-Menten based quantification; the latter accurately quantified ligand efficacies despite large difference in ligand affinities. Together, these results showed that the V2R is promiscuous in its ability to engage several G proteins and that its' signaling profile is biased by small structural changes in the ligand. SIGNIFICANCE STATEMENT: By modelling the G protein activation as Michaelis-Menten reaction, we developed a novel way of quantifying signalling bias. V2R activates, or at least engages, G proteins from all G protein subfamilies, including Gi2, Gz, Gq, G12, and G13. Their relative activation may explain its Gs-independent signalling.

Abstract 5670: TRPM8 ion channel role in prostate cancer: Actions as a rapid testosterone signaling receptor

Abstract The transient receptor potential melastatin 8 (TRPM8) is well-known for its role in cold sensation in somatosensory neurons. TRPM8 was first identified as a prostate epithelial cell-specific gene, however, its role was unclear due to the absence of its endogenous agonist. We are the first to discover the novel role of TRPM8 as a rapid testosterone receptor. The TRPM8 mRNA is highly expressed in prostate cancer (PC) and is lost during the transition to androgen-independent prostate cancer (AIPC). Although emerging studies have shed light regarding androgen regulation of TRPM8 mRNA expression, we found that the addition of androgen receptor (AR) on the lipid bilayers inhibited testosterone-TRPM8 induced Ca2+ uptake. Additionally, our IHC revealed increased internalization of the TRPM8 protein in high-grade PC and that TRPM8 protein was targeted for proteasomal degradation in PC. We observed that inhibition of AR and UBA1 promoted the stabilization of TRPM8 on the plasma membrane, triggering Ca2+-induced cytotoxicity and apoptosis in both androgen dependent and androgen independent PC cells. Furthermore, in line with previous studies, we showed that testosterone-induced TRPM8 activation on the planar lipid bilayers also required phosphatidylinositol 4,5-bisphosphate (PIP2). Loss of PTEN is associated with tumor recurrence and the transition to AIPC. Thus, PTEN loss mediated PIP2 deficiency may be an important mechanism of TRPM8 desensitization in PC. Therefore, we propose that the rescue of TRPM8 activity on the plasma membrane combined with AR targeting may be an effective therapy for PC. Citation Format: Kiran Velpula, Katherine Shishido, Susovon Bayen, Swapna Asuthkar. TRPM8 ion channel role in prostate cancer: Actions as a rapid testosterone signaling receptor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5670.

Abstract 4234: ONM-501: A polyvalent STING agonist for oncology immunotherapy

Abstract Background: The stimulator of interferon genes (STING) plays a central role in innate immune response against infection and cancer. Several cyclic di-nucleotide (CDN) and non-CDN small molecule STING agonists have demonstrated effectiveness against cancer in preclinical animal models, however their clinical trials showed limited therapeutic efficacy. ONM-501, a dual-activating STING agonist employs PC7A, a synthetic polymer that induces polyvalent STING condensation and prolongs innate immune activation has been recently developed. ONM-501 encapsulates the endogenous STING agonist cGAMP with the PC7A micelles offering dual ‘burst’ and ‘sustained’ STING activation. The mechanism and effectiveness of intratumorally delivered ONM-501 as an immunotherapy against solid tumors has been demonstrated in preclinical models. Methods: ONM-501 was recently evaluated for STING activation across different species: STING related IFNB1 and CXCL10 gene expression after ONM-501 treatment was measured by RT-qPCR in PBMCs from rat, cynomolgus monkey, and human. ONM-501 antitumor efficacy was evaluated in murine syngeneic tumor models. Abscopal effect was demonstrated by studying antitumor efficacy using both a primary/distal model and a lung metastatic model. STING knockout mice and STING knockout cancer cells were used to clarify the dependence of STING status to the ONM-501 antitumor immunity. Immune cell dependence was further elucidated by depletion of specific immune cell populations. Pilot safety studies including major organ function and systemic cytokine levels were performed in immunocompetent mice. Results: STING activation was observed across different species by measuring IFNB1 and CXCL10 mRNA in PBMCs from rat, cynomolgus monkey, and human after ONM-501 treatment. Antitumor efficacy was demonstrated both as a monotherapy and in combination with anti-PD1 in six different syngeneic tumor models. ONM-501 also induced an abscopal effect - tumor inhibition was observed in both primary and distal MC38 tumors in the same animal. Reduction of lung metastasis in an immune “cold” triple negative orthotopic breast cancer 4T1 model further confirmed the systemic antitumor immunity. Knocking out host STING (i.e. STING KO mice) rather than cancer cell STING KO resulted in abrogation of tumor inhibition suggesting that the host STING status is responsible for ONM-501-mediated anti-tumor immunity. Immune cell depletion studies further clarified that the antitumor immunity is dependent on CD8+ T cell and dendritic cells (DCs). No alteration of main organ function or systemic cytokine storm were observed in the pilot safety study. Conclusions: ONM-501 demonstrated marked anti-tumor efficacy in a panel of syngeneic tumor models. The anti-tumor effect was mediated by host STING and dependent on CD8+ T cells and DCs. These results support further evaluation of ONM-501 as a clinical candidate for the potential treatment of solid tumors. Citation Format: Suxin Li, Jian Wang, Jonathan Wilhelm, Qingtai Su, Gaurav Bharadwaj, Jason Miller, Wei Li, Katy Torres, Ruolan Han, Tian Zhao, Jinming Gao. ONM-501: A polyvalent STING agonist for oncology immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4234.

TGR5 agonists induce peripheral and central hypersensitivity to bladder distension

The mechanisms underlying chronic bladder conditions such as interstitial cystitis/bladder pain syndrome (IC/BPS) and overactive bladder syndrome (OAB) are incompletely understood. However, targeting specific receptors mediating neuronal sensitivity to specific stimuli is an emerging treatment strategy. Recently, irritant-sensing receptors including the bile acid receptor TGR5, have been identified within the viscera and are thought to play a key role in neuronal hypersensitivity. Here, in mice, we identify mRNA expression of TGR5 (Gpbar1) in all layers of the bladder as well as in the lumbosacral dorsal root ganglia (DRG) and in isolated bladder-innervating DRG neurons. In bladder-innervating DRG neurons Gpbar1 mRNA was 100% co-expressed with Trpv1 and 30% co-expressed with Trpa1. In vitro live-cell calcium imaging of bladder-innervating DRG neurons showed direct activation of a sub-population of bladder-innervating DRG neurons with the synthetic TGR5 agonist CCDC, which was diminished in Trpv1−/− but not Trpa1−/− DRG neurons. CCDC also activated a small percentage of non-neuronal cells. Using an ex vivo mouse bladder afferent recording preparation we show intravesical application of endogenous (5α-pregnan-3β-ol-20-one sulphate, Pg5α) and synthetic (CCDC) TGR5 agonists enhanced afferent mechanosensitivity to bladder distension. Correspondingly, in vivo intravesical administration of CCDC increased the number of spinal dorsal horn neurons that were activated by bladder distension. The enhanced mechanosensitivity induced by CCDC ex vivo and in vivo was absent using Gpbar1−/− mice. Together, these results indicate a role for the TGR5 receptor in mediating bladder afferent hypersensitivity to distension and thus may be important to the symptoms associated with IC/BPS and OAB.

Diet-Induced Obesity Disrupts Histamine-Dependent Oleoylethanolamide Signaling in the Mouse Liver

Introduction: Previous work suggests the existence of a paracrine signaling mechanism in which histamine released from visceral mast cells into the portal circulation contributes to fasting-induced ketogenesis by stimulating biosynthesis of the endogenous high-affinity PPAR-α agonist oleoylethanolamide (OEA). Methods: Male C57Bl/6J mice were rendered obese by exposure to a high-fat diet (HFD; 60% fat). We measured histamine, OEA, and other fatty-acid ethanolamides by liquid-chromatography/mass spectrometry, gene transcription by RT-PCR, protein expression by ELISA, neutral lipid accumulation in the liver using Red Oil O and BODIPY staining, and collagen levels using picrosirius red staining. Results: Long-term exposure to HFD suppressed both fasting-induced histamine release into portal blood and histamine-dependent OEA production in the liver. Additionally, subchronic OEA administration reduced lipid accumulation, inflammatory responses, and fibrosis in the liver of HFD-exposed mice. Discussion: The results suggest that disruption of histamine-dependent OEA signaling in the liver might contribute to pathology in obesity-associated liver steatosis.

"Microwave Radiation-Induced Up-Regulation of Inflammatory Cytokines in Sertoli Cells is Triggered by Endogenous TLR Agonists Released By Damaged Spermatogenic Cells and Mediated by the TLRS-Mapks Signaling Pathway"

Hui Wu1*, Dewen Wang2*, Yuhong Meng1, Hui Wang2, Yifan Xie2, Yang Li2, Hongyan Zuo2, Li Zhao2, Cuifang Zhang2 and Ruiyun Peng2 Author Affiliations 1Department of Pathology, Sixth Medical Center of PLA General Hospital, 100048, China 2Beijing Institute of Radiation Medicine. Beijing, China, 100850, China Received: April 04, 2022 | Published: June 03, 2022 Corresponding author: Hui Wu, Department of Pathology, the Sixth Medical Center of PLA General Hospital, 6 Fucheng roads, Beijing 100048, PR China Dewen Wang, Beijing Institute of Radiation Medicine, 27 Taiping roads, Beijing 100850, PR China DOI: 10.26717/BJSTR.2022.44.007029

Discovery of the cyclotide caripe 11 as a ligand of the cholecystokinin-2 receptor

The cholecystokinin-2 receptor (CCK2R) is a G protein-coupled receptor (GPCR) that is expressed in peripheral tissues and the central nervous system and constitutes a promising target for drug development in several diseases, such as gastrointestinal cancer. The search for ligands of this receptor over the past years mainly resulted in the discovery of a set of distinct synthetic small molecule chemicals. Here, we carried out a pharmacological screening of cyclotide-containing plant extracts using HEK293 cells transiently-expressing mouse CCK2R, and inositol phosphate (IP1) production as a readout. Our data demonstrated that cyclotide-enriched plant extracts from Oldenlandia affinis, Viola tricolor and Carapichea ipecacuanha activate the CCK2R as measured by the production of IP1. These findings prompted the isolation of a representative cyclotide, namely caripe 11 from C. ipecacuanha for detailed pharmacological analysis. Caripe 11 is a partial agonist of the CCK2R (Emax = 71%) with a moderate potency of 8.5 µM, in comparison to the endogenous full agonist cholecystokinin-8 (CCK-8; EC50 = 11.5 nM). The partial agonism of caripe 11 is further characterized by an increase on basal activity (at low concentrations) and a dextral-shift of the potency of CCK-8 (at higher concentrations) following its co-incubation with the cyclotide. Therefore, cyclotides such as caripe 11 may be explored in the future for the design and development of cyclotide-based ligands or imaging probes targeting the CCK2R and related peptide GPCRs.

Pharmacology of Antagonism of GPCR.

Agonists are defined as the ligands that activate intracellular signaling and evoke cellular responses. Synthetic and endogenous agonists should bind specific amino acids to activate G protein-coupled receptor (GPCR). Agonists that induce maximal responses are full agonists. Partial agonists cannot induce full responses unlike full agonists. In definition, antagonists inhibit agonist-stimulated responses by binding to orthosteric or allosteric sites. Antagonists modulate agonist-induced responses and are often related with inverse agonist activity. However, the relationship between antagonists and partial agonists is complex. An antagonist behaves as a partial agonist when the constitutive activity of the GPCR is high. In contrast, a partial agonist with very weak intrinsic activity may be classified as an antagonist. Thus, antagonisms of the compounds are influenced by constitutive activity of GPCRs, intrinsic activity and differences in the binding sites of GPCRs. Since "antagonism" has been revealed to have multiple aspects and more complex than previously thought, it may be difficult to classify each compound as simply "agonist" or "antagonist" as before. In this review, we discuss the recent findings and perspectives on the pharmacology of GPCR-binding antagonists, inverse agonists, and signaling.

SCAP deficiency facilitates obesity and insulin resistance through shifting adipose tissue macrophage polarization

IntroductionSterol regulatory element binding protein (SREBP) cleavage-associating protein (SCAP) is a sterol-regulated escort protein that translocates SREBPs from the endoplasmic reticulum to the Golgi apparatus, thereby activating lipid metabolism and cholesterol synthesis. Although SCAP regulates lipid metabolism in metabolic tissues, such as the liver and muscle, the effect of macrophage-specific SCAP deficiency in adipose tissue macrophages (ATMs) of patients with metabolic diseases is not completely understood. ObjectivesHere, we examined the function of SCAP in high-fat/high-sucrose diet (HFHS)-fed mice and investigated its role in the polarization of classical activated macrophages in adipose tissue. MethodsMacrophage-specific SCAP knockout (mKO) mice were generated through crossbreeding lysozyme 2-cre mice with SCAP floxed mice which were then fed HFHS for 12 weeks. Primary macrophages were derived from bone marrow cells and analyzed further. ResultsWe found that fat accumulation and the appearance of proinflammatory M1 macrophages were both higher in HFHS-fed SCAP mKO mice relative to floxed control mice. We traced the effect to a defect in the lipopolysaccharide-mediated increase in SREBP-1a that occurs in control but not SCAP mKO mice. Mechanistically, SREBP-1a increased expression of cholesterol 25-hydroxylase transcription, resulting in an increase in the production of 25-hydroxycholesterol (25-HC), an endogenous agonist of liver X receptor alpha (LXRα) which increased expression of cholesterol efflux to limit cholesterol accumulation and M1 polarization. In the absence of SCAP mediated activation of SREBP-1a, increased M1 macrophage polarization resulted in reduced cholesterol efflux downstream from 25-HC-dependent LXRα activation. ConclusionOverall, the activation of the SCAP-SREBP-1a pathway in macrophages may provide a novel therapeutic strategy that ameliorates obesity by controlling cholesterol homeostasis in ATMs.