Search Results (976)

CC chemokine receptor 2 (CCR2) has been linked to many inflammatory and immune diseases, making it a relevant drug target. Yet, all CCR2 antagonists developed so far have failed in clinical trials; thus, novel strategies are needed to target this receptor. Targeted protein degradation represents a novel approach to inhibit protein function by hijacking the cellular degradation machinery, such as the proteasome, to degrade the protein of interest. Here, we aimed to determine the amenability of CCR2 to chemically induced degradation by using a CCR2 fusion protein containing a HaloTag7 and HiBiT tag (CCR2-HaloTag-HiBiT). After characterization of the CCR2 construct, we used luminescence-based assays and immunofluorescence to quantify CCR2 levels, as well as a label-free, phenotypic assay to investigate the functional effect of CCR2 degradation. Treatment with HaloPROTAC3, which selectively degrades HaloTag fusion proteins, led to concentration- and time-dependent degradation of CCR2-HaloTag-HiBiT. HaloPROTAC3 induced degradation via the proteasome, as degradation was fully blocked with proteasomal inhibitors. Finally, functional assays showed that degradation of CCR2-HaloTag-HiBiT leads to a reduced functional response after agonist stimulation. Overall, our results indicate that CCR2 is amenable to targeted degradation, paving the way for the future development of CCR2 chemical degraders. Full article

The P2Y₆ receptor (P2Y₆R), a G_q-coupled receptor, is a potential drug discovery target for various inflammatory and degenerative conditions. Antagonists have been shown to attenuate colitis, acute lung injury, etc. In the search for competitive antagonists, we have investigated the SAR of 3-nitro-2-(trifluoromethyl)-2H-chromene derivatives, although high affinity is lacking. We now reveal that long-chain amino-functionalized congeners display greatly enhanced affinity in the antagonism of UDP-induced Ca²⁺ mobilization in human (h) P2Y₆R-transfected 1321N1 astrocytoma cells. A 6-(Boc-amino-n-heptylethynyl) analogue 30 (MRS4940) had an IC₅₀ of 162 nM, which was a 123-fold greater affinity than the corresponding unprotected primary alkylamine, 107-fold greater than the corresponding pivaloyl derivative 30, and 132-fold selective compared to the P2Y₁₄R. However, similar Boc-amino chains attached at the 8-position produced weak µM affinity. Thus, the P2Y₆R affinity depended on the chain length, attachment point, and terminal functionality. Off-target activities, at 45 sites, were tested for acylamino derivatives 20, 24, 26, 30, 31, and 37, which showed multiple interactions, particularly at the biogenic amine receptors. The more potent analogues may be suitable for evaluation in inflammation and cancer models, which will be performed in future studies. Full article

Embryonic diapause is a common evolutionary adaptation observed across a wide range of organisms. Artemia is one of the classic animal models for diapause research. The current studies of Artemia diapause mainly focus on the induction and maintenance of the embryonic diapause, with little research on the molecular regulatory mechanism of Artemia embryonic reactivation. The first 5 h after embryonic diapause breaking has been proved to be most important for embryonic reactivation in Artemia. In this work, two high-throughput sequencing methods, ATAC-seq and RNA-seq, were integrated to study the signal regulation process in embryonic reactivation of Artemia at 5 h after diapause breaking. Through the GO and KEGG enrichment analysis of the high-throughput datasets, it was showed that after 5 h of diapause breaking, the metabolism and regulation of Artemia cyst were quite active. Several signal transduction pathways were identified in the embryonic reactivation process, such as G-protein-coupled receptor (GPCR) signaling pathway, cell surface receptor signaling pathway, hormone-mediated signaling pathway, Wnt, Notch, mTOR signaling pathways, etc. It indicates that embryonic reactivation is a complex process regulated by multiple signaling pathways. With the further protein structure analysis and RT-qPCR verification, 11 GPCR genes were identified, in which 5 genes function in the embryonic reactivation stage and the other 6 genes contribute to the diapause stage. The results of this work reveal the signal transduction pathways and GPCRs involved in the embryonic reactivation process of Artemia cysts. These findings offer significant clues for in-depth research on the signal regulatory mechanisms of the embryonic reactivation process and valuable insights into the mechanism of animal embryonic diapause. Full article

The receptor–receptor interaction (RRI) of G protein-coupled receptors (GPCRs) leads to new functional entities that are conceptually distinct from the simple addition of signals mediated by the activation of the receptors that form the heteromers. Focusing on astrocytes, there is evidence for the existence of inhibitory and facilitatory RRIs, including the heteromers formed by the adenosine A2A and the dopamine D2 receptors, by A2A and the oxytocin receptor (OTR), and the D2-OTR heteromers. The possible involvement of these receptors in mosaicism has never been investigated in striatal astrocytes. By biophysical and functional approaches, we focused our attention on the existence of an A2A-D2-OTR high-order receptor complex and its role in modulating cytosolic calcium levels and endogenous glutamate release, when striatal astrocyte processes were stimulated with 4-aminopyridine. Functional data indicate a permissive role of OTR on dopamine signaling in the regulation of the glutamatergic transmission, and an inhibitory control mediated by A2A on both the D2-mediated signaling and on the OTR-facilitating effect on D2. Imaging biochemical and bioinformatic evidence confirmed the existence of the A2A-D2-OTR complex and its ternary structure in the membrane. In conclusion, the D2 receptor appears to be a hotspot in the control of the glutamate release from the astrocytic processes and may contribute to the regulation and integration of different neurotransmitter-mediated signaling in the striatum by the A2A-D2-OTR heterotrimers. Considering the possible selectivity of allosteric interventions on GPCRs organized as receptor mosaics, A2A-D2-OTR heterotrimers may offer selective pharmacological targets in neuropsychiatric disorders and neurodegenerative diseases. Full article

Human endocannabinoid signaling is primarily mediated by the cannabinoid receptors, CB1 and CB2, which are G protein-coupled receptors (GPCRs). These receptors have been linked to a variety of physiological processes and are being pursued as prospective drug targets due to their potential in treating pain and inflammation. However, because of their homology and shared signaling mechanisms, investigating the individual physiological roles of these receptors and designing subtype-selective ligands has been challenging. Using active-state CB1 and CB2 structures as guides, homologous residues within the orthosteric pocket of each receptor were mutated to alanine to test whether they equally impair CB1 and CB2 activity in response to two high-affinity, nonselective agonists (CP55,940 and AM12033). Interestingly, mutating the Y5.39 position impairs CB1 but not CB2 function. Conversely, mutating residue C6.47 improves CB1 but impairs CB2 signaling. The F7.35A mutation leads to a decrease in CP55,940 potency at CB1 and impairs internalization; however, AM12033 gains potency and promotes CB1 internalization. In CB2, mutation of F7.35A decreases the potency of CP55,940 and neither agonist induces internalization. These observations provide some insight into functional sensitivity of CB1 and CB2 to different agonists when conserved residues are mutated in the orthosteric pocket. Full article

Eosinophilic esophagitis (EoE) and inflammatory bowel disease (IBD) are chronic inflammatory disorders of the gastrointestinal tract, with EoE predominantly provoked by food and aeroallergens, whereas IBD is driven by a broader spectrum of immunopathological and environmental triggers. This review presents a comprehensive comparison of the pathophysiological and therapeutic strategies for EoE and IBD. We examine the current understanding of their underlying mechanisms, particularly the interplay between environmental factors and genetic susceptibility. A crucial element in both diseases is the integrity of the epithelial barrier, whose disruption plays a central role in their pathogenesis. The involvement of eosinophils, mast cells, B cells, T cells, dendritic cells, macrophages, and their associated cytokines is examined, highlighting the importance of targeting cytokine signaling pathways to modulate immune–epithelial interactions. We propose that advances in computation tools will uncover the significance of G-protein coupled receptors (GPCRs) in connecting immune and epithelial cells, leading to novel therapies for EoE and IBD. Full article

2-arachidonoylglycerol (2-AG) is the most abundant endocannabinoid (EC), acting as a full agonist at both CB1 and CB2 cannabinoid receptors. It is synthesized on demand in postsynaptic membranes through the sequential action of phosphoinositide-specific phospholipase Cβ1 (PLCβ1) and diacylglycerol lipase α (DAGLα), contributing to retrograde signaling upon interaction with presynaptic CB1. However, 2-AG production might also involve various combinations of PLC and DAGL isoforms, as well as additional intracellular pathways implying other enzymes and substrates. Three other alternative pathways of 2-AG synthesis rest on the extracellular cleavage of 2-arachidonoyl-lysophospholipids by three different hydrolases: glycerophosphodiesterase 3 (GDE3), lipid phosphate phosphatases (LPPs), and two members of ecto-nucleotide pyrophosphatase/phosphodiesterases (ENPP6–7). We propose the names of AlterAG-1, -2, and -3 for three pathways sharing an ectocellular localization, allowing them to convert extracellular lysophospholipid mediators into 2-AG, thus inducing typical signaling switches between various G-protein-coupled receptors (GPCRs). This implies the critical importance of the regioisomerism of both lysophospholipid (LPLs) and 2-AG, which is the object of deep analysis within this review. The precise functional roles of AlterAGs are still poorly understood and will require gene invalidation approaches, knowing that both 2-AG and its related lysophospholipids are involved in numerous aspects of physiology and pathology, including cancer, inflammation, immune defenses, obesity, bone development, neurodegeneration, or psychiatric disorders. Full article

G-protein-coupled receptors (GPCRs) represent a family of druggable targets when treating several diseases and continue to be a leading part of the drug discovery process. Trace amine-associated receptors (TAARs) are GPCRs involved in many physiological functions with TAAR1 having important roles within the central nervous system (CNS). By using homology modeling methods, the responsiveness of TAAR1 to endogenous and synthetic ligands has been explored. In addition, the discovery of different chemo-types as selective murine and/or human TAAR1 ligands has helped in the understanding of the species-specificity preferences. The availability of TAAR1–ligand complexes sheds light on how different ligands bind TAAR1. TAAR5 is considered an olfactory receptor but has specific involvement in some brain functions. In this case, the drug discovery effort has been limited. Here, we review the successful computational efforts developed in the search for novel TAAR1 and TAAR5 ligands. A specific focus on applying structure-based and/or ligand-based methods has been done. We also give a perspective of the experimental data available to guide the future drug design of new ligands, probing species-specificity preferences towards more selective ligands. Hints for applying repositioning approaches are also discussed. Full article

Cancer cells depend on specific oncogenic pathways or present a genetic alteration that leads to a particular disturbance. Still, personalized and targeted biological therapy remains challenging, with current efforts generally yielding disappointing results. Carefully assessing onco-target molecular pathways can, however, potently assist with such efforts for the selection of patient populations that would best respond to a given drug treatment. RNF43, an E3 ubiquitin ligase that negatively regulates Wnt/frizzled (FZD) receptors by their ubiquitination, internalization, and degradation, controls a key pathway in cancer. Recently, additional target proteins of RNF43 were described, including p85 of the PI3K/AKT/mTOR signaling pathway and protease-activated receptor 2 (PAR₂), a G-protein-coupled receptor that potently induces β-catenin stabilization, independent of Wnts. RNF43 mutations with impaired E3 ligase activity were found in several types of cancers (e.g., gastrointestinal system tumors and endometrial and ovarian cancer), pointing to a high dependency on FZD receptors and possibly PAR₂ and the PI3K/AKT/mTOR signaling pathway. The development of drugs toward these targets is essential for improved treatment of cancer patients. Full article

The melanocortin-4 receptor (MC4R) is a G protein-coupled receptor (GPCR) that is expressed in several brain locations encompassing the hypothalamus and the brainstem, where the receptor controls several body functions, including metabolism. In a well-defined pathway to decrease appetite, hypothalamic proopiomelanocortin (POMC) neurons localized in the arcuate nucleus (Arc) project to MC4R neurons in the paraventricular nuclei (PVN) to release the natural MC4R agonist α-melanocyte-stimulating hormone (α-MSH). Arc neurons also project excitatory glutamatergic fibers to the MC4R neurons in the PVN for a fast synaptic transmission to regulate a satiety pathway potentiated by α-MSH. By using super-resolution microscopy, we found that in hypothalamic neurons in a primary culture, postsynaptic density protein 95 (PSD95) colocalizes with GluN1, a subunit of the ionotropic N-methyl-D-aspartate receptor (NMDAR). Thus, hypothalamic neurons form excitatory postsynaptic specializations. To study the MC4R distribution at these sites, tagged HA-MC4R under the synapsin promoter was expressed in neurons by adeno-associated virus (AAV) gene transduction. HA-MC4R immunofluorescence peaked at the center and in proximity to the PSD95- and NMDAR-expressing sites. These data provide morphological evidence that MC4R localizes together with glutamate receptors at postsynaptic and peri-postsynaptic sites. Full article

The prokineticin system plays a role in hypothalamic neurons in the control of energy homeostasis. Prokineticin receptors (PKR1 and PKR2), like other G-protein-coupled receptors (GPCRs) are involved in the regulation of energy intake and expenditure and are modulated by the accessory membrane protein 2 of the melanocortin receptor (MRAP2). The aim of this work is to characterise the interaction and regulation of the non-melanocortin receptor PKR1 by MRAP2a in zebrafish (zMRAP2a) in order to use zebrafish as a model for the development of drugs targeting accessory proteins that can alter the localisation and activity of GPCRs. To this end, we first showed that zebrafish PKR1 (zPKR1) is able to interact with both zMRAP2a and human MRAP2 (hMRAP2). This interaction occurs between the N-terminal region of zPKR1 and the C-terminal domain of zMRAP2a, which shows high sequence identity with hMRAP2 and a similar propensity for dimer formation. Moreover, we demonstrated that in Chinese hamster ovary (CHO) cells, zMRAP2a or hMRAP2 are able to modulate zPKR1 activation induced by zebrafish PK2 (zPK2) resulting in an impaired ERK and STAT3 activation. Full article

A key step in platelet production is the migration of megakaryocytes to the vascular sinusoids within the bone marrow. This homing is mediated by the chemokine CXCL12 and its receptor CXCR4. CXCR4 is also a positive regulator of platelet activation and thrombosis. Pim-1 kinase has been shown to regulate CXCR4 signalling in other cell types, and we have previously described how Pim kinase inhibitors attenuate platelet aggregation to CXCL12. However, the mechanism by which Pim-1 regulates CXCR4 signalling in platelets and megakaryocytes has yet to be elucidated. Using human platelets, murine bone marrow-derived megakaryocytes, and the megakaryocyte cell line MEG-01, we demonstrate that pharmacological Pim kinase inhibition leads to reduced megakaryocyte and platelet function responses to CXCL12, including reduced megakaryocyte migration and platelet granule secretion. Attenuation of CXCL12 signalling was found to be attributed to the reduced surface expression of CXCR4. The decrease in CXCR4 surface levels was found to be mediated by rapid receptor internalisation, in the absence of agonist stimulation. We demonstrate that pharmacological Pim kinase inhibition disrupts megakaryocyte and platelet function by reducing constitutive CXCR4 surface expression, decreasing the number of receptors available for agonist stimulation and signalling. These findings have implications for the development and use of Pim kinase inhibitors for the treatment of conditions associated with elevated circulating levels of CXCL12/SDF1α and increased thrombotic risk. Full article

Depression is the most common chronic mental illness and is characterized by low mood, insomnia, and affective disorders. However, its pathologic mechanisms remain unclear. Numerous studies have suggested that the ghrelin/GHSR system may be involved in the pathophysiologic process of depression. Ghrelin plays a dual role in experimental animals, increasing depressed behavior and decreasing anxiety. By combining several neuropeptides and traditional neurotransmitter systems to construct neural networks, this hormone modifies signals connected to depression. The present review focuses on the role of ghrelin in neuritogenesis, astrocyte protection, inflammatory factor production, and endocrine disruption in depression. Furthermore, ghrelin/GHSR can activate multiple signaling pathways, including cAMP/CREB/BDNF, PI3K/Akt, Jak2/STAT3, and p38-MAPK, to produce antidepressant effects, given which it is expected to become a potential therapeutic target for the treatment of depression. Full article

The melanocortin-4 receptor (MC4R) is a key player in the hypothalamic leptin–melanocortin pathway that regulates satiety and hunger. MC4R belongs to the G protein-coupled receptors (GPCRs), which are known to form heterodimers with other membrane proteins, potentially modulating receptor function or characteristics. Like MC4R, thyroid hormones (TH) are also essential for energy homeostasis control. TH transport across membranes is facilitated by the monocarboxylate transporter 8 (MCT8), which is also known to form heterodimers with GPCRs. Based on the finding in single-cell RNA-sequencing data that both proteins are simultaneously expressed in hypothalamic neurons, we investigated a putative interplay between MC4R and MCT8. We developed a novel staining protocol utilizing a fluorophore-labeled MC4R ligand and demonstrated a co-localization of MC4R and MCT8 in human brain tissue. Using in vitro assays such as BRET, IP1, and cAMP determination, we found that MCT8 modulates MC4R-mediated phospholipase C activation but not cAMP formation via a direct interaction, an effect that does not require a functional MCT8 as it was not altered by a specific MCT8 inhibitor. This suggests an extended functional spectrum of MCT8 as a GPCR signaling modulator and argues for the investigation of further GPCR-protein interactions with hitherto underrepresented physiological functions. Full article

Short chain fatty acids (SCFAs), mainly including acetate, propionate and butyrate, are produced by intestinal bacteria during the fermentation of partially digested and indigestible polysaccharides. SCFAs play an important role in regulating intestinal energy metabolism and maintaining the homeostasis of the intestinal environment and also play an important regulatory role in organs and tissues outside the gut. In recent years, many studies have shown that SCFAs can regulate inflammation and affect host health, and two main signaling mechanisms have also been identified: the activation of G-protein coupled receptors (GPCRs) and inhibition of histone deacetylase (HDAC). In addition, a growing body of evidence highlights the importance of every SCFA in influencing health maintenance and disease development. In this review, we summarized the recent advances concerning the biological properties of SCFAs and their signaling pathways in inflammation and body health. Hopefully, it can provide a systematic theoretical basis for the nutritional prevention and treatment of human diseases. Full article