G Protein-coupled Receptors Ligands Research Articles

Antibodies expand the scope of angiotensin receptor pharmacology.

G-protein-coupled receptors (GPCRs) are key regulators of human physiology and are the targets of many small-molecule research compounds and therapeutic drugs. While most of these ligands bind to their target GPCR with high affinity, selectivity is often limited at the receptor, tissue and cellular levels. Antibodies have the potential to address these limitations but their properties as GPCR ligands remain poorly characterized. Here, using protein engineering, pharmacological assays and structural studies, we develop maternally selective heavy-chain-only antibody ('nanobody') antagonists against the angiotensin II type I receptor and uncover the unusual molecular basis of their receptor antagonism. We further show that our nanobodies can simultaneously bind to angiotensin II type I receptor with specific small-molecule antagonists and demonstrate that ligand selectivity can be readily tuned. Our work illustrates that antibody fragments can exhibit rich and evolvable pharmacology, attesting to their potential as next-generation GPCR modulators.

Analgesic Peptides: From Natural Diversity to Rational Design.

Pain affects one-third of the global population and is a significant public health issue. The use of opioid drugs, which are the strongest painkillers, is associated with several side effects, such as tolerance, addiction, overdose, and even death. An increasing demand for novel, safer analgesic agents is a driving force for exploring natural sources of bioactive peptides with antinociceptive activity. Since the G protein-coupled receptors (GPCRs) play a crucial role in pain modulation, the discovery of new peptide ligands for GPCRs is a significant challenge for novel drug development. The aim of this review is to present peptides of human and animal origin with antinociceptive potential and to show the possibilities of their modification, as well as the design of novel structures. The study presents the current knowledge on structure-activity relationship in the design of peptide-based biomimetic compounds, the modification strategies directed at increasing the antinociceptive activity, and improvement of metabolic stability and pharmacodynamic profile. The procedures employed in prolonged drug delivery of emerging compounds are also discussed. The work summarizes the conditions leading to the development of potential morphine replacements.

2023 Julius Axelrod Symposium: Plant-Derived Molecules Acting on G Protein-Coupled Receptors.

Plant extracts have played a significant role in traditional medicine for centuries, contributing to improved health and the treatment of various human illnesses. G protein-coupled receptors (GPCRs) are crucial in numerous physiologic functions, and there is growing evidence suggesting their involvement in the therapeutic effects of many plant extracts. In recent years, scientists have identified an expanding number of isolated molecules responsible for the biologic activity of these extracts, with many believed to act on GPCRs. This article critically reviews the evidence supporting the modulation of GPCR function by these plant-derived molecules through direct binding. Structural information is now available for some of these molecules, allowing for a comparison of their binding mode with that of endogenous GPCR ligands. The final section explores future trends and challenges, focusing on the identification of new plant-derived molecules with both orthosteric and allosteric binding modes, as well as innovative strategies for designing GPCR ligands inspired by these plant-derived compounds. In conclusion, plant-derived molecules are anticipated to play an increasingly vital role as therapeutic drugs and serve as templates for drug design. SIGNIFICANCE STATEMENT: This minireview summarizes the most pertinent publications on isolated plant-derived molecules interacting with G protein-coupled receptors (GPCRs) and comments on available structural information on GPCR/plant-derived ligand pairs. Future challenges and trends for the isolation and characterization of plant-derived molecules and drug design are discussed.

Orphan G protein-coupled receptors: the ongoing search for a home.

G protein-coupled receptors (GPCRs) make up the largest receptor superfamily, accounting for 4% of protein-coding genes. Despite the prevalence of such transmembrane receptors, a significant number remain orphans, lacking identified endogenous ligands. Since their conception, the reverse pharmacology approach has been used to characterize such receptors. However, the multifaceted and nuanced nature of GPCR signaling poses a great challenge to their pharmacological elucidation. Considering their therapeutic relevance, the search for native orphan GPCR ligands continues. Despite limited structural input in terms of 3D crystallized structures, with advances in machine-learning approaches, there has been great progress with respect to accurate ligand prediction. Though such an approach proves valuable given that ligand scarcity is the greatest hurdle to orphan GPCR deorphanization, the future pairings of the remaining orphan GPCRs may not necessarily take a one-size-fits-all approach but should be more comprehensive in accounting for numerous nuanced possibilities to cover the full spectrum of GPCR signaling.

Cryo-electron microscopy for GPCR research and drug discovery in endocrinology and metabolism.

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors, with many GPCRs having crucial roles in endocrinology and metabolism. Cryogenic electron microscopy (cryo-EM) has revolutionized the field of structural biology, particularly regarding GPCRs, over the past decade. Since the first pair of GPCR structures resolved by cryo-EM were published in 2017, the number of GPCR structuresresolved by cryo-EM has surpassed the number resolved by X-ray crystallography by 30%, reaching >650, and the number has doubled every ~0.63 years for the past 6 years. At this pace, it is predicted that the structure of 90% of all human GPCRs will be completed within the next 5-7 years. This Review highlights the general structural features and principles that guide GPCR ligand recognition, receptor activation, Gprotein coupling, arrestin recruitment and regulation by GPCR kinases. The Review also highlights the diversity of GPCR allosteric binding sites and how allosteric ligands could dictate biased signalling that is selective for a G protein pathwayor an arrestin pathway. Finally, the authors use the examples of glycoprotein hormone receptors and glucagon-like peptide 1 receptor to illustrate the effect of cryo-EM on understanding GPCR biology in endocrinology and metabolism, as well as on GPCR-related endocrine diseases and drug discovery.

GPCR-IPL score: multilevel featurization of GPCR-ligand interaction patterns and prediction of ligand functions from selectivity to biased activation.

G-protein-coupled receptors (GPCRs) mediate diverse cell signaling cascades after recognizing extracellular ligands. Despite the successful history of known GPCR drugs, a lack of mechanistic insight into GPCR challenges both the deorphanization of some GPCRs and optimization of the structure-activity relationship of their ligands. Notably, replacing a small substituent on a GPCR ligand can significantly alter extracellular GPCR-ligand interaction patterns and motion of transmembrane helices in turn to occur post-binding events of the ligand. In this study, we designed 3D multilevel features to describe the extracellular interaction patterns. Subsequently, these 3D features were utilized to predict the post-binding events that result from conformational dynamics from the extracellular to intracellular areas. To understand the adaptability of GPCR ligands, we collected the conformational information of flexible residues during binding and performed molecular featurization on a broad range of GPCR-ligand complexes. As a result, we developed GPCR-ligand interaction patterns, binding pockets, and ligand features as score (GPCR-IPL score) for predicting the functional selectivity of GPCR ligands (agonism versus antagonism), using the multilevel features of (1) zoomed-out 'residue level' (for flexible transmembrane helices of GPCRs), (2) zoomed-in 'pocket level' (for sophisticated mode of action) and (3) 'atom level' (for the conformational adaptability of GPCR ligands). GPCR-IPL score demonstrated reliable performance, achieving area under the receiver operating characteristic of 0.938 and area under the precision-recall curve of 0.907 (available in gpcr-ipl-score.onrender.com). Furthermore, we used the molecular features to predict the biased activation of downstream signaling (Gi/o, Gq/11, Gs and β-arrestin) as well as the functional selectivity. The resulting models are interpreted and applied to out-of-set validation with three scenarios including the identification of a new MRGPRX antagonist.

Butyrate acts as a G-protein-coupled receptor ligand that prevents high glucose-induced amyloidogenesis in N2a cells through the protein kinase B/glycogen synthase kinase-3β pathway.

To the Editor: Alzheimer's disease (AD) and type 2 diabetes mellitus (T2DM) are major causes of morbidity and mortality in older individuals, and their epidemiological link generates substantial challenges for the management of these devastating diseases. Abnormal glucose metabolism, accompanied by inhibition of the protein kinase B (Akt)/glycogen synthase kinase-3β (GSK-3β) signaling pathway, is a key factor in amyloid precursor protein (APP) accumulation and amyloid β (Aβ) formation in neuronal cells.[1-3] Therefore, activation of the Akt/GSK-3β pathway may be a promising target for AD treatment. Several lines of evidence indicated that a major microbial metabolite, butyrate (NaB) downregulated the amyloidogenic pathway and improved learning or memory ability in vivo or in vitro models of AD. NaB improved glucose metabolism in T2DM models through the Akt/GSK-3β pathway, which suggests its beneficial effect in the management of T2DM and AD.[4] However, there are few studies evaluating the mechanism of NaB on T2DM-related AD. Therefore, we used mouse neuroblastoma-derived N2a cells (serial TCM29, obtained from National Collection of Authenticated Cell Cultures of China), a cell model extensively used for studying AD-related signal transduction, to investigate the potential impact and mechanism of NaB on high glucose-induced amyloidogenesis. We hypothesized that high glucose exposure would inhibit Akt and GSK-3β phosphorylation and induce amyloidogenesis in N2a cells, and NaB would downregulate pathological changes via activation of the Akt/GSK-3β pathway. To establish a cell model of abnormal glucose metabolism, N2a cells were exposed to different concentrations of glucose (25, 50, 75, 100, 125, 150 mmol/L) for 24, 48, or 72 h, and mannitol was used to exclude the effect of high osmolarity [Supplementary Figure 1A–1E, https://links.lww.com/CM9/B383]. Compared to 5.5 mmol/L glucose (control), cell viability within 24 h decreased significantly from 90.15 ± 2.21% to 55.87 ± 1.22%. We detected markers of glucose uptake and metabolism, that is, glucose transporter 3 (GLUT3), glucose transporter 4 (GLUT4), and phosphorylated insulin receptor substrate 1 (p-IRS1). After 24 h of high glucose treatment, we found that the expression levels of GLUT3, GLUT4, and p-IRS1 were statistically significantly reduced in the 75 mmol/L glucose group. Furthermore, the transcription and protein levels of APP and beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) were examined to upregulate in the 75 and 100 mmol/L glucose groups [Supplementary Figure 1F–1H, https://links.lww.com/CM9/B383]. These findings suggest that 75 mmol/L glucose treatment for 24 h induces amyloidogenesis, and the cell model was used in the subsequent investigations. Next, we investigated the effect of NaB on high glucose-induced amyloidogenesis and compared to acetate (NaA) and propionate (NaP), the other two major short-chain fatty acids (SCFAs). Different concentrations ranging from 0.5 to 8.0 mmol/L NaA, NaB, and NaP did not statistically significantly decrease cell activity. To further determine the effects of SCFAs on amyloidogenesis under high glucose conditions, related mRNA and protein levels in N2a cells under pretreatment with 0.5, 1.0, or 2.0 mmol/L NaA, NaB, or NaP for 2 h before 75 mmol/L glucose stimulation for 24 h were assessed. As shown in Supplementary Figure 2, https://links.lww.com/CM9/B383, NaB and NaP, but not NaA decreased APP and BACE1 mRNA levels, and APP and BACE1 protein concentrations were decreased by 1 mmol/L and 500 μmol/L NaB treatment, and 500 μmol/L NaB showed the most significant inhibitory effect. Similar results were obtained from immunostaining: cells in the hyperglycemia model exhibited a higher level of Aβ than control cells, and pretreatment with 500 μmol/L NaB decreased the high glucose-induced accumulation of Aβ [Supplementary Figure 3A, https://links.lww.com/CM9/B383]. These results indicated that the inhibitory effects of NaA, NaB, and NaP on AD-related pathology were different despite their similar structure, which was documented in other studies and partially related to different water solubilities and affinities for cell receptors. However, the exact underlying mechanisms require further investigation. Moreover, Akt and GSK-3β phosphorylation levels were assessed in N2a cells treated with various glucose concentrations and SCFAs. High glucose (50, 75, and 100 mmol/L) lowered the relative p-Akt expression in cells in a concentration-dependent manner [Supplementary Figure 4A, https://links.lww.com/CM9/B383]. Only NaB treatment increased the level of p-Akt by a small margin in the 2 mmol/L and 1 mmol/L NaB-treated groups and significantly reversed the glucose-stimulated reduction of p-Akt to 96.39 ± 7.11% in the 500 μmol/L NaB-treated group [Supplementary Figure 4B–4D, https://links.lww.com/CM9/B383]. Meanwhile, the p-GSK-3β level shown in immunofluorescence, a downstream signal of Akt, was lowest in the glucose-treated group and was higher in the NaB-treated group than the glucose-treated group [Supplementary Figure 3B, https://links.lww.com/CM9/B383]. In agreement, the p-GSK-3β protein level in the high glucose group showed a significant reduction to 56.45 ± 6.62% compared to the control, and pretreatment with 500 μmol/L NaB significantly prevented their reduction in the glucose-induced group [Figure 1A]. These results showed that high glucose diminished Akt-mediated GSK-3β deactivation and enhanced amyloidogenesis in N2a cells.Figure 1: Inhibitory effect of NaB on high glucose-induced amyloidogenesis through inhibition of GSK-3β pathway. N2a cells were pretreated with NaB and ARA (5 μmol/L) for 2 h prior to treatment of high glucose for 24 h. The expression of p-GSK-3β (A), APP (B), BACE1 (C) were measured by Western blotting. GAPDH was used as a loading control. n = 6. Data are represented as individual points, ∗ P < 0.05, † P < 0.01 compared to the control group, ‡ P < 0.05 compared to the treatment of high glucose and NaB group. APP: amyloid precursor protein; BACE1: Beta-site amyloid precursor protein cleaving enzyme 1; GAPDH: Glyceraldehyde-3phosphate dehydrogenase; GSK-3β: glycogen synthase kinase-3β.To further determine the internal signaling pathway, we used the GSK-3β inhibitor N-(4-methoxybenyl)-N′-(5-nitro-1,3-thiazol-2-yl) urea (Sigma-Aldrich; AR-A014418) to investigate whether the Akt/GSK-3β pathway interfered with this progress. Compared to the glucose-treated group, N2a cells exposed to 5 μmol/L GSK-3b inhibitor or NaB before glucose treatment had significantly reduced APP and BACE1 levels [Figure 1B and 1C], which showed that high glucose-induced upregulation of APP and BACE1 was partially dependent on the inhibition of GSK-3β. Pretreatment with 500 μmol/L NaB in the presence of 5 μmol/L GSK-3b inhibitor significantly decreased the levels of APP and BACE1 compared to NaB pretreatment, indicating that the effect of NaB on high glucose-induced amyloidogenesis occurred through activation of the Akt/GSK-3β pathway. NaB affects cell signaling pathways through two different approaches, inhibition of class I and most class II histone deacetylase (HDAC) or interaction with G-protein-coupled receptor (GPCR), especially the Gαi family of G proteins. In this study, we examined the activated way of NaB by using pertussis toxin (PTX) to inhibit Gαi and ibuprofen to inhibit sodium-coupled monocarboxylate transporter 1, which is the co-transporter for the entry of HDAC inhibitors. As shown in Supplementary Figure 5, https://links.lww.com/CM9/B383, cells in the presence of NaB and PTX prevented the ability of NaB to affect high glucose-induced alterations in APP and BACE1 expression; in contrast, when cells were pretreated with NaB and ibuprofen, NaB decreased high glucose-induced APP and BACE1 upregulation. These findings suggest that NaB was absorbed into N2a cells via GPCRs, which activated Gαi to attenuate high glucose-induced amyloidogenesis. The present study provides several important findings on the effects of NaB on high glucose-induced amyloidogenesis. First, N2a cells treated with a high concentration of glucose (75 mmol/L, more than threefold the normal concentration) for 24 h exhibited significant insulin resistance and induced amyloidogenesis. Similar conclusions were reported in the correlation between long-term hyperglycemia and insulin resistance of T2DM and AD-related pathology and cognitive impairment. Second, our study found that high glucose treatment decreased activated Akt, which activated GSK-3β and enhanced APP processing, suggesting that high glucose induces amyloidogenesis via GSK-3β signaling. Akt is a serine/threonine-protein kinase that is an important upstream regulator of the inhibitory phosphorylation of GSK-3β involved in insulin receptor signaling. Several studies evaluated the underlying mechanisms of AD and T2DM and suggested that impaired insulin signaling played a critical role in the pathogenesis of both disorders. Third, we found the involvement of the Akt/GSK-3β pathway in NaB-mediated anti-amyloidogenesis effect in neuronal cells under high glucose conditions. Increasing evidence suggests that Akt/GSK-3β signaling is linked to age-related structural and functional changes in humans and is crucial to protect against several stress stimuli in neuronal cells. GSK-3β activity may play a central role in the development of AD, and GSK-3β activity was implicated in APP processing and Aβ production.[5] Overexpression of GSK-3β resulted in amyloidogenesis and neuronal death, and inhibition of GSK-3β activity decreased Aβ production and plaque accumulation in transgenic models of AD.[6] Other biological substances or drugs such as neuroglobin, ethosuximide, and ghrelin, were also found to alleviate amyloidogenesis by activating the Akt/GSK-3β pathway in cell and animal models of AD. Among them, NaB may be the most promising compound to target high glucose-induced amyloidogenesis. Firstly, humans could obtain NaB supplementation by consuming dietary carbohydrates, and diets rich in fiber were reported to decrease brain Aβ and improve memory in AD models and older adults.[7] Besides, gut microbiota generates NaB, and prebiotics and probiotics to increase NaB production would be a rather innovative approach for AD treatment. Taken together, we revealed that NaB inhibited high glucose-induced amyloidogenesis in N2a cells by acting as a GPCR ligand and mediating the Akt/GSK-3β pathway. Our findings provide new insights into the prevention and treatment of T2DM-associated AD pathogenesis. Funding This study was supported by grants from the Active Health and Aging Technologic Solutions Major Project of the National Key R&D Program (No. 2020YFC2006300), the Department of Science and Technology of Sichuan Province (No. 2021YJ0156), and the West China Nursing Discipline Development Special Fund Project of Sichuan University (No. HXHL20006). Conflicts of interest None.

Interplay between G protein-coupled receptors and nanotechnology.

G protein-coupled receptors (GPCRs), as the largest family of membrane receptors, actively modulate plasma membrane and endosomal signalling. Importantly, GPCRs are naturally nanosized, and spontaneously formed nanoaggregates of GPCRs (natural nano-GPCRs) may enhance GPCR-related signalling and functions. Although GPCRs are the molecular targets of the majority of marketed drugs, the poor pharmacokinetics and physicochemical properties of GPCR ligands greatly limit their clinical applicability. Nanotechnology, as versatile techniques, can encapsulate GPCR ligands to assemble synthetic nano-GPCRs to overcome their obstacles, robustly elevating drug efficacy and safety. Moreover, endosomal delivery of GPCR ligands by nanoparticles can precisely initiate sustained endosomal signal transduction, while nanotechnology has been widely utilized for isolation, diagnosis, and detection of GPCRs. In turn, due to overexpression of GPCRs on the surface of various types of cells, GPCR ligands can endow nanoparticles with active targeting capacity for specific cells via ligand-receptor binding and mediate receptor-dependent endocytosis of nanoparticles. This significantly enhances the potency of nanoparticle delivery systems. Therefore, emerging evidence has revealed the interplay between GPCRs and nanoparticles, although investigations into their relationship have been inadequate. This review aims to summarize the interaction between GPCRs and nanotechnology for understanding their mutual influences and utilizing their interplay for biomedical applications. It will provide a fundamental platform for developing powerful and safe GPCR-targeted drugs and nanoparticle systems. STATEMENT OF SIGNIFICANCE: GPCRs as molecular targets for the majority of marketed drugs are naturally nanosized, and even spontaneously form nano aggregations (nano-GPCRs). Nanotechnology has also been applied to construct synthetic nano-GPCRs or detect GPCRs, while endosomal delivery of GPCR ligands by nanoparticles can magnify endosomal signalling. Meanwhile, molecular engineering of nanoparticles with GPCRs or their ligands can modulate membrane binding and endocytosis, powerfully improving the efficacy of nanoparticle system. However, there are rare summaries on the interaction between GPCRs and nanoparticles. This review will not only provide a versatile platform for utilizing nanoparticles to modulate or detect GPCRs, but also facilitate better understanding of the designated value of GPCRs for molecular engineering of biomaterials with GPCRs in therapeutical application.

Accessible and Generalizable in Vitro Luminescence Assay for Detecting GPCR Activation.

G protein-coupled receptors (GPCRs) serve critical physiological roles as the most abundant family of receptors. Here, we describe the design of a generalizable and cell lysate-based method that leverages the interaction between an agonist-activated GPCR and a conformation-specific binder to reconstitute split nanoluciferase (NanoLuc) in vitro. This tool, In vitro GPCR split NanoLuc ligand Triggered Reporter (IGNiTR), has broad applications. We have demonstrated IGNiTR's use with three Gs-coupled GPCRs, two Gi-coupled GPCRs and three classes of conformation-specific binders: nanobodies, miniG proteins, and G protein peptidomimetics. As an in vitro method, IGNiTR enables the use of synthetic G protein peptidomimetics and provides easily scalable and portable reagents for characterizing GPCRs and ligands. We tested three diverse applications of IGNiTR: (1) proof-of-concept GPCR ligand screening using dopamine receptor D1 IGNiTR; (2) detection of opioids for point-of-care testing; and (3) characterizing GPCR functionality during Nanodisc-based reconstitution processes. Due to IGNiTR's unique advantages and the convenience of its cell lysate-based format, this tool will find extensive applications in GPCR ligand detection, screening, and GPCR characterization.

SOCS3 inhibits the mesenchymal stromal cell secretory factor SDF-1-mediated improvement of islet function in non-obese diabetic mice

BackgroundIslet transplantation is used therapeutically in a minority of patients with type 1 diabetes (T1D). However, successful outcomes are hampered by early islet β-cell loss caused by immune rejection and autoimmunity. Recent studies have demonstrated that mesenchymal stromal cells can enhance islet function both in vitro and in vivo by secreting ligands that activate islet G-protein coupled receptors (GPCRs). Stromal cell-derived factor 1 (SDF-1) is an MSC-secreted GPCR ligand, whereas the suppressor of cytokine signaling 3 (SOCS3) is a negative regulator of STAT3-activating cytokines. Here, we determined whether improvement in islet function mediated by exogenous SDF-1 is impaired by SOCS3 in experimental models of T1D.MethodsIsolated islets were cultured for 48 h with SDF-1. Cytokine-induced apoptosis was measured immediately. Islets from Socs3−/− mice were pre-cultured with exogenous SDF-1 and transplanted underneath the kidney capsule of C57BL/6 mice with streptozotocin-induced diabetes. Blood glucose levels were monitored for 28 days. AMD3100, an antagonist of the SDF-1 ligand CXCR4, was administered subcutaneously to islet transplanted mice to inhibit CXCR4 before and after transplantation.ResultsSDF-1 protected islet cells from cytokine-induced apoptosis in vitro. SOCS3-knockout (KO) islets pretreated with SDF-1 were effective in reducing blood glucose in non-obese diabetic mice in vivo. We found that SDF-1 elicits localized immunosuppression in transplanted SOCS3-KO islets. Immunomodulation was observed when SOCS-KO islets were preconditioned with SDF-1. Gene expression and flow cytometric analyses revealed significantly decreased immune cell infiltration, inflammatory cytokines, and concomitant increases in FOXP3+ regulatory T cells, alternatively activated M2 macrophages, and dendritic cell phenotypes. Administration of AMD3100 impaired the SDF-1-mediated improvement in SOCS3-KO islet function and local immune suppression.ConclusionSDF-1 improves the function of islet grafts in autoimmune diabetes through regulation by CXCR4; however, the presence of SOCS3 reverses the protective effect of SDF-1 on islet grafts. These data reveal a molecular pathway that can elicit localized immunosuppression and delay graft destruction in transplanted islets.

Insight into the Mode of Action of 8-Hydroxyquinoline-Based Blockers on the Histamine Receptor 2

Histamine receptor 2 (HRH2) blockers are used to treat peptic ulcers and gastric reflux. Chlorquinaldol and chloroxine, which contain an 8-hydroxyquinoline (8HQ) core, have recently been identified as blocking HRH2. To gain insight into the mode of action of 8HQ-based blockers, here, we leverage an HRH2-based sensor in yeast to evaluate the role of key residues in the HRH2 active site on histamine and 8HQ-based blocker binding. We find that the HRH2 mutations D98A, F254A, Y182A, and Y250A render the receptor inactive in the presence of histamine, while HRH2:D186A and HRH2:T190A retain residual activity. Based on molecular docking studies, this outcome correlates with the ability of the pharmacologically relevant histamine tautomers to interact with D98 via the charged amine. Docking studies also suggest that, unlike established HRH2 blockers that interact with both ends of the HRH2 binding site, 8HQ-based blockers interact with only one end, either the end framed by D98/Y250 or T190/D186. Experimentally, we find that chlorquinaldol and chloroxine still inactivate HRH2:D186A by shifting their engagement from D98 to Y250 in the case of chlorquinaldol and D186 to Y182 in the case of chloroxine. Importantly, the tyrosine interactions are supported by the intramolecular hydrogen bonding of the 8HQ-based blockers. The insight gained in this work will aid in the development of improved HRH2 therapeutics. More generally, this work demonstrates that Gprotein-coupled receptor (GPCR)-based sensors in yeast can help elucidate the mode of action of novel ligands for GPCRs, a family of receptors that bind 30% of FDA therapeutics.

Design of Drug Efficacy Guided by Free Energy Simulations of the β2-Adrenoceptor.

G protein-coupled receptors (GPCR) play important roles in physiological processes and are modulated by drugs that either activate or block signaling. Rational design of the pharmacological efficacy profiles of GPCR ligands could enable the development of more efficient drugs, but is challenging even if high-resolution receptor structures are available. We performed molecular dynamics simulations of the β2 adrenergic receptor (β2R) in active and inactive conformations to assess if binding free energy calculations can predict differences in ligand efficacy for closely related compounds. Previously identified ligands were successfully classified into groups with comparable efficacy profiles based on the calculated shift in ligand affinity upon activation. A series of ligands were then predicted and synthesized, leading to the discovery of partial agonists with nanomolar potencies and novel scaffolds. Our results demonstrate that free energy simulations enable design of ligand efficacy and the same approach can be applied to other GPCR drug targets.

Structure-based pharmacophore modeling 2. Developing a novel framework for structure-based pharmacophore model generation and selection

Pharmacophore models are three-dimensional arrangements of molecular features required for biological activity that are used in ligand identification efforts for many biological targets, including G protein-coupled receptors (GPCR). Though GPCR are integral membrane proteins of considerable interest as targets for drug development, many of these receptors lack known ligands or experimentally determined structures necessary for ligand- or structure-based pharmacophore model generation, respectively. Thus, we here present a structure-based pharmacophore modeling approach that uses fragments placed with Multiple Copy Simultaneous Search (MCSS) to generate high-performing pharmacophore models in the context of experimentally determined, as well as modeled GPCR structures. Moreover, we have addressed the oft-neglected topic of pharmacophore model selection via development of a cluster-then-predict machine learning workflow. Herein score-based pharmacophore models were generated in experimentally determined and modeled structures of 13 class A GPCR and resulted in pharmacophore models exhibiting high enrichment factors when used to search a database containing 569 class A GPCR ligands. In addition, classification of pharmacophore models with the best performing cluster-then-predict logistic regression classifier resulted in positive predictive values (PPV) of 0.88 and 0.76 for selecting high enrichment pharmacophore models from among those generated in experimentally determined and modeled structures, respectively.

Constrained catecholamines gain β2AR selectivity through allosteric effects on pocket dynamics

G protein-coupled receptors (GPCRs) within the same subfamily often share high homology in their orthosteric pocket and therefore pose challenges to drug development. The amino acids that form the orthosteric binding pocket for epinephrine and norepinephrine in the β1 and β2 adrenergic receptors (β1AR and β2AR) are identical. Here, to examine the effect of conformational restriction on ligand binding kinetics, we synthesized a constrained form of epinephrine. Surprisingly, the constrained epinephrine exhibits over 100-fold selectivity for the β2AR over the β1AR. We provide evidence that the selectivity may be due to reduced ligand flexibility that enhances the association rate for the β2AR, as well as a less stable binding pocket for constrained epinephrine in the β1AR. The differences in the amino acid sequence of the extracellular vestibule of the β1AR allosterically alter the shape and stability of the binding pocket, resulting in a marked difference in affinity compared to the β2AR. These studies suggest that for receptors containing identical binding pocket residues, the binding selectivity may be influenced in an allosteric manner by surrounding residues, like those of the extracellular loops (ECLs) that form the vestibule. Exploiting these allosteric influences may facilitate the development of more subtype-selective ligands for GPCRs.

Abstract 2868: A chemogenetic approach reveals a GPCR-Gαs-PKA signaling axis promoting T cell dysfunction and cancer immunotherapy failure

Abstract Recent advances in immune checkpoint blockade (ICB) inhibiting programmed death-1 (PD-1) and cytotoxic T-lymphocyte-associated protein (CTLA-4) have revolutionized the standard of care for cancer treatment. However, the limited response rates to ICB across multiple cancer types suggest that new approaches and targets are clearly needed to fully elucidate the underlying biology of dysfunctional and exhausted CD8 T cells in cancer in order to achieve durable responses (cure). G protein-coupled receptors (GPCRs) are the most intensively studied drug targets, primarily due to their druggability and relevance to most physiological processes and disease conditions. However, the role of GPCRs in cancer and particularly in cancer immunology has been largely underexplored. We have applied a new computational pipeline to cross-integrate hundreds of thousands of CD8 T cells from multiple single cell RNA-seq datasets from 13 distinct cancer types. Using this approach, we identified many GPCRs that are overrepresented in exhausted CD8 T cells. A new computational strategy enabled us to predict the G protein coupling specificity of most GPCRs. This led to the discovery that Gαs-coupled GPCRs are significantly enriched in exhausted CD8 T cells. These include EP2, EP4, A2AR, β1AR, and β2AR, and we found that all of these receptors promote CD8 T cell dysfunction by inhibiting cytotoxicity and cytokine secretion. However, these receptors are also expressed in other immune cells within the tumor microenvironment. To address the direct impact of these receptors on T cell function we used a novel synthetic biology approach, involving the development of a chemogenetic CD8-restricted Gαs-DREADD (Designer Receptor Exclusively Activated by A Designer Drug) transgenic mouse model in which activation of Gαs signaling is temporally and spatially controlled. Utilizing this Gαs-DREADD model, we discovered that the Gαs-signaling axis represents a previously uncharacterized signaling axis that dampens the anti-tumor CD8 T cell activity and leads to ICB immunotherapy failure. Our central hypothesis is that secretion of GPCR ligands in the tumor microenvironment and their actions on CD8 T cells lead to the activation of the Gαs signaling cascade leading to T cell dysfunction, by decreasing cytotoxic and migratory activity that abolished the effectiveness of ICB. Our findings reveal that Gαs-coupled GPCRs may represent new targetable immune checkpoints that can be combined with ICB as part of novel multimodal precision approaches to enhance the response to immunotherapies. Citation Format: Bryan S. Yung, Victoria H. Wu, Farhoud Faraji, Robert Saddawi-Konefka, Zhiyong Wang, Alexander T. Wenzel, Miranda Song, Meghana S. Pagadala, Lauren M. Clubb, Joshua Chiou, Sanju Sinha, Marin Matic, Francesco Raimondi, Thomas Hoang, Rebecca Berdeaux, Dario A. Vignali, Ramiro Iglesias-Bartolome, Hannah Carter, Eytan Ruppin, Jill P. Mesirov, J. Silvio Gutkind. A chemogenetic approach reveals a GPCR-Gαs-PKA signaling axis promoting T cell dysfunction and cancer immunotherapy failure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2868.

Loss of biased signaling at a G protein-coupled receptor in overexpressed systems.

G protein-coupled receptors (GPCRs) regulate cellular signaling pathways by coupling to two classes of transducers: heterotrimeric G proteins and β-arrestins. [Sarcosine1Ile4Ile8]-angiotensin II (SII), an analog of the endogenous ligand angiotensin II (AngII) for the angiotensin II type 1 receptor (AT1R), fails to activate G protein in physiologically relevant models. Despite this, SII and several derivatives induce cellular signaling outcomes through β-arrestin-2-dependent mechanisms. However, studies reliant on exogenous AT1R overexpression indicate that SII is a partial agonist for G protein signaling and lacks β-arrestin-exclusive functional specificity. We investigated this apparent discrepancy by profiling changes in functional specificity at increasing expression levels of AT1R using a stably integrated tetracycline-titratable expression system stimulated with AngII, SII, and four other AngII analogs displaying different signaling biases. Unbiased and G protein-biased ligands activated dose-dependent calcium responses at all tested receptor concentrations. In contrast, β-arrestin-biased ligands induced dose-dependent calcium signaling only at higher AT1R overexpression levels. Using inhibitors of G proteins, we demonstrated that both Gi and Gq/11 mediated overexpression-dependent calcium signaling by β-arrestin-biased ligands. Regarding β-arrestin-mediated cellular events, the β-arrestin-biased ligand TRV026 induced receptor internalization at low physiological receptor levels insufficient for it to initiate calcium signaling. In contrast, unbiased AngII exhibited no relative preference between these outcomes under such low receptor conditions. However, with high receptor overexpression, TRV026 lost its functional selectivity. These results suggest receptor overexpression misleadingly distorts the bias of AT1R ligands and highlight the risks of using overexpressed systems to infer the signaling bias of GPCR ligands in physiologically relevant contexts.

Direct Selection of DNA-Encoded Libraries for Biased Agonists of GPCRs on Live Cells.

G protein-coupled receptors (GPCRs) are the largest superfamily of human membrane target proteins for approved drugs. GPCR ligands can have a complex array of pharmacological activities. Among these activities, biased agonists have potential to serve as both chemical probes to understand specific aspects of receptor signaling and therapeutic leads with more specific, desired activity. Challenges exist, however, in the development of new biased activators due, in part, to the low throughput of traditional screening approaches. DNA-encoded chemical libraries (DELs) dramatically improve the throughput of drug discovery by allowing a collective selection, rather than discrete screening, of large compound libraries. The use of DELs has been largely limited to affinity-based selections against purified protein targets, which identify binders only. Herein, we report a split protein complementation approach that allows direct identification of DNA-linked molecules that induce the dimerization of two proteins. We used this selection with a DEL against opioid receptor GPCRs on living cells for the identification of small molecules that possess the specific function of activation of either β-arrestin or G protein signaling pathways. This approach was applied to δ-, μ-, and κ-opioid receptors and enabled the discovery of compound [66,66], a selective, G-protein-biased agonist of the κ-opioid receptor (EC50 = 100 nM, E max = 82%, Gi bias factor = 6.6). This approach should be generally applicable for the direct selection of chemical inducers of dimerization from DELs and expand the utility of DELs to enrich molecules with a specific and desired biochemical function.

Single nucleotide variations encoding missense mutations in G protein-coupled receptors may contribute to autism.

Autism is a neurodevelopmental condition with a range of symptoms that vary in intensity and severity from person to person. Genetic sequencing has identified thousands of genes containing mutations in autistic individuals, which may contribute to the development of autistic symptoms. Several of these genes encode G protein-coupled receptors (GPCRs), which are cell surface expressed proteins that transduce extracellular messages to the intracellular space. Mutations in GPCRs can impact their function, resulting in aberrant signalling within cells and across neurotransmitter systems in the brain. This review summarises the current knowledge on autism-associated single nucleotide variations encoding missense mutations in GPCRs and the impact of these genetic mutations on GPCR function. For some autism-associated mutations, changes in GPCR expression levels, ligand affinity, potency and efficacy have been observed. However, for many the functional consequences remain unknown. Thus, further work to characterise the functional impacts of the genetically identified mutations is required.

Bioactive phytoconstituents and hemostatic and angiogenetic activities of Chromolaena odorata L. leaf extract gel on an animal epistaxis model

Introduction: Epistaxis occurs in approximately 60% of the general population globally and herbal medicine for its treatment including Chromolaena odorata L. could be one of the alternatives. The aims of this study were: (a) to determine the putative compounds and the bioactivities of C. odorata leaf extract; and (b) to assess their hemostatic and angiogenesis properties in an animal epistaxis model. Methods: The putative compounds of C. odorata extract were determined using gas chromatography and mass spectroscopy (GC-MS) and the bioactivities were determined using the Molinspiration Cheminformatics 2018 software. The hemostatic and angiogenesis activities were assessed using an epistaxis model of male rabbits (Oryctolagus cuniculus), on which three concentrations of C. odorata were tested. The gel was applied twice a day until 21 days. The hemostatic activity was evaluated by measuring the clotting time and the angiogenesis activity was evaluated by measuring the serum blood electrolyte, serum CD34 and CD68 levels and the histopathology of fibroblast and new blood vessels. Results: Eight putative compounds with activities that increased immune responses and angiogenesis by having antithrombotic, antioxidant and anti-inflammatory effects were identified. Those compounds had a range of bioactivity to G-protein coupled receptor ligand, ion channel modulator, kinase inhibitor, nuclear receptor ligand, protease inhibitor and enzyme inhibitor. Our data suggested that C. odorata extract had an effect on the levels of sodium, potassium and chloride. There was no significant difference in the mean levels of CD34 and CD68 among treatment and control groups, p=0.443 and p&gt;0.050, respectively. The extract had no significant effect in inducing the growth of fibroblasts. Our data indicated that C. odorata extract induced angiogenesis significantly (p=0.018). Conclusions: The C. odorata extract gel consisted of the compounds that contribute in antithrombotic, antioxidant and anti-inflammatory activities and these compounds increased the angiogenesis during wound healing in the epistaxis model.

Application of computational methods for class A GPCR Ligand discovery

G protein-coupled receptors (GPCR) are integral membrane proteins of considerable interest as targets for drug development due to their role in transmitting cellular signals in a multitude of biological processes. Of the six classes categorizing GPCR (A, B, C, D, E, and F), class A contains the largest number of therapeutically relevant GPCR. Despite their importance as drug targets, many challenges exist for the discovery of novel class A GPCR ligands serving as drug precursors. Though knowledge of the structural and functional characteristics of GPCR has grown significantly over the past 20 years, a large portion of GPCR lack reported, experimentally determined structures. Furthermore, many GPCR have no known endogenous and/or synthetic ligands, limiting further exploration of their biochemical, cellular, and physiological roles. While many successes in GPCR ligand discovery have resulted from experimental high-throughput screening, computational methods have played an increasingly important role in GPCR ligand identification in the past decade. Here we discuss computational techniques applied to GPCR ligand discovery. This review summarizes class A GPCR structure/function and provides an overview of many obstacles currently faced in GPCR ligand discovery. Furthermore, we discuss applications and recent successes of computational techniques used to predict GPCR structure as well as present a summary of ligand- and structure-based methods used to identify potential GPCR ligands. Finally, we discuss computational hit list generation and refinement and provide comprehensive workflows for GPCR ligand identification.