Activation and Signaling Mechanism Revealed by Cannabinoid Receptor-Gi Complex Structures.

Human endocannabinoid systems modulate multiple physiological processes mainly through the activation of cannabinoid receptors CB1 and CB2. Their high sequence similarity, low agonist selectivity, and lack of activation and G protein-coupling knowledge have hindered the development of therapeutic applications. Importantly, missing structural information has significantly held back the development of promising CB2-selective agonist drugs for treating inflammatory and neuropathic pain without the psychoactivity of CB1. Here, we report the cryoelectron microscopy structures of synthetic cannabinoid-bound CB2 and CB1 in complex with Gi, as well as agonist-bound CB2 crystal structure. Of important scientific and therapeutic benefit, our results reveal a diverse activation and signaling mechanism, the structural basis of CB2-selective agonists design, and the unexpected interaction of cholesterol with CB1, suggestive of its endogenous allosteric modulating role.

Crystal Structure of the Human Cannabinoid Receptor CB2.

The cannabinoid receptor CB2 is predominately expressed in the immune system, and selective modulation of CB2 without the psychoactivity of CB1 has therapeutic potential in inflammatory, fibrotic, and neurodegenerative diseases. Here, we report the crystal structure of human CB2 in complex with a rationally designed antagonist, AM10257, at 2.8 Å resolution. The CB2-AM10257 structure reveals a distinctly different binding pose compared with CB1. However, the extracellular portion of the antagonist-bound CB2 shares a high degree of conformational similarity with the agonist-bound CB1, which led to the discovery of AM10257's unexpected opposing functional profile of CB2 antagonism versus CB1 agonism. Further structural analysis using mutagenesis studies and molecular docking revealed the molecular basis of their function and selectivity for CB2 and CB1. Additional analyses of our designed antagonist and agonist pairs provide important insight into the activation mechanism of CB2. The present findings should facilitate rational drug design toward precise modulation of the endocannabinoid system.

5-HT2C Receptor Structures Reveal the Structural Basis of GPCR Polypharmacology.

Drugs frequently require interactions with multiple targets-via a process known as polypharmacology-to achieve their therapeutic actions. Currently, drugs targeting several serotonin receptors, including the 5-HT2C receptor, are useful for treating obesity, drug abuse, and schizophrenia. The competing challenges of developing selective 5-HT2C receptor ligands or creating drugs with a defined polypharmacological profile, especially aimed at G protein-coupled receptors (GPCRs), remain extremely difficult. Here, we solved two structures of the 5-HT2C receptor in complex with the highly promiscuous agonist ergotamine and the 5-HT2A-C receptor-selective inverse agonist ritanserin at resolutions of 3.0 Å and 2.7 Å, respectively. We analyzed their respective binding poses to provide mechanistic insights into their receptor recognition and opposing pharmacological actions. This study investigates the structural basis of polypharmacology at canonical GPCRs and illustrates how understanding characteristic patterns of ligand-receptor interaction and activation may ultimately facilitate drug design at multiple GPCRs.

Crystal Structure of the Human Cannabinoid Receptor CB1.

Cannabinoid receptor 1 (CB1) is the principal target of Δ9-tetrahydrocannabinol (THC), a psychoactive chemical from Cannabis sativa with a wide range of therapeutic applications and a long history of recreational use. CB1 is activated by endocannabinoids and is a promising therapeutic target for pain management, inflammation, obesity, and substance abuse disorders. Here, we present the 2.8 Å crystal structure of human CB1 in complex with AM6538, a stabilizing antagonist, synthesized and characterized for this structural study. The structure of the CB1-AM6538 complex reveals key features of the receptor and critical interactions for antagonist binding. In combination with functional studies and molecular modeling, the structure provides insight into the binding mode of naturally occurring CB1 ligands, such as THC, and synthetic cannabinoids. This enhances our understanding of the molecular basis for the physiological functions of CB1 and provides new opportunities for the design of next-generation CB1-targeting pharmaceuticals.

Bitter taste TAS2R14 activation by intracellular tastants and cholesterol.

Bitter taste receptors, particularly TAS2R14, play central roles in discerning a wide array of bitter substances, ranging from dietary components to pharmaceutical agents1,2. TAS2R14 is also widely expressed in extragustatory tissues, suggesting its extra roles in diverse physiological processes and potential therapeutic applications3. Here we present cryogenic electron microscopy structures of TAS2R14 in complex with aristolochic acid, flufenamic acid and compound 28.1, coupling with different G-protein subtypes. Uniquely, a cholesterol molecule is observed occupying what is typically an orthosteric site in class A G-protein-coupled receptors. The three potent agonists bind, individually, to the intracellular pockets, suggesting a distinct activation mechanism for this receptor. Comprehensive structural analysis, combined with mutagenesis and molecular dynamic simulation studies, elucidate the broad-spectrum ligand recognition and activation of the receptor by means of intricate multiple ligand-binding sites. Our study also uncovers the specific coupling modes of TAS2R14 with gustducin and Gi1 proteins. These findings should be instrumental in advancing knowledge of bitter taste perception and its broader implications in sensory biology and drug discovery.

Structural basis of ligand recognition and self-activation of orphan GPR52.

GPR52 is a class-A orphan G-protein-coupled receptor that is highly expressed in the brain and represents a promising therapeutic target for the treatment of Huntington's disease and several psychiatric disorders1,2. Pathological malfunction of GPR52 signalling occurs primarily through the heterotrimeric Gs protein2, but it is unclear how GPR52 and Gs couple for signal transduction and whether a native ligand or other activating input is required. Here we present the high-resolution structures of human GPR52 in three states: a ligand-free state, a Gs-coupled self-activation state and a potential allosteric ligand-bound state. Together, our structures reveal that extracellular loop 2 occupies the orthosteric binding pocket and operates as a built-in agonist, conferring an intrinsically high level of basal activity to GPR523. A fully active state is achieved when Gs is coupled to GPR52 in the absence of an external agonist. The receptor also features a side pocket for ligand binding. These insights into the structure and function of GPR52 could improve our understanding of other self-activated GPCRs, enable the identification of endogenous and tool ligands, and guide drug discovery efforts that target GPR52.

Crystal structure of the Frizzled 4 receptor in a ligand-free state.

Frizzled receptors (FZDs) are class-F G-protein-coupled receptors (GPCRs) that function in Wnt signalling and are essential for developing and adult organisms1,2. As central mediators in this complex signalling pathway, FZDs serve as gatekeeping proteins both for drug intervention and for the development of probes in basic and in therapeutic research. Here we present an atomic-resolution structure of the human Frizzled 4 receptor (FZD4) transmembrane domain in the absence of a bound ligand. The structure reveals an unusual transmembrane architecture in which helix VI is short and tightly packed, and is distinct from all other GPCR structures reported so far. Within this unique transmembrane fold is an extremely narrow and highly hydrophilic pocket that is not amenable to the binding of traditional GPCR ligands. We show that such a pocket is conserved across all FZDs, which may explain the long-standing difficulties in the development of ligands for these receptors. Molecular dynamics simulations on the microsecond timescale and mutational analysis uncovered two coupled, dynamic kinks located at helix VII that are involved in FZD4 activation. The stability of the structure in its ligand-free form, an unfavourable pocket for ligand binding and the two unusual kinks on helix VII suggest that FZDs may have evolved a novel ligand-recognition and activation mechanism that is distinct from that of other GPCRs.

Study on the Design, Synthesis, Bioactivity and Translocation of the Conjugates of Phenazine-1-carboxylic Acid and N-Phenyl Alanine Ester.

The natural pesticide phenazine-1-carboxylic acid (PCA) is known to lack phloem mobility, whereas Metalaxyl is a representative phloem systemic fungicide. In order to endow PCA with phloem mobility and also enhance its antifungal activity, thirty-two phenazine-1-carboxylic acid-N-phenylalanine esters conjugates were designed and synthesized by conjugating PCA with the active structure N-acylalanine methyl ester of Metalaxyl. All target compounds were characterized by 1H NMR, 13C NMR and HRMS. The antifungal evaluation results revealed that several target compounds exhibited moderate to potent antifungal activities against Sclerotinia sclerotiorum, Bipolaris sorokiniana, Phytophthora parasitica, Phytophthora citrophthora. In particular, compound F7 displayed excellent antifungal activity against S. sclerotiorum with an EC50 value of 6.57 µg/mL, which was superior to that of Metalaxyl. Phloem mobility study in castor bean system indicated good phloem mobility for the target compounds F1-F16. Particularly, compound F2 exhibited excellent phloem mobility; the content of compound F2 in the phloem sap of castor bean was 19.12 µmol/L, which was six times higher than Metalaxyl (3.56 µmol/L). The phloem mobility tests under different pH culture solutions verified the phloem translocation of compounds related to the "ion trap" effect. The distribution of the compound F2 in tobacco plants further suggested its ambimobility in the phloem, exhibiting directional accumulation towards the apical growth point and the root. These results provide valuable insights for developing phloem mobility fungicides mediated by exogenous compounds.

Design, Synthesis, Antibacterial, and Antifungal Evaluation of Phenylthiazole Derivatives Containing a 1,3,4-Thiadiazole Thione Moiety.

To effectively control the infection of plant pathogens, we designed and synthesized a series of phenylthiazole derivatives containing a 1,3,4-thiadiazole thione moiety and screened for their antibacterial potencies against Ralstonia solanacearum, Xanthomonas oryzae pv. oryzae, as well as their antifungal potencies against Sclerotinia sclerotiorum, Rhizoctonia solani, Magnaporthe oryzae and Colletotrichum gloeosporioides. The chemical structures of the target compounds were characterized by 1H NMR, 13C NMR and HRMS. The bioassay results revealed that all the tested compounds exhibited moderate-to-excellent antibacterial and antifungal activities against six plant pathogens. Especially, compound 5k possessed the most remarkable antibacterial activity against R. solanacearum (EC50 = 2.23 µg/mL), which was significantly superior to that of compound E1 (EC50 = 69.87 µg/mL) and the commercial agent Thiodiazole copper (EC50 = 52.01 µg/mL). Meanwhile, compound 5b displayed the most excellent antifungal activity against S. sclerotiorum (EC50 = 0.51 µg/mL), which was equivalent to that of the commercial fungicide Carbendazim (EC50 = 0.57 µg/mL). The preliminary structure-activity relationship (SAR) results suggested that introducing an electron-withdrawing group at the meta-position and ortho-position of the benzene ring could endow the final structure with remarkable antibacterial and antifungal activity, respectively. The current results indicated that these compounds were capable of serving as promising lead compounds.

Human GLP-1 receptor transmembrane domain structure in complex with allosteric modulators.

The glucagon-like peptide-1 receptor (GLP-1R) and the glucagon receptor (GCGR) are members of the secretin-like class B family of G-protein-coupled receptors (GPCRs) and have opposing physiological roles in insulin release and glucose homeostasis. The treatment of type 2 diabetes requires positive modulation of GLP-1R to inhibit glucagon secretion and stimulate insulin secretion in a glucose-dependent manner. Here we report crystal structures of the human GLP-1R transmembrane domain in complex with two different negative allosteric modulators, PF-06372222 and NNC0640, at 2.7 and 3.0 Å resolution, respectively. The structures reveal a common binding pocket for negative allosteric modulators, present in both GLP-1R and GCGR and located outside helices V-VII near the intracellular half of the receptor. The receptor is in an inactive conformation with compounds that restrict movement of the intracellular tip of helix VI, a movement that is generally associated with activation mechanisms in class A GPCRs. Molecular modelling and mutagenesis studies indicate that agonist positive allosteric modulators target the same general region, but in a distinct sub-pocket at the interface between helices V and VI, which may facilitate the formation of an intracellular binding site that enhances G-protein coupling.

Crystal structures of agonist-bound human cannabinoid receptor CB1.

The cannabinoid receptor 1 (CB1) is the principal target of the psychoactive constituent of marijuana, the partial agonist Δ9-tetrahydrocannabinol (Δ9-THC). Here we report two agonist-bound crystal structures of human CB1 in complex with a tetrahydrocannabinol (AM11542) and a hexahydrocannabinol (AM841) at 2.80 Å and 2.95 Å resolution, respectively. The two CB1-agonist complexes reveal important conformational changes in the overall structure, relative to the antagonist-bound state, including a 53% reduction in the volume of the ligand-binding pocket and an increase in the surface area of the G-protein-binding region. In addition, a 'twin toggle switch' of Phe2003.36 and Trp3566.48 (superscripts denote Ballesteros-Weinstein numbering) is experimentally observed and appears to be essential for receptor activation. The structures reveal important insights into the activation mechanism of CB1 and provide a molecular basis for predicting the binding modes of Δ9-THC, and endogenous and synthetic cannabinoids. The plasticity of the binding pocket of CB1 seems to be a common feature among certain class A G-protein-coupled receptors. These findings should inspire the design of chemically diverse ligands with distinct pharmacological properties.

Preventive effects of arctigenin from Arctium lappa L against LPS-induced neuroinflammation and cognitive impairments in mice.

Arctigenin (Arc) is a phenylpropanoid dibenzylbutyrolactone lignan in Arctium lappa L, which has been widely applied as a traditional Chinese herbal medicine for treating inflammation. In the present study, we explored the neuroprotective effect and the potential mechanisms of arctigenin against LPS-evoked neuroinflammation, neurodegeneration, and memory impairments in the mice hippocampus. Daily administration of arctigenin (50 mg/kg per day, i.g.) for 28 days revealed noticeable improvements in spatial learning and memory deficits after exposure to LPS treatment. Arctigenin prevented LPS-induced neuronal/synaptic injury and inhibited the increases in Abeta (Aß) generation and the levels of amyloid precursor protein (APP) and ß-site amyloid precursor protein cleavage enzyme 1 (BACE1). Moreover, arctigenin treatment also suppressed glial activation and reduced the production of proinflammatory cytokines. In LPS-treated BV-2 microglial cells and mice, activation of the TLR4 mediated NF-κB signaling pathway was significantly suppressed by arctigenin administration. Mechanistically, arctigenin reduced the LPS-induced interaction of adiponectin receptor 1 (AdipoR1) with TLR4 and its coreceptor CD14 and inhibited the TLR4-mediated downstream inflammatory response. The outcomes of the current study indicate that arctigenin mitigates LPS-induced apoptotic neurodegeneration, amyloidogenesis and neuroinflammation as well as cognitive impairments, and suggest that arctigenin may be a potential therapeutic candidate for neuroinflammation/neurodegeneration-related diseases.

Lysophosphatidic Acid Induced Apoptosis, DNA Damage, and Oxidative Stress in Spinal Cord Neurons by Upregulating LPA4/LPA6 Receptors.

Lysophosphatidic acid (LPA) has disruptive effects on lumbar spinal stenosis (LSS). Recently, LPA has been reported to be involved in spinal cord neuronal injury and toxicity, promoting the pathogenesis of LSS. However, the exact effects of LPA on spinal cord neurons remain unknown. The purpose of this study is to investigate the effects of LPA (18 : 1) on spinal cord neuronal cytotoxicity, apoptosis, DNA damage, and oxidative stress. After clinical detection of LPA secretion, spinal cord neurons were treated with LPA (18 : 1); cell viability was analyzed by MTT assay, and LDH leakage was detected by LDH kit; cell apoptosis was detected by flow cytometry; ROS production was measured by DCFDA staining and MitoSOX Red Staining; the activation of the Gα12/Gα13 signaling pathway was detected by serum response factor response element (SRF-RE) luciferase reporter gene; the relationship among LPA, LPA4/6, and ROCK was examined by western blotting. In spinal cord neurons treated with LPA (18 : 1), cellular activity decreased and LDH release increased. The Rho kinase inhibitor (Y-27632) can attenuate LPA-induced apoptosis, DNA damage, and oxidative stress in spinal cord neurons. Moreover mechanistic investigation indicated that LPA (18 : 1) activates Gα12/13-Rho-ROCK2-induced apoptosis, DNA damage, and oxidative stress in spinal cord neurons by upregulating LPA4/LPA6 receptors. Further, the Rho kinase inhibitor Y-27632 attenuates the effects of LPA by downregulating LPA4/LPA6 receptors. Taken together, the possible mechanism by which LPA secretion in LSS patients aggravates patient injury was further elucidated using an LPA-induced spinal cord neuronal injury cell model in vitro.

Sensitive and Facile HCOOH Fluorescence Sensor Based on Highly Active Ir Complexes' Catalytic Transfer Hydrogen Reaction.

With several major polarity and weak optical properties, the sensitive detection of HCOOH remains a major challenge. Given the special role of HCOOH in assisting in the catalytic hydrogenation process of Ir complexes, HCOOH (as a hydrogen source) could rapidly activate Ir complexes as catalysts and further reduce the substrates. This work developed a facile and sensitive HCOOH fluorescence sensor utilizing an optimal catalytic fluorescence generation system, which consists of the phenyl-pyrazole-type Ir-complex PP-Ir-Cl and the coumarin-type fluorescence probe P-coumarin. The sensor demonstrates excellent sensitivity and specificity for HCOOH and formates; the limits of detection for HCOOH, HCOONa, and HCOOEt3N were tested to be 50.6 ppb, 68.0 ppb, and 146.0 ppb, respectively. Compared to previous methods, the proposed sensor exhibits good detection accuracy and excellent sensitivity. Therefore, the proposed HCOOH sensor could be used as a new detection method for HCOOH and could provide a new design path for other sensors.

Research progress in fluid and semifluid microwave heating technology in food processing.

Microwave is a form of electromagnetic radiation that has high penetration and heating efficiency in food processing. Uneven heating is the main problem of microwave processing, especially in solid foods. Fluid and semifluid media, which are good carriers in microwave processing, have uniform dielectric properties and good material fluidity. Herein, we review the development, application prospects, and limitations of microwave in fluid and semifluid food processing and the research progress in microwave heating with steam as carrier. The mixture of generated steam and tiny micro droplets from food material under the action of microwave can absorb microwave and transfer heat evenly, which effectively improves the uniformity of microwave heating. Due to the relatively uniform dielectric properties and consistent texture of fluid and semifluid food materials, uneven heating phenomenon during their microwave processing can be significantly inhibited. Based on the development of microwave heating technology and equipment design, the microbial inactivation and enzyme inhibition in fluid and semifluid food were improved and food product with better retention of nutrients and sensory profile were produced. Also, microwave radiation can be used to prepare the printing material or process the printed product for 3D food printing, which enhances the added value of 3D printed products and the personalization of food manufacturing. In future research, intelligent control technology can be applied in the microwave processing of fluid and semifluid food materials for various applications. Therefore, the processing conditions can be adjusted automatically.

Uniformity improvement on received optical power for an indoor visible light communication system with an angle diversity receiver.

In this work, an angle diversity receiver (ADR) structure is proposed to optimize the uniformity of the received optical power distribution in an indoor visible light communication (VLC) system. Taking the rectangular and hybrid layouts with 16 light-emitting diodes as examples, different inclination angles and the number of side detectors are investigated with three diversity combining techniques in a typical room, where the primary reflection of the wall is considered. Simulation results showed that the inclination angles and the number of side detectors would affect the variance and average of the received optical power, and the variance would decrease with the increase of the number of side detectors. In addition, maximal ratio combining is more suitable for the ADR when the variance and average of the received optical power are considered simultaneously. By applying the ADR with five side detectors, the variances of the received optical power will decrease by 81.34% and 86.09% under the rectangle layout and the hybrid layout, respectively. This work will benefit the design and development of the VLC system.

Probing the CB1 Cannabinoid Receptor Binding Pocket with AM6538, a High-Affinity Irreversible Antagonist.

Cannabinoid receptor 1 (CB1) is a potential therapeutic target for the treatment of pain, obesity and obesity-related metabolic disorders, and addiction. The crystal structure of human CB1 has been determined in complex with the stabilizing antagonist AM6538. In the present study, we characterize AM6538 as a tight-binding/irreversible antagonist of CB1, as well as two derivatives of AM6538 (AM4112 and AM6542) as slowly dissociating CB1 antagonists across binding simulations and cellular signaling assays. The long-lasting nature of AM6538 was explored in vivo wherein AM6538 continues to block CP55,940-mediated behaviors in mice up to 5 days after a single injection. In contrast, the effects of SR141716A abate in mice 2 days after injection. These studies demonstrate the functional outcome of CB1 antagonist modification and open the path for development of long-lasting CB1 antagonists.

The structure-based traceless specific fluorescence labeling of the smoothened receptor.

The smoothened receptor (SMO) mediates the hedgehog (Hh) signaling pathway and plays a vital role in embryonic development and tumorigenesis. The visualization of SMO has the potential to provide new insights into its enigmatic mechanisms and associated disease pathogenesis. Based on recent progress in structural studies of SMO, we have designed and characterized a group of affinity probes to facilitate the turn-on fluorescence labeling of SMO at the ε-amine of K395. These chemical probes were derived from a potent SMO antagonist skeleton by the conjugation of a small non-fluorescent unit, O-nitrobenzoxadiazole (O-NBD). In this context, optimal probes were developed to be capable of efficiently and selectively lighting up SMO regardless of whether it is in micelles or in native membranes. More importantly, the resulting labeled SMO only bears a very small fluorophore and allows for the recovery of the unoccupied pocket by dissociation of the residual ligand module. These advantages should allow the probe to serve as a potential tool for monitoring SMO trafficking, understanding Hh activation mechanisms, and even the diagnosis of tumorigenesis in the future.

Improving long-term stability of retinol in dried blood spots and quantification of its levels via a novel LC-MS/MS method.

Vitamin A deficiency (VAD) is a major micronutrient deficiency in children. Although plasma and serum retinol levels are proposed as the key indicators of VAD, collecting and transporting plasma and serum are difficult and inconvenient in field studies. Dried blood spot (DBS) retinol has been used as an alternative to plasma retinol in several epidemiological and clinical studies. A limitation of methods that use DBS retinol is the instability and apparent loss of retinol in DBSs. Therefore, an accurate, reliable method for stabilizing retinol in DBSs and quantifying and comparing DBS retinol concentrations with equivalent plasma retinol levels is required. In this study, antioxidants on paper combined with vacuum treatment were found to greatly increase the stability of DBS retinol during 120 min of air drying and 30 days of room-temperature storage. A surrogate matrix of whole blood prepared using a mixture of human erythrocytes and 2% BSA in PBS was firstly used in DBS retinol determination based on the fact that retinol is excluded from erythrocytes. The method was linear in the concentration range of 0.04-300 µg/mL. Both the between-run (n = 5) and within-run (n = 6) precision (relative standard deviations, RSD%) were below 8.42%. The spiked recoveries at 3 concentrations ranged from 86.48 to 98.13%. The internal standard (IS)-normalized matrix factor (MF) was 99.72% with a RSD% of 10.50% (n = 3). The accuracy was calibrated using two National Institute of Standards and Technology (NIST) serum-generated calibrants at concentrations of 0.1962 and 0.3948 g/mL, and relative errors (RE% values) of 0.07% and 4.95% were found, respectively. A simple calibration model was first developed to convert DBS retinol concentration to the equivalent plasma retinol concentration, thereby enabling comparisons with clinical reference ranges and with studies using serum or plasma samples. Graphical abstract.

GPCR Allosteric Modulator Discovery.

G protein-coupled receptors (GPCRs) influence virtually every aspect of human physiology; about one-third of all marketed drugs target members of this family. GPCR allosteric ligands hold the promise of improved subtype selectivity, spatiotemporal sensitivity, and possible biased property over typical orthosteric ligands. However, only a small number of GPCR allosteric ligands have been approved as drugs or in clinical trials since the discovery process is very challenging. The rapid development of GPCR structural biology leads to the discovery of several allosteric sites and sheds light on understanding the mechanism of GPCR allosteric ligands, which is critical for discovering novel therapeutics. This book chapter summarized different GPCR allosteric modulating mechanisms and discussed validated mechanisms based on allosteric modulator-GPCR complex structures.