Tetrahydrocannabinol (THC) is the principal psychoactive compound derived from the cannabis plant Cannabis sativa and approved for emetic conditions, appetite stimulation and sleep apnea relief. THC's psychoactive actions are mediated primarily by the cannabinoid receptor CB. Here, we determine the cryo-EM structure of HU210, a THC analog and widely used tool compound, bound to CB and its primary transducer, G.
View Article and Find Full Text PDFUnlabelled: Peptide-based treatments represent an expanding area and require innovative approaches to enhance bioavailability. Combination with cell-penetrating peptides (CPPs) is an attractive strategy to improve non-invasive delivery across nasal epithelial barriers for systemic and direct nose-to-brain transport. We previously developed a modified CPP system termed Glycosaminoglycan-binding Enhanced Transduction (GET) that improves insulin delivery across gastrointestinal epithelium.
View Article and Find Full Text PDFThe endocannabinoid system (ECS) is a critical regulatory network composed of endogenous cannabinoids (eCBs), their synthesizing and degrading enzymes, and associated receptors. It is integral to maintaining homeostasis and orchestrating key functions within the central nervous and immune systems. Given its therapeutic significance, we have launched a series of drug discovery endeavors aimed at ECS targets, including peroxisome proliferator-activated receptors (PPARs), cannabinoid receptors types 1 (CB1R) and 2 (CB2R), and monoacylglycerol lipase (MAGL), addressing a wide array of medical needs.
View Article and Find Full Text PDFThere are two main families of G protein-coupled receptors that detect odours in humans, the odorant receptors (ORs) and the trace amine-associated receptors (TAARs). Their amino acid sequences are distinct, with the TAARs being most similar to the aminergic receptors such as those activated by adrenaline, serotonin, dopamine and histamine. To elucidate the structural determinants of ligand recognition by TAARs, we have determined the cryo-EM structure of a murine receptor, mTAAR7f, coupled to the heterotrimeric G protein G and bound to the odorant N,N-dimethylcyclohexylamine (DMCHA) to an overall resolution of 2.
View Article and Find Full Text PDFWe report a blueprint for the rational design of G protein coupled receptor (GPCR) ligands with a tailored functional response. The present study discloses the structure-based design of cannabinoid receptor type 2 (CBR) selective inverse agonists ()- and ()-, which were derived from privileged agonist HU-308 by introduction of a phenyl group at the -dimethylheptyl side chain. Epimer ()- exhibits high affinity for CBR with = 39.
View Article and Find Full Text PDFThe concept of agonist-independent signalling that can be attenuated by inverse agonists is a fundamental element of the cubic ternary complex model of G protein-coupled receptor (GPCR) activation. This model shows how a GPCR can exist in two conformational states in the absence of ligands; an inactive R state and an active R* state that differ in their affinities for agonists, inverse agonists, and G-protein alpha subunits. The proportion of R* receptors that exist in the absence of agonists determines the level of constitutive receptor activity.
View Article and Find Full Text PDFMeasurements of membrane protein thermostability reflect ligand binding. Current thermostability assays often require protein purification or rely on pre-existing radiolabelled or fluorescent ligands, limiting their application to established targets. Alternative methods, such as fluorescence-detection size exclusion chromatography thermal shift, detect protein aggregation but are not amenable to high-throughput screening.
View Article and Find Full Text PDFThe cannabinoid receptor (CBR) subtypes 1 (CBR) and 2 (CBR) are key components of the endocannabinoid system (ECS), playing a central role in the control of peripheral pain, inflammation and the immune response, with further roles in the endocrine regulation of food intake and energy balance. So far, few medicines targeting these receptors have reached the clinic, suggesting that a better understanding of the receptor signalling properties of existing tool compounds and clinical candidates may open the door to the development of more effective and safer treatments. Both CBR and CBR are Gα protein-coupled receptors but detecting Gα protein signalling activity reliably and reproducibly is challenging.
View Article and Find Full Text PDFPharmacological modulation of cannabinoid receptor type 2 (CBR) holds promise for the treatment of neuroinflammatory disorders, such as Alzheimer's disease. Despite the importance of CBR, its expression and downstream signaling are insufficiently understood in disease- and tissue-specific contexts. Herein, we report the first ligand-directed covalent (LDC) labeling of CBR enabled by a novel synthetic strategy and application of platform reagents.
View Article and Find Full Text PDFBackground And Aim: Standard pharmacological analysis of agonist activity utilises measurements of receptor-mediated responses at a set time-point, or at the peak response level, to characterise ligands. However, the occurrence of non-equilibrium conditions may dramatically impact the properties of the response being measured. Here we have analysed the initial kinetic phases of cAMP responses to β -adrenoceptor agonists in HEK293 cells expressing the endogenous β -adrenoceptor at extremely low levels.
View Article and Find Full Text PDFG protein-coupled receptors (GPCRs) are valuable therapeutic targets for many diseases. A central question of GPCR drug discovery is to understand what determines the agonism or antagonism of ligands that bind them. Ligands exert their action via the interactions in the ligand binding pocket.
View Article and Find Full Text PDFActivation of G protein-coupled receptors by agonists may result in the activation of one or more G proteins and recruitment of arrestins. The extent of the activation of each of these pathways depends on the intrinsic efficacy of the ligand. Quantification of intrinsic efficacy relative to a reference compound is essential for the development of novel compounds.
View Article and Find Full Text PDFIn this study, we report the β -adrenoceptor binding kinetics of several clinically relevant β -adrenoceptor (β AR) agonists and antagonists. [ H]-DHA was used to label CHO-β AR for binding studies. The kinetics of ligand binding was assessed using a competition association binding method.
View Article and Find Full Text PDFTwo-thirds of human hormones and one-third of clinical drugs activate ~350 G-protein-coupled receptors (GPCR) belonging to four classes: A, B1, C and F. Whereas a model of activation has been described for class A, very little is known about the activation of the other classes, which differ by being activated by endogenous ligands bound mainly or entirely extracellularly. Here we show that, although they use the same structural scaffold and share several 'helix macroswitches', the GPCR classes differ in their 'residue microswitch' positions and contacts.
View Article and Find Full Text PDFIn this work, we examine methyl nuclear magnetic resonance (NMR) spectra of the methionine ε-[CH] labelled thermostabilized β adrenergic receptor from turkey in association with a variety of different effectors, including mini-G and nanobody 60 (Nb60), which have not been previously studied in complex with β adrenergic receptor (βAR) by NMR. Complexes with pindolol and Nb60 induce highly similar inactive states of the receptor, closely resembling the resting state conformational ensemble. We show that, upon binding of mini-Gs or nanobody 80 (Nb80), large allosteric changes throughout the receptor take place.
View Article and Find Full Text PDFTo study the localisation of G protein-coupled receptors (GPCR) in their native cellular environment requires their visualisation through fluorescent labelling. To overcome the requirement for genetic modification of the receptor or the limitations of dissociable fluorescent ligands, here we describe rational design of a compound that covalently and selectively labels a GPCR in living cells with a fluorescent moiety. We designed a fluorescent antagonist, in which the linker incorporated between pharmacophore (ZM241385) and fluorophore (sulfo-cyanine5) is able to facilitate covalent linking of the fluorophore to the adenosine A receptor.
View Article and Find Full Text PDFPharmacological modulation of cannabinoid type 2 receptor (CBR) holds promise for the treatment of numerous conditions, including inflammatory diseases, autoimmune disorders, pain, and cancer. Despite the significance of this receptor, researchers lack reliable tools to address questions concerning the expression and complex mechanism of CBR signaling, especially in cell-type and tissue-dependent contexts. Herein, we report for the first time a versatile ligand platform for the modular design of a collection of highly specific CBR fluorescent probes, used successfully across applications, species, and cell types.
View Article and Find Full Text PDFG protein-coupled receptors (GPCRs) are versatile membrane proteins involved in the regulation of many physiological processes and pathological conditions, making them interesting pharmacological targets. In order to study their structure and function, GPCRs are traditionally extracted from membranes using detergents. However, due to their hydrophobic nature, intrinsic instability in aqueous solutions, and their denaturing effects, the isolation of properly folded and functional GPCRs is not trivial.
View Article and Find Full Text PDFSite-directed scanning mutagenesis is a useful tool applied in studying protein function and designing proteins with new properties, such as increased stability or enzymatic activity. Creating a systematic library of hundreds of site-directed mutants is still a demanding and expensive task. The established protocols for making such libraries include PCR amplification of the recombinant DNA using a pair of primers carrying a target mutation in the same PCR.
View Article and Find Full Text PDFCellular functions of arrestins are determined in part by the pattern of phosphorylation on the G protein-coupled receptors (GPCRs) to which arrestins bind. Despite high-resolution structural data of arrestins bound to phosphorylated receptor C-termini, the functional role of each phosphorylation site remains obscure. Here, we employ a library of synthetic phosphopeptide analogues of the GPCR rhodopsin C-terminus and determine the ability of these peptides to bind and activate arrestins using a variety of biochemical and biophysical methods.
View Article and Find Full Text PDFIn the absence of orthosteric ligands, most G protein-coupled receptors (GPCRs) exist in an equilibrium of different conformational states. This equilibrium is shifted by an agonist towards the active state or by an inverse agonist towards the inactive state. The basal activity of the receptor, and its ability to activate intracellular signaling pathways, is defined by the probability that a fraction of the receptor adopts the active state in the absence of ligand.
View Article and Find Full Text PDFThe various oligomeric states of the M2 isoform of pyruvate kinase (PKM2) were distinguished using native mass spectrometry. The effect of PKM2 concentration on its dimer-tetramer equilibrium was monitored, and a value for the dissociation constant ( K) of the two species was estimated to be 0.95 μM.
View Article and Find Full Text PDFSite-directed scanning mutagenesis is a powerful protein engineering technique which allows studies of protein functionality at single amino acid resolution and design of stabilized proteins for structural and biophysical work. However, creating libraries of hundreds of mutants remains a challenging, expensive and time-consuming process. The efficiency of the mutagenesis step is the key for fast and economical generation of such libraries.
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