Water molecules in the cannabinoid receptor 2 binding site crucially impact the discovery of novel ligands.

The cannabinoid receptor 2 (CBR) is of considerable therapeutic and scientific interest. Hence, the discovery of novel molecules that target and modulate this receptor, ideally selectively over its closest relative, the cannabinoid receptor 1, is of great importance. In this study, we aimed to discover novel ligands targeting the CBR using large library in silico docking screens.

Biased Signaling and Its Role in the Genesis of Short- and Long-Acting β-Adrenoceptor Agonists.

Drug discovery is a costly and time-intensive process that is often limited by efficacy issues and unforeseen side effects. GPCR-targeting ligands, which account for one-third of marketed drugs, have been shown to exhibit biased signaling and preferential activation of one signaling pathway over another. While designing biased ligands is a recent advancement, their therapeutic benefits remain uncertain.

Multiple intramolecular triggers converge to preferential G protein coupling in the CBR.

G protein-coupled receptors (GPCRs) are important therapeutic drug targets for a wide range of diseases. Upon activation, GPCRs can initiate several signaling pathways, each with unique therapeutic implications. Therefore, understanding how drugs selectively engage specific signaling pathways becomes paramount.

A universal cannabinoid CB1 and CB2 receptor TR-FRET kinetic ligand-binding assay.

Introduction: The kinetics of ligand binding to G protein-coupled receptors (GPCRs) is an important optimization parameter in drug discovery. Traditional radioligand assays are labor-intensive, preventing their application at the early stages of drug discovery. Fluorescence-based assays offer several advantages, including a possibility to develop a homogeneous format, continuous data collection, and higher throughput.

Agonist efficacy at the βAR is driven by the faster association rate of the G protein.

Introduction: The β-adrenoceptor (βAR) is a class A G protein-coupled receptor (GPCR). It is therapeutically relevant in asthma and chronic obstructive pulmonary disease (COPD), where βAR agonists relieve bronchoconstriction. The βAR is a prototypical GPCR for structural and biophysical studies.

Profiling Allosteric Modulators of CBR with an Allosteric Fluoroprobe.

Allosteric modulation of cannabinoid receptor type 1 (CBR) offers a promising alternative to conventional therapeutic approaches using orthosteric ligands (OLs). Currently, CBR allosteric modulators (AMs) are characterized based on their ability to modulate binding or functional response of OLs, preventing isolation of individual contributions by allosteric and orthosteric ligands. Herein, we develop the first allosteric fluoroprobe and attendant FRET-based assay allowing for the direct profiling of CBR AMs without coincubation with an OL.

Structural basis of THC analog activity at the Cannabinoid 1 receptor.

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.

A Modified Cell-Penetrating Peptide Enhances Insulin and Oxytocin Delivery across an RPMI 2650 Nasal Epithelial Cell Barrier In Vitro.

Unlabelled: 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.

Enhancing Drug Discovery and Development through the Integration of Medicinal Chemistry, Chemical Biology, and Academia-Industry Partnerships: Insights from Roche's Endocannabinoid System Projects.

The 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.

Molecular recognition of an odorant by the murine trace amine-associated receptor TAAR7f.

There 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.

Flipping the GPCR Switch: Structure-Based Development of Selective Cannabinoid Receptor 2 Inverse Agonists.

We 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.

Mechano-sensitivity of β2-adrenoceptors enhances constitutive activation of cAMP generation that is inhibited by inverse agonists.

The 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.

ThermoBRET: A Ligand-Engagement Nanoscale Thermostability Assay Applied to GPCRs.

Measurements 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.

Development of a membrane-based Gi-CASE biosensor assay for profiling compounds at cannabinoid receptors.

The 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.

Platform Reagents Enable Synthesis of Ligand-Directed Covalent Probes: Study of Cannabinoid Receptor 2 in Live Cells.

Pharmacological 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.

Kinetic analysis of endogenous β -adrenoceptor-mediated cAMP GloSensor™ responses in HEK293 cells.

Background 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.

Combined docking and machine learning identify key molecular determinants of ligand pharmacological activity on β2 adrenoceptor.

G 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.

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

Activation of G protein-coupled receptors by agonists may result in the activation of one or more G proteins and recruitment of arrestins. The extent of the activation of each of these pathways depends on the intrinsic efficacy of the ligand. Quantification of intrinsic efficacy relative to a reference compound is essential for the development of novel compounds.

Exploring the kinetic selectivity of drugs targeting the β -adrenoceptor.

In 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.

GPCR activation mechanisms across classes and macro/microscales.

Two-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.

GPCR Activation States Induced by Nanobodies and Mini-G Proteins Compared by NMR Spectroscopy.

In 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.

Ligand-directed covalent labelling of a GPCR with a fluorescent tag in live cells.

To 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.

Development of High-Specificity Fluorescent Probes to Enable Cannabinoid Type 2 Receptor Studies in Living Cells.

Pharmacological 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.