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The diversity of physiological roles of the endocannabinoid system has turned it into an attractive yet elusive therapeutic target. However, chemical probes with various functionalities could pave the way for a better understanding of the endocannabinoid system at the cellular level. Notably, inverse agonists of CB 2 R - a key receptor of the endocannabinoid system - lagged behind despite the evidence regarding the therapeutic potential of its antagonism. Herein, we report a matched fluorescent probe pair based on a common chemotype to address and visualize both the active and inactive states of CB 2 R, selectively. Alongside extensive cross-validation by flow cytometry, time-lapse confocal microscopy, and super-resolution microscopy, we successfully visualize the intracellular localization of CB 2 R pools in live cells. The synthetic simplicity, together with the high CB 2 R-selectivity and specificity of our probes, turns them into valuable tools in chemical biology and drug development that can benefit the clinical translatability of CB 2 R-based drugs., Competing Interests: The authors declare no competing financial interest., (This journal is © The Royal Society of Chemistry.)

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. The pursuit of new therapeutic agents has been enhanced by the creation of specialized labeled chemical probes, which aid in target localization, mechanistic studies, assay development, and the establishment of biomarkers for target engagement. By fusing medicinal chemistry with chemical biology in a comprehensive, translational end-to-end drug discovery strategy, we have expedited the development of novel therapeutics. Additionally, this strategy promises to foster highly productive partnerships between industry and academia, as will be illustrated through various examples., (Copyright 2024 Johannes Aebi, Kenneth Atz, Simon M. Ametamey, Jörg Benz, Julie Blaising, Stefania Butini, Giuseppe Campiani, Erick M. Carreira, Ludovic Collin, Eva de Lago, Thais Gazzi, Jürg Gertsch, Luca Gobbi, Wolfgang Guba, Javier Fernández-Ruiz, Jürgen Fingerle, Ahmed Haider, Yingfang He, Laura H. Heitman, Michael Honer, Daniel Hunziker, Bernd Kuhn, Mauro Maccarrone, Hans Peter Märki, Rainer E. Martin, Peter Mohr, Linjing Mu, Marc Nazaré, David F. Nippa, Sergio Oddi, Fionn O’Hara, Pal Pacher, Julian Romero, Stephan Röver, Arne C. Rufer, Roger Schibli, Gisbert Schneider, Antonia F. Stepan, David A. Sykes, Christoph Ullmer, Mario van der Stelt, Dmitry B. Veprintsev, Matthias B. Wittwer, Uwe Grether. License: This work is licensed under a Creative Commons Attribution 4.0 International License.)

Introduction: Preclinical studies suggest that cannabinoid receptor type 2 (CB2R) activation has a therapeutic effect in animal models on chronic inflammation and vascular permeability, which are key pathological features of diabetic retinopathy (DR). A novel CB2R agonist, triazolopyrimidine RG7774, was generated through lead optimization of a high-throughput screening hit. The aim of this study was to characterize the pharmacology, absorption, distribution, metabolism, elimination, and toxicity (ADMET) profile of RG7774, and to explore its potential for managing the key pathological features associated with retinal disease in rodents., Methods: The in vitro pharmacology of RG7774 was investigated for CB2R binding and receptor activation using recombinant human and mouse CB2R expression in Chinese hamster ovary cells, and endogenous CB2R expression in human Jurkat cells, and rat and mouse spleen cells. The ADMET profile was evaluated and the effects of RG7774 on retinal permeability, leukocyte adhesion, and choroidal neovascularization (CNV) were investigated in rodent models of retinal disease. Pharmacokinetic (PK) parameters and the exposure-response relationship were characterized in healthy animals and in animals with laser-induced CNV., Results: RG7774 was found to be a potent (EC 50 : 2.8 nM and K i : 51.3 nM), selective, and full CB2R agonist with no signs of cannabinoid receptor type 1 (CB1R) binding or activation. The ligand showed a favorable ADMET profile and exhibited systemic and ocular exposure after oral delivery. Functional potency in vitro translated from recombinant to endogenous expression systems. In vivo , orally administered RG7774 reduced retinal permeability and leukocyte adhesion in rodents with lipopolysaccharide (LPS)-induced uveitis and streptozotocin (STZ)-induced DR, and reduced lesion areas in rats with laser-induced CNV with an ED 50 of 0.32 mg/kg. Anatomically, RG7774 reduced the migration of retinal microglia to retinal lesions., Discussion: RG7774 is a novel, highly selective, and orally bioavailable CB2R agonist, with an acceptable systemic and ocular PK profile, and beneficial effects on retinal vascular permeability, leukocyte adhesion, and ocular inflammation in rodent animal models. Results support the development of RG7774 as a potential treatment for retinal diseases with similar pathophysiologies as addressed by the animal models., Competing Interests: UG, RF, SG, CK, AO, EZ, SU, MN, RM, CB, WG, AA, FR, AP, CP, AC, SF, CU: Employee: F. Hoffmann-La Roche Ltd. AK: Employee: Chugai Pharmaceuticals Co. Ltd. JT, NC, MR-E, SR, J-MA, JF: Employee: F. Hoffmann-La Roche Ltd., at the time of writing. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Grether, Foxton, Gruener, Korn, Kimbara, Osterwald, Zirwes, Uhles, Thoele, Colé, Rogers-Evans, Röver, Nettekoven, Martin, Adam, Fingerle, Bissantz, Guba, Alker, Szczesniak, Porter, Toguri, Revelant, Poirier, Perret, Winther, Caruso, Fezza, Maccarrone, Kelly, Fauser and Ullmer.)

Background: The elevation of endocannabinoid levels through inhibiting their degradation afforded neuroprotection in CaMKIIα-TDP-43 mice, a conditional transgenic model of frontotemporal dementia. However, which cannabinoid receptors are mediating these benefits is still pending to be elucidated., Methods: We have investigated the involvement of the CB 1 and the CB 2 receptor using chronic treatments with selective ligands in CaMKIIα-TDP-43 mice, analysis of their cognitive deterioration with the Novel Object Recognition test, and immunostaining for neuronal and glial markers in two areas of interest in frontotemporal dementia., Results: Our results confirmed the therapeutic value of activating either the CB 1 or the CB 2 receptor, with improvements in the animal performance in the Novel Object Recognition test, preservation of pyramidal neurons, in particular in the medial prefrontal cortex, and attenuation of glial reactivity, in particular in the hippocampus. In addition, the activation of both CB 1 and CB 2 receptors reduced the elevated levels of TDP-43 in the medial prefrontal cortex of CaMKIIα-TDP-43 mice, an effect exerted by mechanisms that are currently under investigation., Conclusions: These data reinforce the notion that the activation of CB 1 and CB 2 receptors may represent a promising therapy against TDP-43-induced neuropathology in frontotemporal dementia. Future studies will have to confirm these benefits, in particular with one of the selective CB 2 agonists used here, which has been thoroughly characterized for clinical development., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: CGC, ISG, LGT, CRC, JFR and EdL declare that they have no conflicts of interest, whereas MBW, PD and UG are employees of the company F. Hoffmann-La Roche Ltd., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The cannabinoid CB2 receptor (CB2R) is a potential therapeutic target for distinct forms of tissue injury and inflammatory diseases. To thoroughly investigate the role of CB2R in pathophysiological conditions and for target validation in vivo , optimal pharmacological tool compounds are essential. Despite the sizable progress in the generation of potent and selective CB2R ligands, pharmacokinetic parameters are often neglected for in vivo studies. Here, we report the generation and characterization of a tetra-substituted pyrazole CB2R full agonist named RNB-61 with high potency ( K i 0.13-1.81 nM, depending on species) and a peripherally restricted action due to P-glycoprotein mediated efflux from the brain. 3 H and 14 C labelled RNB-61 showed apparent K d values < 4 nM towards human CB2R in both cell and tissue experiments. The >6000-fold selectivity over CB1 receptors and negligible off-targets in vitro , combined with high oral bioavailability and suitable systemic pharmacokinetic (PK) properties, prompted the assessment of RNB-61 in a mouse ischemia-reperfusion model of acute kidney injury (AKI) and in a rat model of chronic kidney injury/inflammation and fibrosis (CKI) induced by unilateral ureteral obstruction. RNB-61 exerted dose-dependent nephroprotective and/or antifibrotic effects in the AKI/CKI models. Thus, RNB-61 is an optimal CB2R tool compound for preclinical in vivo studies with superior biophysical and PK properties over generally used CB2R ligands., Competing Interests: Declaration of competing interest The authors declare the following competing financial interest(s): Christoph Ullmer, Antonello Caruso, Thomas Hartung, Roland Degen, Matthias Muller, and Uwe Grether are employees and shareholders of F. Hoffmann-La Roche AG. The remaining authors declare no potential competing interests.

De novo drug design aims to generate molecules from scratch that possess specific chemical and pharmacological properties. We present a computational approach utilizing interactome-based deep learning for ligand- and structure-based generation of drug-like molecules. This method capitalizes on the unique strengths of both graph neural networks and chemical language models, offering an alternative to the need for application-specific reinforcement, transfer, or few-shot learning. It enables the "zero-shot" construction of compound libraries tailored to possess specific bioactivity, synthesizability, and structural novelty. In order to proactively evaluate the deep interactome learning framework for protein structure-based drug design, potential new ligands targeting the binding site of the human peroxisome proliferator-activated receptor (PPAR) subtype gamma are generated. The top-ranking designs are chemically synthesized and computationally, biophysically, and biochemically characterized. Potent PPAR partial agonists are identified, demonstrating favorable activity and the desired selectivity profiles for both nuclear receptors and off-target interactions. Crystal structure determination of the ligand-receptor complex confirms the anticipated binding mode. This successful outcome positively advocates interactome-based de novo design for application in bioorganic and medicinal chemistry, enabling the creation of innovative bioactive molecules., (© 2024. The Author(s).)

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 (CB 2 R) selective inverse agonists ( S )- 1 and ( R )- 1 , which were derived from privileged agonist HU-308 by introduction of a phenyl group at the gem -dimethylheptyl side chain. Epimer ( R )- 1 exhibits high affinity for CB 2 R with K d = 39.1 nM and serves as a platform for the synthesis of a wide variety of probes. Notably, for the first time these fluorescent probes retain their inverse agonist functionality, high affinity, and selectivity for CB 2 R independent of linker and fluorophore substitution. Ligands ( S )- 1 , ( R )- 1 , and their derivatives act as inverse agonists in CB 2 R-mediated cAMP as well as G protein recruitment assays and do not trigger β-arrestin-receptor association. Furthermore, no receptor activation was detected in live cell ERK 1/2 phosphorylation and Ca 2+ -release assays. Confocal fluorescence imaging experiments with ( R )- 7 (Alexa488) and ( R )- 9 (Alexa647) probes employing BV-2 microglial cells visualized CB 2 R expressed at endogenous levels. Finally, molecular dynamics simulations corroborate the initial docking data in which inverse agonists restrict movement of toggle switch Trp258 6.48 and thereby stabilize CB 2 R in its inactive state., Competing Interests: The authors declare the following competing financial interest(s): M.K., R.C.S., B.K., W.G., U.G., and E.M.C. have filed a patent on CB2R selective modulators and fluorescent probes., (© 2024 The Authors. Published by American Chemical Society.)

This study aimed to evaluate ( R )-[ 18 F]YH134 as a novel PET tracer for imaging monoacylglycerol lipase (MAGL). Considering the ubiquitous expression of MAGL throughout the whole body, the impact of various MAGL inhibitors on ( R )-[ 18 F]YH134 brain uptake and its application in brain-periphery crosstalk were explored. Methods: MAGL knockout and wild-type mice were used to evaluate ( R )-[ 18 F]YH134 in in vitro autoradiography and PET experiments. To explore the impact of peripheral MAGL occupancy on ( R )-[ 18 F]YH134 brain uptake, PET kinetics with an arterial input function were studied in male Wistar rats under baseline and blocking conditions. Results: In in vitro autoradiography, ( R )-[ 18 F]YH134 revealed a heterogeneous distribution pattern with high binding to MAGL-rich brain regions in wild-type mouse brain slices, whereas the radioactive signal was negligible in MAGL knockout mouse brain slices. The in vivo brain PET images of ( R )-[ 18 F]YH134 in wild-type and MAGL knockout mice demonstrated its high specificity and selectivity in mouse brain. A Logan plot with plasma input function was applied to estimate the distribution volume ( V T ) of ( R )-[ 18 F]YH134. V T was significantly reduced by a brain-penetrant MAGL inhibitor but was unchanged by a peripherally restricted MAGL inhibitor. The MAGL target occupancy in the periphery was estimated using ( R )-[ 18 F]YH134 PET imaging data from the brain. Conclusion: ( R )-[ 18 F]YH134 is a highly specific and selective PET tracer with favorable kinetic properties for imaging MAGL in rodent brain. Our results showed that blocking of the peripheral target influences brain uptake but not the V T of ( R )-[ 18 F]YH134. ( R )-[ 18 F]YH134 can be used for estimating the dose of MAGL inhibitor at half-maximal peripheral target occupancy., (© 2024 by the Society of Nuclear Medicine and Molecular Imaging.)

Monoacylglycerol lipase (MAGL) regulates endocannabinoid 2-arachidonoylglycerol (2-AG) and eicosanoid signalling. MAGL inhibition provides therapeutic opportunities but clinical potential is limited by central nervous system (CNS)-mediated side effects. Here, we report the discovery of LEI-515, a peripherally restricted, reversible MAGL inhibitor, using high throughput screening and a medicinal chemistry programme. LEI-515 increased 2-AG levels in peripheral organs, but not mouse brain. LEI-515 attenuated liver necrosis, oxidative stress and inflammation in a CCl 4 -induced acute liver injury model. LEI-515 suppressed chemotherapy-induced neuropathic nociception in mice without inducing cardinal signs of CB 1 activation. Antinociceptive efficacy of LEI-515 was blocked by CB 2 , but not CB 1 , antagonists. The CB 1 antagonist rimonabant precipitated signs of physical dependence in mice treated chronically with a global MAGL inhibitor (JZL184), and an orthosteric cannabinoid agonist (WIN55,212-2), but not with LEI-515. Our data support targeting peripheral MAGL as a promising therapeutic strategy for developing safe and effective anti-inflammatory and analgesic agents., (© 2023. The Author(s).)

Enhancing the properties of advanced drug candidates is aided by the direct incorporation of specific chemical groups, avoiding the need to construct the entire compound from the ground up. Nevertheless, their chemical intricacy often poses challenges in predicting reactivity for C-H activation reactions and planning their synthesis. We adopted a reaction screening approach that combines high-throughput experimentation (HTE) at a nanomolar scale with computational graph neural networks (GNNs). This approach aims to identify suitable substrates for late-stage C-H alkylation using Minisci-type chemistry. GNNs were trained using experimentally generated reactions derived from in-house HTE and literature data. These trained models were then used to predict, in a forward-looking manner, the coupling of 3180 advanced heterocyclic building blocks with a diverse set of sp 3 -rich carboxylic acids. This predictive approach aimed to explore the substrate landscape for Minisci-type alkylations. Promising candidates were chosen, their production was scaled up, and they were subsequently isolated and characterized. This process led to the creation of 30 novel, functionally modified molecules that hold potential for further refinement. These results positively advocate the application of HTE-based machine learning to virtual reaction screening., (© 2023. The Author(s).)

The cannabis derivative marijuana is the most widely used recreational drug in the Western world and is consumed by an estimated 83 million individuals (∼3% of the world population). In recent years, there has been a marked transformation in society regarding the risk perception of cannabis, driven by its legalization and medical use in many states in the United States and worldwide. Compelling research evidence and the Food and Drug Administration cannabis-derived cannabidiol approval for severe childhood epilepsy have confirmed the large therapeutic potential of cannabidiol itself, Δ 9 -tetrahydrocannabinol and other plant-derived cannabinoids (phytocannabinoids). Of note, our body has a complex endocannabinoid system (ECS) - made of receptors, metabolic enzymes, and transporters - that is also regulated by phytocannabinoids. The first endocannabinoid to be discovered 30 years ago was anandamide ( N -arachidonoyl-ethanolamine); since then, distinct elements of the ECS have been the target of drug design programs aimed at curing (or at least slowing down) a number of human diseases, both in the central nervous system and at the periphery. Here a critical review of our knowledge of the goods and bads of the ECS as a therapeutic target is presented to define the benefits of ECS-active phytocannabinoids and ECS-oriented synthetic drugs for human health. SIGNIFICANCE STATEMENT: The endocannabinoid system plays important roles virtually everywhere in our body and is either involved in mediating key processes of central and peripheral diseases or represents a therapeutic target for treatment. Therefore, understanding the structure, function, and pharmacology of the components of this complex system, and in particular of key receptors (like cannabinoid receptors 1 and 2) and metabolic enzymes (like fatty acid amide hydrolase and monoacylglycerol lipase), will advance our understanding of endocannabinoid signaling and activity at molecular, cellular, and system levels, providing new opportunities to treat patients., (Copyright © 2023 by The American Society for Pharmacology and Experimental Therapeutics.)

Introduction: The cannabinoid receptor (CBR) subtypes 1 (CB 1 R) and 2 (CB 2 R) 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 CB 1 R and CB 2 R are Gα i protein-coupled receptors but detecting Gα i protein signalling activity reliably and reproducibly is challenging. This is due to the inherent variability in live cell-based assays and restrictions around the use of radioactive [ 35 S]-GTPγS, a favoured technology for developing higher-throughput membrane-based Gα i protein activity assays. Methods: Here, we describe the development of a membrane-based Gα i signalling system, produced from membrane preparations of HEK293TR cells, stably overexpressing CB 1 R or CB 2 R, and components of the Gα i -CASE biosensor. This BRET-based system allows direct detection of Gα i signalling in both cells and membranes by monitoring bioluminescence resonance energy transfer (BRET) between the α and the βγ subunits. Cells and membranes were subject to increasing concentrations of reference cannabinoid compounds, with 10 μM furimazine added to generate RET signals, which were detected on a PHERAstar FSX plate reader, then processed using MARS software and analysed in GraphPad PRISM 9.2. Results: In membranes expressing the Gi-CASE biosensor, the cannabinoid ligands profiled were found to show agonist and inverse agonist activity. Agonist activity elicited a decrease in the BRET signal, indicative of receptor activation and G protein dissociation. Inverse agonist activity caused an increase in BRET signal, indicative of receptor inactivation, and the accumulation of inactive G protein. Our membrane-based Gi-CASE NanoBRET system successfully characterised the potency (pEC 50 ) and efficacy (E max ) of CBR agonists and inverse agonists in a 384-well screening format. Values obtained were in-line with whole-cell Gi-CASE assays and consistent with literature values obtained in the GTPγS screening format. Discussion: This novel, membrane-based Gα i protein activation assay is applicable to other Gα i -coupled GPCRs, including orphan receptors, allowing real-time higher-throughput measurements of receptor activation., Competing Interests: Authors WG, AR, and UG was/were employed by F Hoffmann-La Roche Ltd. Authors DV and DS was/were employed by Z7 Biotech Limited. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Scott-Dennis, Rafani, Yi, Perera, Harwood, Guba, Rufer, Grether, Veprintsev and Sykes.)

Chronic inflammation and blood-brain barrier dysfunction are key pathological hallmarks of neurological disorders such as multiple sclerosis, Alzheimer's disease and Parkinson's disease. Major drivers of these pathologies include pro-inflammatory stimuli such as prostaglandins, which are produced in the central nervous system by the oxidation of arachidonic acid in a reaction catalyzed by the cyclooxygenases COX1 and COX2. Monoacylglycerol lipase hydrolyzes the endocannabinoid signaling lipid 2-arachidonyl glycerol, enhancing local pools of arachidonic acid in the brain and leading to cyclooxygenase-mediated prostaglandin production and neuroinflammation. Monoacylglycerol lipase inhibitors were recently shown to act as effective anti-inflammatory modulators, increasing 2-arachidonyl glycerol levels while reducing levels of arachidonic acid and prostaglandins, including PGE2 and PGD2. In this study, we characterized a novel, highly selective, potent and reversible monoacylglycerol lipase inhibitor (MAGLi 432) in a mouse model of lipopolysaccharide-induced blood-brain barrier permeability and in both human and mouse cells of the neurovascular unit: brain microvascular endothelial cells, pericytes and astrocytes. We confirmed the expression of monoacylglycerol lipase in specific neurovascular unit cells in vitro, with pericytes showing the highest expression level and activity. However, MAGLi 432 did not ameliorate lipopolysaccharide-induced blood-brain barrier permeability in vivo or reduce the production of pro-inflammatory cytokines in the brain. Our data confirm monoacylglycerol lipase expression in mouse and human cells of the neurovascular unit and provide the basis for further cell-specific analysis of MAGLi 432 in the context of blood-brain barrier dysfunction caused by inflammatory insults., Competing Interests: All authors, (except Britta Engelhardt) Alicia M Kemble, Benoit Hornsperger, Iris Ruf, Hans Richter, Jörg Benz Bernd Kuhn, Dominik Heer, Matthias Wittwer, Uwe Grether, Ludovic Collin are paid employment by the company F. Hoffmann-La Roche. Britta Engelhardt is an employee of the University of Bern. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials.

Labeled chemical probes are of utmost importance to bring drugs from the laboratory through the clinic and ultimately to market. They support and impact all research and discovery phases: target verification and validation; assay development; lead optimization; and biomarker engagement in the context of preclinical studies and human trials. Probes should display high potency and selectivity as well as fulfill specific criteria in connection with absorption, distribution, metabolism, excretion and toxicology (ADMET) profile. Progress in fields such as imaging and proteomics increased the need for specialized probes to support drug discovery. Labeled probes carrying an additional reporter group are valuable tools to meet specific application requirements, but pose significant challenges in design and construction. In the reverse-design approach, small molecules previously optimized in medicinal chemistry programs form the basis for the generation of such high-quality probes. We discuss the reverse design concept for the generation of labeled probes targeting the endocannabinoid system (ECS), a complex lipid signaling network that plays a key role in many human health and disease conditions. The examples highlighted include diverse reporter units for a range of applications. In several cases the reported probes were the product of mutually rewarding and highly cross-fertilizing collaborations among academic and industry research programs, a strategy that can serve as a blueprint for future probe generation efforts., (Copyright 2022 Mónica Guberman, Miroslav Kosar, Anahid Omran, Erick M. Carreira, Marc Nazaré, Uwe Grether. License: This work is licensed under a Creative Commons Attribution 4.0 International License.)

The distribution and roles of the cannabinoid CB 2 receptor in the CNS are still a matter of debate. Recent data suggest that, in addition to its presence in microglial cells, the CB 2 receptor may be also expressed at low levels, yet biologically relevant, in other cell types such as neurons. It is accepted that the expression of CB 2 receptors in the CNS is low under physiological conditions and is significantly elevated in chronic neuroinflammatory states associated with neurodegenerative diseases such as Alzheimer's disease. By using a novel mouse model (CB 2 EGFP/f/f ), we studied the distribution of cannabinoid CB 2 receptors in the 5xFAD mouse model of Alzheimer's disease (by generating 5xFAD/CB 2 EGFP/f/f mice) and explored the roles of CB 2 receptors in microglial function. We used a novel selective and brain penetrant CB 2 receptor agonist (RO6866945) as well as mice lacking the CB 2 receptor (5xFAD/CB 2 -/- ) for these studies. We found that CB 2 receptors are expressed in dystrophic neurite-associated microglia and that their modulation modifies the number and activity of microglial cells as well as the metabolism of the insoluble form of the amyloid peptide. These results support microglial CB 2 receptors as potential targets for the development of amyloid-modulating therapies., Competing Interests: Authors CK, CR and UG were employed by the company F. Hoffmann-La Roche Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ruiz de Martín Esteban, Benito-Cuesta, Terradillos, Martínez-Relimpio, Arnanz, Ruiz-Pérez, Korn, Raposo, Sarott, Westphal, Elezgarai, Carreira, Hillard, Grether, Grandes, Grande and Romero.)

Despite its essential role in the (patho)physiology of several diseases, CB 2 R tissue expression profiles and signaling mechanisms are not yet fully understood. We report the development of a highly potent, fluorescent CB 2 R agonist probe employing structure-based reverse design. It commences with a highly potent, preclinically validated ligand, which is conjugated to a silicon-rhodamine fluorophore, enabling cell permeability. The probe is the first to preserve interspecies affinity and selectivity for both mouse and human CB 2 R. Extensive cross-validation (FACS, TR-FRET and confocal microscopy) set the stage for CB 2 R detection in endogenously expressing living cells along with zebrafish larvae. Together, these findings will benefit clinical translatability of CB 2 R based drugs., Competing Interests: The authors have no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The G-protein-coupled cannabinoid receptor type 2 (CB 2 R) is a key element of the endocannabinoid (EC) system. EC/CB 2 R signaling has significant therapeutic potential in major pathologies affecting humans such as allergies, neurodegenerative disorders, inflammation or ocular diseases. CB 2 R agonism exerts anti-inflammatory and tissue protective effects in preclinical animal models of cardiovascular, gastrointestinal, liver, kidney, lung and neurodegenerative disorders. Existing ligands can be subdivided into endocannabinoids, cannabinoid-like and synthetic CB 2 R ligands that possess various degrees of potency on and selectivity against the cannabinoid receptor type 1. This review is an account of granted CB 2 R ligand patents from 2010 up to the present, which were surveyed using Derwent Innovation ® .

(1) Background: The cannabinoid 2 receptor (CB 2 R) is a promising anti-inflammatory drug target and development of selective CB 2 R ligands may be useful for treating sight-threatening ocular inflammation. (2) Methods: This study examined the pharmacology of three novel chemically-diverse selective CB 2 R ligands: CB 2 R agonists, RO6871304, and RO6871085, as well as a CB 2 R inverse agonist, RO6851228. In silico molecular modelling and in vitro cell-based receptor assays were used to verify CB 2 R interactions, binding, cell signaling (ß-arrestin and cAMP) and early absorption, distribution, metabolism, excretion, and toxicology (ADMET) profiling of these receptor ligands. All ligands were evaluated for their efficacy to modulate leukocyte-neutrophil activity, in comparison to the reported CB 2 R ligand, HU910, using an in vivo mouse model of endotoxin-induced uveitis (EIU) in wild-type (WT) and CB 2 R -/- mice. The actions of RO6871304 on neutrophil migration and adhesion were examined in vitro using isolated neutrophils from WT and CB 2 R -/- mice, and in vivo in WT mice with EIU using adoptive transfer of WT and CB 2 R -/- neutrophils, respectively. (3) Results: Molecular docking studies indicated that RO6871304 and RO6871085 bind to the orthosteric site of CB 2 R. Binding studies and cell signaling assays for RO6871304 and RO6871085 confirmed high-affinity binding to CB 2 R and selectivity for CB 2 R > CB 1 R, with both ligands acting as full agonists in cAMP and ß-arrestin assays (EC 50 s in low nM range). When tested in EIU, topical application of RO6871304 and RO6871085 decreased leukocyte-endothelial adhesion and this effect was antagonized by the inverse agonist, RO6851228. The CB 2 R agonist, RO6871304, decreased in vitro neutrophil migration of WT neutrophils but not neutrophils from CB 2 R -/- , and attenuated adhesion of adoptively-transferred leukocytes in EIU. (4) Conclusions: These unique ligands are potent and selective for CB 2 R and have good immunomodulating actions in the eye. RO6871304 and RO6871085, as well as HU910, decreased leukocyte adhesion in EIU through inhibition of resident ocular immune cells. The data generated with these three structurally-diverse and highly-selective CB 2 R agonists support selective targeting of CB 2 R for treating ocular inflammatory diseases.

Astrocytes are involved in non-cell-autonomous pathogenic cascades in amyotrophic lateral sclerosis (ALS); however, their role is still debated. We show that astrocytic NF-κB activation drives microglial proliferation and leukocyte infiltration in the SOD1 (G93A) ALS model. This response prolongs the presymptomatic phase, delaying muscle denervation and decreasing disease burden, but turns detrimental in the symptomatic phase, accelerating disease progression. The transition corresponds to a shift in the microglial phenotype showing two effects that can be dissociated by temporally controlling NF-κB activation. While NF-κB activation in astrocytes induced a Wnt-dependent microglial proliferation in the presymptomatic phase with neuroprotective effects on motoneurons, in later stage, astrocyte NF-κB-dependent microglial activation caused an accelerated disease progression. Notably, suppression of the early microglial response by CB 2 R agonists had acute detrimental effects. These data identify astrocytes as important regulators of microglia expansion and immune response. Therefore, stage-dependent microglia modulation may be an effective therapeutic strategy in ALS., (© 2018 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

The cannabinoid CB 2 receptor (CB 2 R) represents a promising therapeutic target for various forms of tissue injury and inflammatory diseases. Although numerous compounds have been developed and widely used to target CB 2 R, their selectivity, molecular mode of action and pharmacokinetic properties have been poorly characterized. Here we report the most extensive characterization of the molecular pharmacology of the most widely used CB 2 R ligands to date. In a collaborative effort between multiple academic and industry laboratories, we identify marked differences in the ability of certain agonists to activate distinct signalling pathways and to cause off-target effects. We reach a consensus that HU910, HU308 and JWH133 are the recommended selective CB 2 R agonists to study the role of CB 2 R in biological and disease processes. We believe that our unique approach would be highly suitable for the characterization of other therapeutic targets in drug discovery research., Competing Interests: Industry authors U.G., J.F., C.U., B.R. and C.P. are full-time employees of Hoffmann-La Roche and P.P. is full time employee of NIH. All academic authors state that they have no conflict of interest.