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Monoacylglycerol lipase (MAGL) is a 33 kDa cytosolic serine hydrolase that is widely distributed in the central nervous system and peripheral tissues. MAGL hydrolyzes monoacylglycerols into fatty acids and glycerol, playing a crucial role in endocannabinoid degradation. Inhibition of MAGL in the brain elevates levels of 2-arachidonoylglycerol and leads to decreased pro-inflammatory prostaglandin and thromboxane production. As such, MAGL is considered a potential target for treating neuropsychiatric disorders, metabolic syndromes, and cancer. Based on a novel spirocyclic system, we synthesized two fluorinated carbamate scaffolds as reversible MAGL inhibitors (epimers: (R)-6, IC 50  = 18.6 nM and (S)-6, IC 50  = 1.6 nM). In vitro autoradiography studies of [ 18 F](R)-6 (codenamed [ 18 F]MAGL-2304) and [ 18 F](S)-6 (codenamed [ 18 F]MAGL-2305) demonstrated heterogeneous distribution and specific binding affinity to MAGL-rich brain regions. Autoradiography with MAGL knockout mouse brain tissues confirmed the binding specificity of [ 18 F](S)-6. Dynamic PET imaging studies revealed that [ 18 F](S)-6 exhibited limited brain uptake and homogenous distribution in rat brains. In vivo P-gp inhibition enhanced [ 18 F](S)-6 uptake in the brain, suggesting that [ 18 F](S)-6 constitutes a P-gp efflux substrate. This research could provide new directions in the design of MAGL PET ligands that are based on spirocyclic scaffolds., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Steven Liang reports financial support was provided by Emory University. Ming-Rong Zhang reports financial support was provided by National Institutes for Quantum Science and Technology. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier Masson SAS. All rights reserved.)

Monoacylglycerol lipase (MAGL) is a key enzyme for signal termination in the endocannabinoid system (ECS). MAGL inhibition results in indirect activation of the cannabinoid receptors, which offers unique advantages for the treatment of, e.g., multiple sclerosis, epilepsy, and other neurological disorders. Molecular imaging techniques are valuable tools to overcome the current poor understanding of MAGL's distribution and role in patho- and physiological processes within ECS signaling. Herein, we report the design, synthesis, and validation of highly selective versatile fluorescent and click-chemistry probes for MAGL. Structure-based design combined with a reverse-design approach allowed the development of a structural unit that selectively and effectively recognizes MAGL while offering a versatile platform to attach different fluorophores and further reporter units. In this way, labeled probes with sub-nanomolar potency carrying diverse fluorescent dyes were obtained. Probe affinity and selectivity remained invariant to changes in the fluorophore subunit, showing the remarkable robustness of this platform in delivering tailor-made probes. Highly consistent inhibition across species supports pharmacological model translatability. Extensive profiling and validation in various cellular systems shows the ability of these highly potent and selective probes to elucidate the complex role of MAGL in ECS cellular signaling, inflammatory processes, and disease progression., (© 2024 The Author(s). Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Purpose: The monoacylglycerol lipase (MAGL) plays a pivotal role in modulating the endocannabinoid system and is considered an attractive therapeutic target for diseases in both the central nervous system and periphery. The current study aimed to develop and evaluate a suitable carbon-11 labeled tracer for imaging MAGL in preclinical studies., Methods: (R)-YH168 was synthesized via a multi-step pathway and its half-maximal inhibitory concentration (IC 50 ) values were measured using an enzymatic assay. Radiosynthesis of (R)-[ 11 C]YH168 was accomplished by 11 C-methylation via Suzuki cross-coupling of a pinacol boron precursor. In vitro autoradiography was performed using brain tissues from MAGL knockout and the corresponding wild-type mice. The metabolic stability of (R)-[ 11 C]YH168 in mouse brain and plasma was assessed 5 min after injection. Dynamic PET scans were conducted on anesthetized mice and rhesus monkey. For studies in non-human primates, arterial blood samples were analyzed to obtain the input function for kinetic modeling. Blocking studies with the irreversible MAGL inhibitor PF-06795071 were performed to assess the binding specificity of (R)-[ 11 C]YH168., Results: (R)-[ 11 C]YH168 was synthesized via Suzuki coupling of the phenyl boronic ester with [ 11 C]CH 3 I in the presence of palladium catalyst. In vitro autoradiography revealed a heterogeneous distribution pattern of (R)-[ 11 C]YH168 with higher binding to MAGL-rich brain regions in wild-type mouse brain slices compared to that of MAGL knockout mice. Dynamic PET imaging in wild-type and MAGL knockout mice confirmed its high specificity and selectivity in mouse brains. In the rhesus monkey, (R)-[ 11 C]YH168 displayed good brain permeability. High levels of radioactivity uptake were seen in the cingulate cortex, frontal cortex, cerebellum, occipital cortex, and hippocampus, consistent with MAGL expression. The one-tissue compartment model was appropriate for fitting the regional time-activity curves and provided reliable volume of distribution values across all brain regions. Pretreatment with PF-06795071 (0.1 mg/kg) resulted in almost complete blockade (> 95%) of radioactivity uptake, demonstrating binding specificity of (R)-[ 11 C]YH168 to MAGL in the non-human primate brain. The regional non-displaceable binding potential follows the rank order of cingulate cortex ~ frontal cortex ~ insula > putamen > temporal cortex > caudate ~ occipital cortex ~ thalamus > nucleus accumbens ~ hippocampus ~ cerebellum ~ globus pallidus > substantia nigra > amygdala., Conclusion: (R)-[ 11 C]YH168 is a promising PET probe for imaging and quantifying MAGL in the brains of mice and non-human primates. This 11 C-labeled tracer holds great potential for translation into human subjects and offers the possibility of performing multiple PET scans on the same subject within a single day., Competing Interests: Declarations. Ethical approval: This study was performed in agreement with the international and national law and institutional guidelines of radiation safety protection. In vivo experiments were approved by the local veterinarian department and ethics committee and conducted in accordance with the internal and national law of animal protections. Institutional review board statement: All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. In particular, all animal experiments were carried out according to the guidelines of the Swiss Regulations for Animal Welfare. The preclinical studies have been ethically approved by the Veterinary Office of Canton Zurich (License N° (ZH28/ 2018). The protocol of PET study in rhesus monkey (Macaca mulatta) was approved by the Yale University Institutional Animal Care and Use Committee. Conflict of interest: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024. The Author(s).)

Allosteric modulation of cannabinoid receptor type 1 (CB1R) offers a promising alternative to conventional therapeutic approaches using orthosteric ligands (OLs). Currently, CB1R 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 CB1R AMs without coincubation with an OL. Our allosteric tracer enables differentiation of allosteric and orthosteric ligands as well as their pharmacological profiling at CB1R. The utility of this work is highlighted by addressing ambiguities surrounding the binding of cannabidiol (CBD). CBD was found to interact with both allosteric and orthosteric sites of CB1R with comparable affinity (pKi = 5.34 and 5.67, respectively)., (© 2025 Wiley‐VCH GmbH.)

In this study, we employed a novel fluorescent probe, RO7304924-which selectively targets cannabinoid 2 receptor (CB2R)-to assess the lateral mobility of CB2R within the plasma membrane of Chinese hamster ovary cells stably expressing a functional, untagged receptor variant. Utilizing confocal fluorescence recovery after photobleaching (FRAP), we quantified the diffusion coefficient and mobile fraction of CB2R, thereby demonstrating the efficacy of RO7304924 as an innovative tool for elucidating the dynamics of this major endocannabinoid-binding G protein-coupled receptor. Our present findings highlight the potential of combining advanced ligand-based fluorescent probes with FRAP for future investigations into the biochemical details of CB2R mobility in living cells, and its impact on receptor-dependent cellular processes., (© 2025 Wiley‐VCH GmbH.)

Cannabis sativa has been used therapeutically since early civilizations, with key cannabinoids Δ 9 -tetrahydrocannabinol (THC) 3.1 and cannabidiol characterized in the 1960s, leading to the discovery of cannabinoid receptors type 1 (CB 1 R) and type 2 (CB 2 R) and the endocannabinoid system (ECS) in the 1990s. The ECS, involving endogenous ligands like 2-arachidonoylglycerol (2-AG) 1.1, anandamide (N-arachidonoylethanolamine (AEA)) 1.2, and various proteins, regulates vital processes such as sleep, appetite, and memory, and holds significant therapeutic potential, especially for neurological disorders. Small molecule-derived pharmacological tools, or chemical probes, target key components of the ECS and are crucial for target validation, mechanistic studies, pathway elucidation, phenotypic screening, and drug discovery. These probes selectively interact with specific proteins or pathways, enabling researchers to modulate target activity and observe biological effects. When they carry an additional reporter group, they are referred to as labeled chemical probes. Developed through medicinal chemistry, structural biology, and high-throughput screening, effective chemical probes must be selective, potent, and depending on their purpose meet additional criteria such as cell permeability and metabolic stability.This chapter describes high-quality labeled and unlabeled chemical probes targeting ECS constituents that have been successfully applied for various research purposes. CB 1 R and CB 2 R, class A G protein-coupled receptors, are activated by 2-AG 1.1, AEA 1.2, and THC 3.1, with numerous ligands developed for these receptors. Imaging techniques like single-photon emission computed tomography, positron emission tomography, and fluorescently labeled CB 1 R and CB 2 R probes have enhanced CB receptor studies. CB 2 R activation generally results in immunosuppressive effects, limiting tissue injury. AEA 1.2 is mainly degraded by fatty acid amide hydrolase (FAAH) or N-acylethanolamine acid amidase (NAAA) into ethanolamine and arachidonic acid (AA) 1.3. FAAH inhibitors increase endogenous fatty acid amides, providing analgesic effects without adverse effects. NAAA inhibitors reduce inflammation and pain in animal models. Diacylglycerol lipase (DAGL) is essential for 2-AG 1.1 biosynthesis, while monoacylglycerol lipase (MAGL) degrades 2-AG 1.1 into AA 1.3, thus regulating cannabinoid signaling. Multiple inhibitors targeting FAAH and MAGL have been generated, though NAAA and DAGL probe development lags behind. Similarly, advancements in inhibitors targeting endocannabinoid (eCB) cellular uptake or trafficking proteins like fatty acid-binding proteins have been slower. The endocannabinoidome (eCBome) includes the ECS and related molecules and receptors, offering therapeutic opportunities from non-THC cannabinoids and eCBome mediators. Ongoing research aims to refine chemical tools for ECS and eCBome study, addressing unmet medical needs in central nervous system disorders and beyond., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Utilizing the growing wealth of chemical reaction data can boost synthesis planning and increase success rates. Yet, the effectiveness of machine learning tools for retrosynthesis planning and forward reaction prediction relies on accessible, well-curated data presented in a structured format. Although some public and licensed reaction databases exist, they often lack essential information about reaction conditions. To address this issue and promote the principles of findable, accessible, interoperable, and reusable (FAIR) data reporting and sharing, we introduce the Simple User-Friendly Reaction Format (SURF). SURF standardizes the documentation of reaction data through a structured tabular format, requiring only a basic understanding of spreadsheets. This format enables chemists to record the synthesis of molecules in a format that is understandable by both humans and machines, which facilitates seamless sharing and integration directly into machine learning pipelines. SURF files are designed to be interoperable, easily imported into relational databases, and convertible into other formats. This complements existing initiatives like the Open Reaction Database (ORD) and Unified Data Model (UDM). At Roche, SURF plays a crucial role in democratizing FAIR reaction data sharing and expediting the chemical synthesis process., (© 2025 The Author(s). Molecular Informatics published by Wiley-VCH GmbH.)

Cell-free enzymatic assays are highly useful tools in early compound profiling due to their robustness and scalability. However, their inadequacy to reflect the complexity of target engagement in a cellular environment may lead to a significantly divergent pharmacology that is eventually observed in cells. The discrepancy that emerges from properties like permeability and unspecific protein binding may largely mislead lead compound selection to undergo further chemical optimization. We report the development of a new intracellular NanoBRET assay to assess MAGL inhibition in live cells. Based on a reverse design approach, a highly potent, reversible preclinical inhibitor was conjugated to the cell-permeable BODIPY590 acceptor fluorophore while retaining its overall balanced properties. An engineered MAGL-nanoluciferase (Nluc) fusion protein provided a suitable donor counterpart for the facile interrogation of intracellular ligand activity. Validation of assay conditions using a selection of known MAGL inhibitors set the stage for the evaluation of over 1'900 MAGL drug candidates derived from our discovery program. This evaluation enabled us to select compounds for further development based not only on target engagement, but also on favorable physicochemical parameters like permeability and protein binding. This study highlights the advantages of cell-based target engagement assays for accelerating compound profiling and progress at the early stages of drug discovery programs., (© 2024 The Author(s). ChemBioChem published by Wiley-VCH GmbH.)

Monoacylglycerol lipase (MAGL) is a key enzyme involved in the metabolism of the endogenous signaling ligand 2-arachidonoylglycerol, a neuroprotective endocannabinoid intimately linked to central nervous system (CNS) disorders associated with neuroinflammation. In the quest for novel MAGL inhibitors, a focused screening approach on a Roche library subset provided a reversible benzoxazinone hit exhibiting high ligand efficiency. The subsequent design of the three-dimensional cis -hexahydro-pyrido-oxazinone ( cis -HHPO) moiety as benzoxazinone replacement enabled the combination of high MAGL potency with favorable ADME properties. Through enzymatic resolution an efficient synthetic route of the privileged cis -(4 R ,8 S ) HHPO headgroup was established, providing access to the highly potent and selective MAGL inhibitor 7o . Candidate molecule 7o matches the target compound profile of CNS drugs as it achieves high CSF exposures after systemic administration in rodents. It engages with the target in the brain and modulates neuroinflammatory processes, thus holding great promise for the treatment of CNS disorders.

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: Cannabinoid receptor type 2 (CB 2 R), predominantly expressed in immune tissues, is believed to play a crucial role within the body's protective mechanisms. Its modulation holds immense therapeutic promise for addressing a wide spectrum of dysbiotic conditions, including cardiovascular, gastrointestinal, liver, kidney, neurodegenerative, psychiatric, bone, skin, and autoimmune diseases, as well as lung disorders, cancer, and pain management., Areas Covered: This review is an account of patents from 2016 up to 2023 which describes novel CB 2 R ligands, therapeutic applications, synthesis, as well as formulations of CB 2 R modulators., Expert Opinion: The patents cover a vast, structurally diverse chemical space. The focus of CB 2 R ligand development has shifted from unselective dual-cannabinoid receptor type 1 (CB 1 R) and 2 agonists toward agonists with high selectivity over CB 1 R, particularly for indications associated with inflammation and tissue injury. Currently, there are at least eight CB 2 R agonists and one antagonist in active clinical development. A better understanding of the endocannabinoid system (ECS) and in particular of CB 2 R pharmacology is required to unlock the receptor's full therapeutic potential.

The cannabinoid receptor type 1 (CB 1 R) is pivotal within the endocannabinoid system regulating various signaling cascades with effects in appetite regulation, pain perception, memory formation, and thermoregulation. Still, understanding of CB 1 R's cellular signaling, distribution, and expression dynamics is very fragmentary. Real-time visualization of CB 1 R is crucial for addressing these questions. Selective drug-like CB 1 R ligands with a defined pharmacological profile were investigated for the construction of CB 1 R fluorescent probes using a reverse design-approach. A modular design concept with a diethyl glycine-based building block as the centerpiece allowed for the straightforward synthesis of novel probe candidates. Validated by computational docking studies, radioligand binding, and cAMP assay, this systematic approach allowed for the identification of novel pyrrole-based CB 1 R fluorescent probes. Application in fluorescence-based target-engagement studies and live cell imaging exemplify the great versatility of the tailored CB 1 R probes for investigating CB 1 R localization, trafficking, pharmacology, and its pathological implications.

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.)

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 labeled RNB-61 showed apparent K d values of <4 nM toward human CB2R in both cell and tissue experiments. The 6,800-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: The authors declare the following competing financial interest(s): Christoph Ullmer, Antonello Caruso, Thomas Hartung, Roland Degen, Matthias Mller, Sabine Grner and Uwe Grether are employees and shareholders of F. Hoffmann-La Roche AG. The remaining authors declare no potential competing interests., (© 2024 The Authors. Published by American Chemical Society.)

Suzuki cross-coupling reactions are considered a valuable tool for constructing carbon-carbon bonds in small molecule drug discovery. However, the synthesis of chemical matter often represents a time-consuming and labour-intensive bottleneck. We demonstrate how machine learning methods trained on high-throughput experimentation (HTE) data can be leveraged to enable fast reaction condition selection for novel coupling partners. We show that the trained models support chemists in determining suitable catalyst-solvent-base combinations for individual transformations including an evaluation of the need for HTE screening. We introduce an algorithm for designing 96-well plates optimized towards reaction yields and discuss the model performance of zero- and few-shot machine learning. The best-performing machine learning model achieved a three-category classification accuracy of 76.3% (±0.2%) and an F 1 -score for a binary classification of 79.1% (±0.9%). Validation on eight reactions revealed a receiver operating characteristic (ROC) curve (AUC) value of 0.82 (±0.07) for few-shot machine learning. On the other hand, zero-shot machine learning models achieved a mean ROC-AUC value of 0.63 (±0.16). This study positively advocates the application of few-shot machine learning-guided reaction condition selection for HTE campaigns in medicinal chemistry and highlights practical applications as well as challenges associated with zero-shot machine learning., Competing Interests: G. S. declares a potential financial and non-financial conflict of interest as co-founder of https://inSili.com LLC, Zurich and in his role as a scientific consultant to the pharmaceutical industry. K. A., D. F. N., A. T. M., V. J., M. R. U. G., R. E. M., C. K. and G. W. declare a potential financial and non-financial conflict of interest as full employees of F. Hoffmann-La Roche Ltd., (This journal is © The Royal Society of Chemistry.)

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.)

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).)

Monoacylglycerol lipase (MAGL) plays a crucial role in the degradation of 2-arachidonoylglycerol (2-AG), one of the major endocannabinoids in the brain. Inhibiting MAGL could lead to increased levels of 2-AG, which showed beneficial effects on pain management, anxiety, inflammation, and neuroprotection. In the current study, we report the characterization of an enantiomerically pure (R)-[ 11 C]YH132 as a novel MAGL PET tracer. It demonstrates an improved pharmacokinetic profile compared to its racemate. High in vitro MAGL specificity of (R)-[ 11 C]YH132 was confirmed by autoradiography studies using mouse and rat brain sections. In vivo, (R)-[ 11 C]YH132 displayed a high brain penetration, and high specificity and selectivity toward MAGL by dynamic PET imaging using MAGL knockout and wild-type mice. Pretreatment with a MAGL drug candidate revealed a dose-dependent reduction of (R)-[ 11 C]YH132 accumulation in WT mouse brains. This result validates its utility as a PET probe to assist drug development. Moreover, its potential application in neurodegenerative diseases was explored by in vitro autoradiography using brain sections from animal models of Alzheimer's disease and Parkinson's disease., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

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.)

New potent, selective monoacylglycerol lipase (MAGL) inhibitors based on the azetidin-2-one scaffold ((±)- 5a - v , (±)- 6a - j , and (±)- 7a - d ) were developed as irreversible ligands, as demonstrated by enzymatic and crystallographic studies for (±)- 5d , (±)- 5l , and (±)- 5r . X-ray analyses combined with extensive computational studies allowed us to clarify the binding mode of the compounds. 5v was identified as selective for MAGL when compared with other serine hydrolases. Solubility, in vitro metabolic stability, cytotoxicity, and absence of mutagenicity were determined for selected analogues. The most promising compounds ((±)- 5c , (±)- 5d , and (±)- 5v ) were used for in vivo studies in mice, showing a decrease in MAGL activity and increased 2-arachidonoyl- sn -glycerol levels in forebrain tissue. In particular, 5v is characterized by a high eudysmic ratio and (3 R ,4 S )- 5v is one of the most potent irreversible inhibitors of h / m MAGL identified thus far. These results suggest that the new MAGL inhibitors have therapeutic potential for different central and peripheral pathologies.

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.)

Late-stage functionalization is an economical approach to optimize the properties of drug candidates. However, the chemical complexity of drug molecules often makes late-stage diversification challenging. To address this problem, a late-stage functionalization platform based on geometric deep learning and high-throughput reaction screening was developed. Considering borylation as a critical step in late-stage functionalization, the computational model predicted reaction yields for diverse reaction conditions with a mean absolute error margin of 4-5%, while the reactivity of novel reactions with known and unknown substrates was classified with a balanced accuracy of 92% and 67%, respectively. The regioselectivity of the major products was accurately captured with a classifier F-score of 67%. When applied to 23 diverse commercial drug molecules, the platform successfully identified numerous opportunities for structural diversification. The influence of steric and electronic information on model performance was quantified, and a comprehensive simple user-friendly reaction format was introduced that proved to be a key enabler for seamlessly integrating deep learning and high-throughput experimentation for late-stage functionalization., (© 2023. The Author(s).)

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 cannabinoid type 2 receptor (CB2) has been implicated in a variety of central and peripheral inflammatory diseases, prompting significant interest in the development of CB2-targeted diagnostic and therapeutic agents. A validated positron emission tomography (PET) radioligand for imaging CB2 in the living human brain as well as in peripheral tissues is currently lacking. As part of our research program, we have recently identified the trisubstituted pyridine, [ 18 F]RoSMA-18-d 6 , which proved to be highly suitable for in vitro and in vivo mapping of CB2 in rodents. The aim of this study was to assess the performance characteristics of [ 18 F]RoSMA-18-d 6 in nonhuman primates (NHPs) to pave the way for clinical translation. [ 18 F]RoSMA-18-d 6 was synthesized from the respective tosylate precursor according to previously reported procedures. In vitro autoradiograms with NHP spleen tissue sections revealed a high binding of [ 18 F]RoSMA-18-d 6 to the CB2-rich NHP spleen, which was significantly blocked by coincubation with the commercially available CB2 ligand, GW405833 (10 μM). In contrast, no specific binding was observed by in vitro autoradiography with NHP brain sections, which was in agreement with the notion of a CB2-deficient healthy mammalian brain. In vitro findings were corroborated by PET imaging experiments in NHPs, where [ 18 F]RoSMA-18-d 6 uptake in the spleen was dose-dependently attenuated with 1 and 5 mg/kg GW405833, while no specific brain signal was observed. Remarkably, we observed tracer uptake and retention in the NHP spinal cord, which was reduced by GW405833 blockade, pointing toward a potential utility of [ 18 F]RoSMA-18-d 6 in probing CB2-expressing cells in the bone marrow. If these observations are substantiated in NHP models of enhanced leukocyte proliferation in the bone marrow, [ 18 F]RoSMA-18-d 6 may serve as a valuable marker for hematopoietic activity in various pathologies. In conclusion, [ 18 F]RoSMA-18-d 6 proved to be a suitable PET radioligand for imaging CB2 in NHPs, supporting its translation to humans.

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.)

Pharmacological modulation of cannabinoid receptor type 2 (CB 2 R) holds promise for the treatment of neuroinflammatory disorders, such as Alzheimer's disease. Despite the importance of CB 2 R, 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 CB 2 R enabled by a novel synthetic strategy and application of platform reagents. The LDC modification allows visualization and study of CB 2 R while maintaining its ability to bind other ligands at the orthosteric site. We employed in silico docking and molecular dynamics simulations to guide probe design and assess the feasibility of LDC labeling of CB 2 R. We demonstrate selective, covalent labeling of a peripheral lysine residue of CB 2 R by exploiting fluorogenic O -nitrobenzoxadiazole ( O- NBD)-functionalized probes in a TR-FRET assay. The rapid proof-of-concept validation with O- NBD probes inspired incorporation of advanced electrophiles suitable for experiments in live cells. To this end, novel synthetic strategies toward N -sulfonyl pyridone ( N -SP) and N -acyl- N -alkyl sulfonamide (NASA) LDC probes were developed, which allowed covalent delivery of fluorophores suitable for cellular studies. The LDC probes were characterized by a radioligand binding assay and TR-FRET experiments. Additionally, the probes were applied to specifically visualize CB 2 R in conventional and imaging flow cytometry as well as in confocal fluorescence microscopy using overexpressing and endogenously expressing microglial live cells.

Ground-breaking research in disease biology and continuous efforts in method development have uncovered a range of potential new drug targets. Increasingly, the drug discovery process is informed by technologies involving chemical probes as tools. Applications for chemical probes comprise target identification and assessment, as well as the qualification of small molecules as chemical starting points and drug candidates. Progress in probe chemistry has opened the way to novel assay formats and pharmaceutical compound classes. The European Federation of Medicinal Chemistry and Chemical Biology (EFMC) has launched the Chemical Biology Initiative to advance science in the field of medicinal chemistry and chemical biology, while representing all members of this extended scientific community. This review provides an overview of the many important developments in the field of chemical biology that have happened at the lively interface of academic and industrial research., (© 2023 Wiley-VCH GmbH.)

Computational methods in medicinal chemistry facilitate drug discovery and design. In particular, machine learning methodologies have recently gained increasing attention. This chapter provides a structured overview of the current state of computational chemistry and its applications for the interrogation of the endocannabinoid system (ECS), highlighting methods in structure-based drug design, virtual screening, ligand-based quantitative structure-activity relationship (QSAR) modeling, and de novo molecular design. We emphasize emerging methods in machine learning and anticipate a forecast of future opportunities of computational medicinal chemistry for the ECS., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Peroxisome proliferator-activated receptors are a family of nuclear hormone receptors that control the expression of genes involved in a variety of physiologic processes, through heterodimerization with retinoid X receptor and complex formation with various cofactors. The specific cofactors recruited to PPAR-RXR complexes in response to different ligands lead to major differences in the transactivation of target genes. We developed a cofactor recruitment assay that is based on an europium-labeled anti-GST antibody and streptavidin-APC leading to a fluorescence resonance energy transfer signal. This assay allows for the determination of unique agonistic profiles in terms of potency and co-activator motif. Hence, it is a valuable drug discovery tool to support hit finding and lead optimization campaigns, enabling the characterization of next generation PPAR agonists., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Peroxisome proliferator-activated receptors (PPARs) have been exploited as drug targets for combating multiple diseases. Several activators with different selectivity for the PPAR α, γ, and δ subtypes have been introduced into the market or have reached advanced clinical trials. Binding assays are of utmost importance for the discovery and profiling of such PPAR ligands. Binding assays are often based on radioligands, in particular, tritiated molecules are applied. We developed synthetic procedures for tritiating various PPAR agonists and applied these radioligands for setting up a scintillation proximity assay (SPA) for PPAR α, γ, and δ. These SPAs allow to assess the binding affinities of PPAR α, γ, and δ ligands, along with their respective subtype selectivity profiles. Therefore, SPA is an important tool for hit discovery and lead optimization campaigns aimed at identifying next-generation PPAR ligands., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Monoacylglycerol lipase (MAGL) is a gatekeeper in regulating endocannabinoid signaling and has gained substantial attention as a therapeutic target for neurological disorders. We recently discovered a morpholin-3-one derivative as a novel scaffold for imaging MAGL via positron emission tomography (PET). However, its slow kinetics in vivo hampered the application. In this study, structural optimization was conducted and eleven novel MAGL inhibitors were designed and synthesized. Based on the results from MAGL inhibitory potency, in vitro metabolic stability and surface plasmon resonance assays, we identified compound 7 as a potential MAGL PET tracer candidate. [ 11 C]7 was synthesized via direct 11 CO 2 fixation method and successfully mapped MAGL distribution patterns on rodent brains in in vitro autoradiography. PET studies in mice using [ 11 C]7 demonstrated its improved kinetic profile compared to the lead structure. Its high specificity in vivo was proved by using MAGL KO mice. Although further studies confirmed that [ 11 C]7 is a P-glycoprotein (P-gp) substrate in mice, its low P-gp efflux ratio on cells transfected with human protein suggests that it should not be an issue for the clinical translation of [ 11 C]7 as a novel reversible MAGL PET tracer in human subjects. Overall, [ 11 C]7 ([ 11 C]RO7284390) showed promising results warranting further clinical evaluation., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Yingfang He reports financial support was provided by China Scholarship Council. COLLIN LUDOVIC, EDELMANN MARTIN, GOBBI LUCA, GRETHER UWE, HE YINGFANG, HONER MICHAEL, MU LINJING, RICHTER HANS has patent RADIOLABELED COMPOUNDS issued to ETH Zurich, F. Hoffmann-La Roche Ltd., (Copyright © 2022 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

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.