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The cannabis derivative marijuana is the most widely used recreational drug in the Western world and is consumed by an estimated 83 million individuals (â¼3% 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, Δ9-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.
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Canabidiol , Canabinoides , Cannabis , Alucinógenos , Humanos , Criança , Endocanabinoides/metabolismo , Canabidiol/uso terapêutico , Canabinoides/farmacologia , Canabinoides/uso terapêutico , Canabinoides/metabolismo , Dronabinol , Cannabis/química , Cannabis/metabolismo , Proteínas de Transporte , Agonistas de Receptores de CanabinoidesRESUMO
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)-[11C]YH132 as a novel MAGL PET tracer. It demonstrates an improved pharmacokinetic profile compared to its racemate. High inâ vitro MAGL specificity of (R)-[11C]YH132 was confirmed by autoradiography studies using mouse and rat brain sections. In vivo, (R)-[11C]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)-[11C]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 inâ vitro autoradiography using brain sections from animal models of Alzheimer's disease and Parkinson's disease.
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Monoacilglicerol Lipases , Doenças Neurodegenerativas , Ratos , Camundongos , Animais , Monoacilglicerol Lipases/metabolismo , Doenças Neurodegenerativas/diagnóstico por imagem , Doenças Neurodegenerativas/tratamento farmacológico , Tomografia por Emissão de Pósitrons/métodos , Inflamação , Desenvolvimento de Medicamentos , Inibidores Enzimáticos/farmacologiaRESUMO
Pharmacological modulation of cannabinoid receptor type 2 (CB2R) holds promise for the treatment of neuroinflammatory disorders, such as Alzheimer's disease. Despite the importance of CB2R, 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 CB2R enabled by a novel synthetic strategy and application of platform reagents. The LDC modification allows visualization and study of CB2R 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 CB2R. We demonstrate selective, covalent labeling of a peripheral lysine residue of CB2R 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 CB2R in conventional and imaging flow cytometry as well as in confocal fluorescence microscopy using overexpressing and endogenously expressing microglial live cells.
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Corantes Fluorescentes , Transdução de Sinais , Ligantes , Ligação Proteica , Corantes Fluorescentes/química , Receptores de CanabinoidesRESUMO
Breakthroughs in life sciences require multidisciplinary research. Activities in academia and industry are often complementary, so collaborations between both parties hold great potential for achieving superior overall results and accelerating innovation in life sciences. This special collection highlights successful examples of academia industry collaborations in the field of chemical biology and should encourage future teamwork for the benefit of society.
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Biologia , IndústriasRESUMO
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.
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Química Farmacêutica , Descoberta de Drogas , Sistemas de Liberação de Medicamentos , BiologiaRESUMO
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 CB2R 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.
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Esclerose Lateral Amiotrófica/imunologia , Astrócitos/imunologia , NF-kappa B/imunologia , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/terapia , Animais , Astrócitos/patologia , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos , Microglia/imunologia , Microglia/patologia , Neurônios Motores/imunologia , Neurônios Motores/patologia , NF-kappa B/genética , Receptor CB2 de Canabinoide/agonistas , Receptor CB2 de Canabinoide/genética , Receptor CB2 de Canabinoide/imunologia , Superóxido Dismutase/genética , Superóxido Dismutase/imunologia , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/imunologiaRESUMO
N-acylethanolamines (NAEs), which include the endocannabinoid anandamide, represent an important family of signaling lipids in the brain. The lack of chemical probes that modulate NAE biosynthesis in living systems hamper the understanding of the biological role of these lipids. Using a high-throughput screen, chemical proteomics and targeted lipidomics, we report here the discovery and characterization of LEI-401 as a CNS-active N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) inhibitor. LEI-401 reduced NAE levels in neuroblastoma cells and in the brain of freely moving mice, but not in NAPE-PLD KO cells and mice, respectively. LEI-401 activated the hypothalamus-pituitary-adrenal axis and impaired fear extinction, thereby emulating the effect of a cannabinoid CB1 receptor antagonist, which could be reversed by a fatty acid amide hydrolase inhibitor. Our findings highlight the distinctive role of NAPE-PLD in NAE biosynthesis in the brain and suggest the presence of an endogenous NAE tone controlling emotional behavior.
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Comportamento Animal/efeitos dos fármacos , Inibidores Enzimáticos/química , Metabolismo dos Lipídeos/efeitos dos fármacos , Fosfatidiletanolaminas/metabolismo , Fosfolipase D/antagonistas & inibidores , Amidoidrolases/metabolismo , Animais , Proteínas Sanguíneas/metabolismo , Encéfalo/metabolismo , Antagonistas de Receptores de Canabinoides/metabolismo , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacocinética , Medo/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Receptores de Canabinoides/metabolismo , Transdução de SinaisRESUMO
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.
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The utilization of an activatable, substrate-based probe design in combination with a cellular targeting approach has been rarely explored for cancer imaging on a small-molecule basis, although such probes could benefit from advantages of both concepts. Cysteine proteases like cathepsin S are known to be involved in fundamental processes associated with tumor development and progression and thus are valuable cancer markers. We report the development of a combined dual functional DOTAM-based, RGD-targeted internally quenched fluorescent probe that is activated by cathepsin S. The probe exhibits excellent in vitro activation kinetics which can be fully translated to human cancer cell lines. We demonstrate that the targeted, activatable probe is superior to its nontargeted analog, exhibiting improved uptake into ανß3-integrin expressing human sarcoma cells (HT1080) and significantly higher resultant fluorescence staining. However, profound activation was also found in cancer cells with a lower integrin expression level, whereas in healthy cells almost no probe activation could be observed, highlighting the high selectivity of our probe toward cancer cells. These auspicious results show the outstanding potential of the dual functionality concept combining a substrate-based probe design with a targeting approach, which could form the basis for highly sensitive and selective in vivo imaging probes.
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Corantes Fluorescentes/química , Neoplasias/diagnóstico , Linhagem Celular Tumoral , Humanos , Neoplasias/patologia , Imagem Óptica/métodos , Sensibilidade e EspecificidadeRESUMO
The endocannabinoid (eCB) system is implied in various human diseases ranging from central nervous system to autoimmune disorders. Cannabinoid receptorâ 2 (CB2 R) is an integral component of the eCB system. Yet, the downstream effects elicited by this G protein-coupled receptor upon binding of endogenous or synthetic ligands are insufficiently understood-likely due to the limited arsenal of reliable biological and chemical tools. Herein, we report the design and synthesis of CB2 R-selective cannabinoids along with their in vitro pharmacological characterization (binding and functional studies). They combine structural features of HU-308 and AM841 to give chimeric ligands that emerge as potent CB2 R agonists with high selectivity over the closely related cannabinoid receptorâ 1 (CB1 R). The synthesis work includes convenient preparation of substituted resorcinols often found in cannabinoids. The utility of the synthetic cannabinoids in this study is showcased by preparation of the most selective high-affinity fluorescent probe for CB2 R to date.
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Aminas/química , Canabinoides/química , Dronabinol/análogos & derivados , Receptor CB2 de Canabinoide/metabolismo , Sítios de Ligação , Canabinoides/metabolismo , Dronabinol/química , Dronabinol/metabolismo , Humanos , Cinética , Ligantes , Simulação de Acoplamento Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Receptor CB1 de Canabinoide/química , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/químicaRESUMO
(1) Background: The cannabinoid 2 receptor (CB2R) is a promising anti-inflammatory drug target and development of selective CB2R ligands may be useful for treating sight-threatening ocular inflammation. (2) Methods: This study examined the pharmacology of three novel chemically-diverse selective CB2R ligands: CB2R agonists, RO6871304, and RO6871085, as well as a CB2R inverse agonist, RO6851228. In silico molecular modelling and in vitro cell-based receptor assays were used to verify CB2R 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 CB2R ligand, HU910, using an in vivo mouse model of endotoxin-induced uveitis (EIU) in wild-type (WT) and CB2R-/- mice. The actions of RO6871304 on neutrophil migration and adhesion were examined in vitro using isolated neutrophils from WT and CB2R-/- mice, and in vivo in WT mice with EIU using adoptive transfer of WT and CB2R-/- neutrophils, respectively. (3) Results: Molecular docking studies indicated that RO6871304 and RO6871085 bind to the orthosteric site of CB2R. Binding studies and cell signaling assays for RO6871304 and RO6871085 confirmed high-affinity binding to CB2R and selectivity for CB2R > CB1R, with both ligands acting as full agonists in cAMP and ß-arrestin assays (EC50s 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 CB2R agonist, RO6871304, decreased in vitro neutrophil migration of WT neutrophils but not neutrophils from CB2R-/-, and attenuated adhesion of adoptively-transferred leukocytes in EIU. (4) Conclusions: These unique ligands are potent and selective for CB2R 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 CB2R agonists support selective targeting of CB2R for treating ocular inflammatory diseases.
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Anti-Inflamatórios/administração & dosagem , Agonistas de Receptores de Canabinoides/administração & dosagem , Endotoxinas/efeitos adversos , Receptor CB2 de Canabinoide/antagonistas & inibidores , Uveíte/tratamento farmacológico , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Agonistas de Receptores de Canabinoides/química , Agonistas de Receptores de Canabinoides/farmacologia , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Receptor CB2 de Canabinoide/química , Receptor CB2 de Canabinoide/genética , Transdução de Sinais , Uveíte/induzido quimicamente , Uveíte/imunologiaRESUMO
Chemical tools and methods that report on G protein-coupled receptor (GPCR) expression levels and receptor occupancy by small molecules are highly desirable. We report the development of LEI121 as a photoreactive probe to study the type 2 cannabinoid receptor (CB2R), a promising GPCR to treat tissue injury and inflammatory diseases. LEI121 is the first CB2R-selective bifunctional probe that covalently captures CB2R upon photoactivation. An incorporated alkyne serves as ligation handle for the introduction of reporter groups. LEI121 enables target engagement studies and visualization of endogenously expressed CB2R in HL-60 as well as primary human immune cells using flow cytometry. Our findings show that strategically functionalized probes allow monitoring of endogenous GPCR expression and engagement in human cells using tandem photoclick chemistry and hold promise as biomarkers in translational drug discovery.
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Morfolinas/química , Marcadores de Fotoafinidade/química , Piridinas/química , Receptor CB2 de Canabinoide/biossíntese , Receptor CB2 de Canabinoide/metabolismo , Alcinos/química , Células HL-60 , Humanos , Ligantes , Estrutura Molecular , Morfolinas/síntese química , Marcadores de Fotoafinidade/síntese química , Piridinas/síntese químicaRESUMO
The endocannabinoid system, and in particular the cannabinoid type 2 receptor (CB2R), raised the interest of many medicinal chemistry programs for its therapeutic relevance in several (patho)physiologic processes. However, the physico-chemical properties of tool compounds for CB2R (e.g., the radioligand [3H]CP55,940) are not optimal, despite the research efforts in developing effective drugs to target this system. At the same time, the importance of drug-target binding kinetics is growing since the kinetic binding profile of a ligand may provide important insights for the resulting in vivo efficacy. In this context we synthesized and characterized [3H]RO6957022, a highly selective CB2R inverse agonist, as a radiolabeled tool compound. In equilibrium and kinetic binding experiments [3H]RO6957022 showed high affinity for human CB2R with fast association (kon) and moderate dissociation (koff) kinetics. To demonstrate the robustness of [3H]RO6957022 binding, affinity studies were carried out for a wide range of CB2R reference ligands, spanning the range of full, partial, and inverse agonists. Finally, we used [3H]RO6957022 to study the kinetic binding profiles (i.e., kon and koff values) of selected synthetic and endogenous (i.e., 2-arachidonoylglycerol, anandamide, and noladin ether) CB2R ligands by competition association experiments. All tested ligands, and in particular the endocannabinoids, displayed distinct kinetic profiles, shedding more light on their mechanism of action and the importance of association rates in the determination of CB2R affinity. Altogether, this study shows that the use of a novel tool compound, i.e., [3H]RO6957022, can support the development of novel ligands with a repertoire of kinetic binding profiles for CB2R.
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Canabinoides/agonistas , Canabinoides/metabolismo , Agonismo Inverso de Drogas , Receptor CB2 de Canabinoide/agonistas , Receptor CB2 de Canabinoide/metabolismo , Animais , Células CHO , Canabinoides/farmacologia , Cricetinae , Cricetulus , Cicloexanóis/metabolismo , Cicloexanóis/farmacologia , Relação Dose-Resposta a Droga , Humanos , Ligação Proteica/fisiologia , Trítio/metabolismoRESUMO
Upregulation of the cannabinoid type 2 receptors (CB2R) unveils inflammation processes of pathological disorders, such as cancer, pain, or neurodegenerative diseases. Among others, CB2R agonist A-836339 has been labeled with carbon-11 for PET imaging of the CB2R and displayed promising results in a mouse model of Alzheimer's disease. The aim of the present work was to develop fluorinated analogs of A-836339 for labeling with fluorine-18 to design a new PET tracer for CB2R imaging. Seven fluorinated analogs of A-836339 were synthesized in two to three steps and their binding affinities and selectivities for both the human and the mouse CB2R were measured as well as their early ADME profiles. Among them, compound 2f (KihCB2R = 0.1 nM, KihCB1R/KihCB2R = 300) displayed high affinity and selectivity for CB2R but also promising lipophilicity, kinetic solubility, and membrane permeation properties and was further selected for in vitro metabolism studies. Incubation of 2f with human or rat liver microsomes followed by LC/MS analysis revealed the presence of six different metabolites mainly resulting from oxidation reactions. A tosylated precursor of 2f was synthesized in two steps and radiolabeled with fluorine-18 to afford [18F]2f in 15 ± 5% radiochemical yield and a molar activity of 110 ± 30 GBq/µmol. Autoradiographies of rat spleen and biodistribution studies in healthy rats including pretreatments with either CB2R or CB1R-specific compounds suggested that [18F]2f is a specific tracer for the CB2R in vivo. We have therefore demonstrated here that [18F]2f is a promising novel tracer for imaging CB2R in vivo using PET. Further investigation in animal models of inflammation will follow.
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Tomografia por Emissão de Pósitrons/métodos , Animais , Humanos , Cinética , Camundongos , Ratos , Receptor CB2 de Canabinoide/metabolismo , Tiazóis/químicaRESUMO
INTRODUCTION: Cannabinoid receptor type 2 (CB2R), 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 CB2R ligands, therapeutic applications, synthesis, as well as formulations of CB2R modulators. EXPERT OPINION: The patents cover a vast, structurally diverse chemical space. The focus of CB2R ligand development has shifted from unselective dual-cannabinoid receptor type 1 (CB1R) and 2 agonists toward agonists with high selectivity over CB1R, particularly for indications associated with inflammation and tissue injury. Currently, there are at least eight CB2R agonists and one antagonist in active clinical development. A better understanding of the endocannabinoid system (ECS) and in particular of CB2R pharmacology is required to unlock the receptor's full therapeutic potential.
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Agonistas de Receptores de Canabinoides , Desenvolvimento de Medicamentos , Patentes como Assunto , Receptor CB2 de Canabinoide , Humanos , Receptor CB2 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/agonistas , Receptor CB2 de Canabinoide/antagonistas & inibidores , Animais , Ligantes , Agonistas de Receptores de Canabinoides/farmacologia , Antagonistas de Receptores de Canabinoides/farmacologiaRESUMO
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.
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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. 3H and 14C 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.
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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. 3H and 14C 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.
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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.
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Aprendizado Profundo , Ensaios de Triagem em Larga EscalaRESUMO
This study aimed to evaluate (R)-[18F]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)-[18F]YH134 brain uptake and its application in brain-periphery crosstalk were explored. Methods: MAGL knockout and wild-type mice were used to evaluate (R)-[18F]YH134 in in vitro autoradiography and PET experiments. To explore the impact of peripheral MAGL occupancy on (R)-[18F]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)-[18F]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)-[18F]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)-[18F]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)-[18F]YH134 PET imaging data from the brain. Conclusion: (R)-[18F]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)-[18F]YH134. (R)-[18F]YH134 can be used for estimating the dose of MAGL inhibitor at half-maximal peripheral target occupancy.