RESUMO
The cannabinoid receptor type 1 (CB1R) is pivotal within the endocannabinoid system regulating various signaling cascades with effects in appetite regulation, pain perception, memory formation, and thermoregulation. Still, understanding of CB1R's cellular signaling, distribution, and expression dynamics is very fragmentary. Real-time visualization of CB1R is crucial for addressing these questions. Selective drug-like CB1R ligands with a defined pharmacological profile were investigated for the construction of CB1R 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 CB1R fluorescent probes. Application in fluorescence-based target-engagement studies and live cell imaging exemplify the great versatility of the tailored CB1R probes for investigating CB1R localization, trafficking, pharmacology, and its pathological implications.
Assuntos
Corantes Fluorescentes , Receptor CB1 de Canabinoide , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Receptor CB1 de Canabinoide/metabolismo , Humanos , Simulação de Acoplamento Molecular , Células HEK293 , Ligantes , Pirróis/química , Pirróis/farmacologia , Pirróis/síntese química , Relação Estrutura-Atividade , AMP Cíclico/metabolismoRESUMO
Evaluation of kinetic parameters of drug-target binding, kon, koff, and residence time (RT), in addition to the traditional in vitro parameter of affinity is receiving increasing attention in the early stages of drug discovery. Target binding kinetics emerges as a meaningful concept for the evaluation of a ligand's duration of action and more generally drug efficacy and safety. We report the biological evaluation of a novel series of spirobenzo-oxazinepiperidinone derivatives as inhibitors of the human equilibrative nucleoside transporter 1 (hENT1, SLC29A1). The compounds were evaluated in radioligand binding experiments, i.e., displacement, competition association, and washout assays, to evaluate their affinity and binding kinetic parameters. We also linked these pharmacological parameters to the compounds' chemical characteristics, and learned that separate moieties of the molecules governed target affinity and binding kinetics. Among the 29 compounds tested, 28 stood out with high affinity and a long residence time of 87 min. These findings reveal the importance of supplementing affinity data with binding kinetics at transport proteins such as hENT1.
Assuntos
Transportador Equilibrativo 1 de Nucleosídeo , Tioinosina , Humanos , Transporte Biológico , Tioinosina/metabolismo , Tioinosina/farmacologia , Transportador Equilibrativo 1 de Nucleosídeo/química , Transportador Equilibrativo 1 de Nucleosídeo/metabolismoRESUMO
Cannabinoid CB2 receptor (CB2R) is a class A G protein-coupled receptor (GPCR) involved in a broad spectrum of physiological processes and pathological conditions. For that reason, targeting CB2R might provide therapeutic opportunities in neurodegenerative disorders, neuropathic pain, inflammatory diseases, and cancer. The main components from Cannabis sativa, such as Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD), have been therapeutically exploited and synthetically-derived analogs have been generated. One example is cannabidiol-dimethylheptyl (CBD-DMH), which exhibits anti-inflammatory effects. Nevertheless, its pharmacological mechanism of action is not yet fully understood and is hypothesized for multiple targets, including CB2R. The aim of this study was to further investigate the molecular pharmacology of CBD-DMH on CB2R while CBD was taken along as control. These compounds were screened in equilibrium and kinetic radioligand binding studies and various functional assays, including G protein activation, inhibition of cAMP production and ß-arrestin-2 recruitment. In dissociation studies, CBD-DMH allosterically modulated the radioligand binding. Furthermore, CBD-DMH negatively modulated the G protein activation of reference agonists CP55,940, AEA and 2-AG, but not the agonist-induced ß-arrestin-2 recruitment. Nevertheless, CBD-DMH also displayed competitive binding to CB2R and partial agonism on G protein activation, inhibition of cAMP production and ß-arrestin-2 recruitment. CBD did not exhibit such allosteric behavior and only very weakly bound CB2R without activation. This study shows a dual binding mode of CBD-DMH, but not CBD, to CB2R with the suggestion of two different binding sites. Altogether, it encourages further research into this dual mechanism which might provide a new class of molecules targeting CB2R.
Assuntos
Canabidiol , Canabidiol/farmacologia , Receptores de Canabinoides/metabolismo , beta-Arrestina 1/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Dronabinol , Receptor CB1 de Canabinoide/metabolismo , Agonistas de Receptores de CanabinoidesRESUMO
Cannabinoid CB2 receptor (CB2R) agonists are investigated as therapeutic agents in the clinic. However, their molecular mode-of-action is not fully understood. Here, we report the discovery of LEI-102, a CB2R agonist, used in conjunction with three other CBR ligands (APD371, HU308, and CP55,940) to investigate the selective CB2R activation by binding kinetics, site-directed mutagenesis, and cryo-EM studies. We identify key residues for CB2R activation. Highly lipophilic HU308 and the endocannabinoids, but not the more polar LEI-102, APD371, and CP55,940, reach the binding pocket through a membrane channel in TM1-TM7. Favorable physico-chemical properties of LEI-102 enable oral efficacy in a chemotherapy-induced nephropathy model. This study delineates the molecular mechanism of CB2R activation by selective agonists and highlights the role of lipophilicity in CB2R engagement. This may have implications for GPCR drug design and sheds light on their activation by endogenous ligands.
Assuntos
Agonistas de Receptores de Canabinoides , Canabinoides , Agonistas de Receptores de Canabinoides/farmacologia , Receptores de Canabinoides , Canabinoides/farmacologia , Receptor CB1 de Canabinoide/genética , Receptor CB2 de Canabinoide/genéticaRESUMO
Cannabinoid receptor 1 (CB1R) and cannabinoid receptor 2 (CB2R) are G protein-coupled receptors (GPCRs) that activate a variety of pathways upon activation by (partial) agonists including the G protein pathway and the recruitment of ß-arrestins. Differences in the activation level of these pathways lead to biased signaling. Here, we describe a detailed protocol to characterize the potency and efficacy of ligands to induce or inhibit ß-arrestin recruitment to the human CB1R and CB2R using the PathHunter® assay. This is a cellular assay that uses a ß-galactosidase complementation system which has a chemiluminescent read-out and can be performed in 384-well plates. We have successfully used this assay to characterize a set of reference ligands (both agonists, antagonists, and an inverse agonist) on human CB1R and CB2R, of which some examples will be presented here.