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Docking tangible virtual libraries can reveal unexpected chemotypes that complement the structures of biological targets. Seeking new agonists for the cannabinoid-1 receptor (CB1R), we docked 74 million tangible molecules, prioritizing 46 high ranking ones forde novosynthesis and testing. Nine were active by radioligand competition, with > 50% radioligand displacement, a 20% hit-rate. Structure-based optimization of one of the most potent of these (Ki= 731 nM) led to‘3234, a 1.9 nM binder and a full CB1 agonist. A cryo-EM structure of the‘3234-CB1-Gi1complex confirmed its docked pose, providing a template for further optimization. The new agonist was strongly analgesic especially against thermal pain, with a 10-fold therapeutic window over sedation and no observable catalepsy or conditioned place preference or aversion. These findings suggest that new cannabinoid chemotypes may be able to disentangle the characteristic “tetrad” side-effects from its desired analgesic effect, supporting the further development of cannabinoids as pain therapeutics.

We report the development of novel cannabinergic probes that can stabilize the cannabinoid receptors (CBRs) through tight binding interactions. Ligand design involves the introduction of select groups at a judiciously chosen position within the classical hexahydrocannabinol template (monofunctionalized probes). Such groups include the electrophilic isothiocyanato, the photoactivatable azido, and the polar cyano moieties. These groups can also be combined to produce bifunctionalized probes potentially capable of interacting at two distinct sites within the CBR-binding domains. These novel compounds display remarkably high binding affinities for CBRs and are exceptionally potent agonists. A key ligand (27a, AM11245) exhibits exceptionally high potency in both in vitro and in vivo assays and was designated as "megagonist," a property attributed to its tight binding profile. By acting both centrally and peripherally, 27a distinguishes itself from our previously reported "megagonist" AM841, whose functions are restricted to the periphery.

Although the behavioral effects of acute and chronic exposure to cannabinoids have been extensively studied in mice, spontaneous withdrawal following exposure to cannabinoids has not been well characterized in this species. To address this issue, different groups of mice were treated for 5 days with saline, 20-36 mg/kg/day of the CB partial agonist Δ(9)-tetrahydrocannabinol (Δ(9)-THC), or 0.06-0.1 mg/kg/day of the CB high-efficacy agonist AM2389. Initial studies assessed changes in observable behavior (paw tremors) that were scored from the recordings taken at 4 or 24 h after the last injection. Subsequently, radiotelemetry was used to continuously measure body temperature and locomotor activity before (baseline), during, and after the 5-day dosing regimens. Results show that increases in paw tremors occurred following 5-day exposure to AM2389 or Δ(9)-THC. In telemetry studies, acute AM2389 or THC decreased both temperature and activity. Rapid tolerance occurred to the hypothermic effects of the cannabinoids, whereas locomotor activity continued to be suppressed following each drug injection. In contrast, increases in locomotor activity were evident 12-72 h after discontinuing daily injections of either 0.06 or 0.1 mg/kg/day AM2389. Increases in locomotor activity were also noted in mice treated daily with 30 or 36, but not 20 mg/kg/day Δ(9)-THC; these effects were smaller and appeared later than effects seen in AM2389-treated mice. These results indicate that the discontinuation of daily treatment with a CB high-efficacy agonist will yield evidence of spontaneous withdrawal that may reflect prior dependence, and that the degree of cannabinoid dependence may vary in relation to the dose or efficacy of the agonist injected daily.

Cessation of cannabinoid use in humans often leads to a withdrawal state that includes sleep disruption. Despite important health implications, little is known about how cannabinoid abstention affects sleep architecture, in part because spontaneous cannabinoid withdrawal is difficult to model in animals. In concurrent work we report that repeated administration of the high-efficacy cannabinoid 1 (CB1) receptor agonist AM2389 to mice for five days led to heightened locomotor activity and paw tremor following treatment discontinuation, potentially indicative of spontaneous cannabinoid withdrawal. Here, we performed parallel studies to examine effects on sleep. Using implantable electroencephalography (EEG) and electromyography (EMG) telemetry we examined sleep and neurophysiological measures before, during, and after five days of twice-daily AM2389 injections. We report that AM2389 produces decreases in locomotor activity that wane with repeated treatment, whereas discontinuation produces rebound increases in activity that persist for several days. Likewise, AM2389 initially produces profound increases in slow-wave sleep (SWS) and decreases in rapid eye movement (REM) sleep, as well as consolidation of sleep. By the third AM2389 treatment, this pattern transitions to decreases in SWS and total time sleeping. This pattern persists following AM2389 discontinuation and is accompanied by emergence of sleep fragmentation. Double-labeling immunohistochemistry for hypocretin/orexin (a sleep-regulating peptide) and c-Fos (a neuronal activity marker) in lateral hypothalamus revealed decreases in c-Fos/orexin+ cells following acute AM2389 and increases following discontinuation, aligning with the sleep changes. These findings indicate that AM2389 profoundly alters sleep in mice and suggest that sleep disruption following treatment cessation reflects spontaneous cannabinoid withdrawal.

Structure-based virtual ligand screening is emerging as a key paradigm for early drug discovery owing to the availability of high-resolution target structures(1-4) and ultra-large libraries of virtual compounds(5,6). However, to keep pace with the explosive growth of virtual libraries, such as REadily AvaiLable for synthesis (REAL) combinatorial libraries(7), new approaches to compound screening are needed(8,9). Here we introduce a novel synthon-based approach, V-SYNTHES, to perform hierarchical structure-based screening of REAL library of more than 11 billion compounds. V-SYNTHES first identifies the best synthon-scaffold combinations as seeds suitable for further growth, and then iteratively elaborates these seeds to select complete molecules with the best docking scores. This hierarchical combinatorial approach allows rapid detection of the best-scoring compounds in the Giga-scale chemical space while performing docking of only a small fraction (

In earlier work, we explored the SAR for the C3 side chain pharmacophore in the hexahydrocannabinol template represented by the drug nabilone, which resulted in the development of AM2389. In an effort for further optimization, we have merged features of nabilone and AM2389 and explored the C3 side chain with varying chain lengths and terminal substitutions. Of the compounds described here, a nabilone analog, AM8936, with the C6'-cyano-substituted side chain, was identified as the most successful analog capable of serving as a potential candidate for further development and a valuable tool for further in vivo studies. AM8936 behaved as a balanced and potent CB1 agonist in functional assays and was a potent and efficacious CB1 agonist in vivo. Our SAR studies are highlighted with the docking of AM8936 on the crystal structure of the hCB1 receptor.

The two main constituents oficannabis/iare Δsup9/sup-tetrahydrocannabinol (THC) and cannabidiol (CBD). While Δsup9/sup-THC pharmacology has been studied extensively, CBD-long considered inactive-is now the subject of vigorous research related to epilepsy, pain, and inflammation and is popularly embraced as a virtual cure-all. However, our understanding of CBD pharmacology remains limited, although CBD inhibits cannabinoid CBsub1/subreceptor signaling, likely as a negative allosteric modulator.iCannabis/isynthesizes (-)-CBD, but CBD can also exist as an enantiomer, (+)-CBD. We enantioselectively synthesized both CBD enantiomers using established conditions and describe here a new, practical, and reliable, NMR-based method for confirming the enantiomeric purity of two CBD enantiomers. We also investigated the pharmacology of (+)-CBD in autaptic hippocampal neurons, a well-characterized neuronal model of endogenous cannabinoid signaling, and in CHO-K1 cells. We report the inhibition constant for displacing CP55,940 at CBsub1/subby (+)-CBD, is 5-fold lower than (-)-CBD. We find that (+)-CBD is ∼10 times more potent at inhibiting depolarization-induced suppression of excitation (DSE), a form of endogenous cannabinoid-mediated retrograde synaptic plasticity. (+)-CBD also inhibits CBsub1/subsuppression of cAMP accumulation but with less potency, indicating that the signaling profiles of the enantiomers differ in a pathway-specific manner. In addition, we report that (+)-CBD stereoselectively and potently activates the sphingosine-1 phosphate (S1P) receptors, S1Psub1/suband S1Psub3/subThese results provide an attractive method for synthesizing and distinguishing enantiomers of CBD and related phytocannabinoids and provide further evidence that these enantiomers have their own unique and interesting signaling properties. SIGNIFICANCE STATEMENT: Cannabidiol (CBD) is the subject of considerable scientific and popular interest, but we know little of the enantiomers of CBD. We find that the enantiomer (+)-CBD is substantially more potent inhibitor of cannabinoid CBsub1/subreceptors and that it activates sphingosine-1-phosphate receptors in an enantiomer-specific manner; we have additionally developed an improved method for the synthesis of enantiomers of CBD and related compounds.

Structure-based virtual ligand screening is emerging as a key paradigm for early drug discovery owing to the availability of high-resolution target structures

The endocannabinoid system is a complex network of lipids, G protein-coupled receptors (GPCRs), and metabolic enzymes and plays a major role in modulating key physiological processes, including nociception, inflammation, and metabolism. The type-1 cannabinoid receptor (CB1) holds great promise for treating CNS and metabolic disorders, pain and glaucoma. The CB1 receptor shows multiple activation mechanisms and it can signal through distinct pathways each of which potentially exhibiting different physiological outcomes. Naturally occurring compounds from Cannabis sativa (phytocannabinoids) and their synthetic derivatives (classical/non-classical cannabinoids), endogenous ligands (endocannabinoids) and their synthetic derivatives, and synthetic compounds of other chemical structures were developed as agonists of the CB1 receptor to promote its signalling. Also, ligands that inhibit the activation of the CB1 receptor have been shown to hold great promise in the clinical setting. This chapter summarizes the diverse panel of ligands that target the CB1 receptor and highlights their use as probes for investigating the modulation of the receptor's activation both in vitro and in vivo.

A new approach to synthesize cannabilactones using Suzuki cross-coupling reaction followed by one-step demethylation-cyclization is presented. The two key cannabilactone prototypes AM1710 and AM1714 were obtained selectively in high overall yields and in a lesser number of synthetic steps when compared to our earlier synthesis. The new approach expedited the synthesis of cannabilactone analogs with structural modifications at the four potential pharmacophoric regions.

Human endocannabinoid systems modulate multiple physiological processes mainly through the activation of cannabinoid receptors CB1 and CB2. Their high sequence similarity, low agonist selectivity, and lack of activation and G protein-coupling knowledge have hindered the development of therapeutic applications. Importantly, missing structural information has significantly held back the development of promising CB2-selective agonist drugs for treating inflammatory and neuropathic pain without the psychoactivity of CB1. Here, we report the cryoelectron microscopy structures of synthetic cannabinoid-bound CB2 and CB1 in complex with Gi, as well as agonist-bound CB2 crystal structure. Of important scientific and therapeutic benefit, our results reveal a diverse activation and signaling mechanism, the structural basis of CB2-selective agonists design, and the unexpected interaction of cholesterol with CB1, suggestive of its endogenous allosteric modulating role.

G-protein-coupled receptors (GPCRs) mediate the effects of approximately 33% of all marketed drugs. The development of tools to study GPCR pharmacology is urgently needed as it can lead to the discovery of safer and more effective medications. Fluorescent GPCR ligands represent highly sensitive and safe small-molecule tools for real-time exploration of the life of the receptor, cellular signaling, and ligand-/receptor-receptor interactions in cellulo and/or in vivo. Areas covered: This review summarizes relevant information from published literature and provides critical insights into the design of successful small-molecule fluorescent probes for Class A GPCRs as potential major targets for drug development. Expert opinion: Considering the rapid progress of fluorescence technologies, effective small-molecule fluorescent probes represent valuable pharmacological tools for studying GPCRs. However, the design and development of such probes are challenging, largely due to the low affinity/specificity of the probe for its target, inadequate photophysical properties, extensive non-specific binding, and/or low signal-to-noise ratio. Generally speaking, fluorescent and luminescent small-molecule probes, receptors, and G proteins in combination with FRET and BRET technologies hold great promise for studying kinetic profiles of GPCR signaling.