Cannabinoid Receptor Pharmacology: Binding Mechanisms, Brain Targets, and Assay Strategies

The endocannabinoid system (ECS) has become a central focus in neuroscience and drug discovery, offering therapeutic opportunities in pain management, neurodegeneration, inflammation, and psychiatric disorders. At the heart of this system are cannabinoid receptors, which interact with endogenous ligands and exogenous compounds to regulate multiple physiological and cognitive functions. Understanding receptor biology, agonist and antagonist pharmacology, and the right assay strategies is essential for advancing the next generation of cannabinoid-based therapeutics.

What Are Cannabinoid Receptors and How Do They Work?

Cannabinoid receptors are G protein-coupled receptors (GPCR) that mediate the effects of endogenous cannabinoids (endocannabinoids, like anandamide) and external compounds (including phytocannabinoids and synthetic cannabinoids). Their primary role is to regulate neurotransmitter release and maintain homeostasis across several organ systems.

The two primary cannabinoid receptors in the body are CB₁ and CB₂. Both signal primarily through Gi/o proteins, inhibiting adenylyl cyclase, reducing cyclic adenosine monophosphate (cAMP) levels, and modulating ion channel activity. This cannabinoid receptor signaling influences synaptic plasticity, immune response, and metabolic pathways. The complexity of the cannabinoid receptor function explains why they influence such diverse processes, ranging from neuroprotection to inflammation control.

While their general actions are well-documented, research continues to refine our knowledge of cannabinoids, including less common sub-localizations and their impact on various diseases. For instance,  links between cannabinoid receptors in Attention-Deficit/Hyperactivity Disorder (ADHD) and altered endocannabinoid tone are being investigated, highlighting new therapeutic frontiers.

CB₁ and CB₂ Receptor Functions, Locations, and Brain Impact

• CB₁ receptors

The CB₁ receptor function is primarily neuronal, modulating neurotransmission. Its location is enriched in the cortex, basal ganglia, cerebellum, hippocampus, and amygdala, regions linked with memory, motor coordination, reward, and emotional regulation. 

These receptors are highly sensitive to overstimulation by cannabinoids, which can trigger desensitization, a process in which receptor responsiveness decreases even in the continued presence of agonists. Prolonged exposure reduces overall receptor signaling and contributes to tolerance, fueling interest in strategies aimed at optimizing CB₁ receptor recovery time following chronic cannabinoid use. 

• CB₂ receptors

CB₂ receptors, in contrast, are expressed in immune and peripheral tissues, though evidence shows they also exist in glial cells within the central nervous system (CNS). They are strongly implicated in immune modulation, inflammation control, and neuroinflammation-associated diseases. 

The role of cannabinoid receptors in the brain extends beyond acute signaling. CB₁ dysregulation has been linked to psychological and psychiatric conditions such as anxiety, depression, and even ADHD associations in preclinical models. These insights are critical for designing targeted therapies that minimize psychoactive side effects while preserving therapeutic benefits.

Cannabinoid Receptor Agonists: Types, Binding Dynamics, and Therapeutic Roles

Agonists are ligands that activate cannabinoid receptors. They can be endogenous, exogenous, or synthetic:

• Endocannabinoids: naturally produced ligands such as anandamide and 2-arachidonoylglycerol. 
• Phytocannabinoids: plant-derived ligands, such as Δ9-tetrahydrocannabinol (THC), which primarily bind to cannabinoid receptor CB₁, and cannabidiol (CBD), which has more complex modulatory effects.
• Synthetic agonists: designed for research or therapeutic use, some with high receptor selectivity. They provide valuable insights into receptor pharmacodynamics.

The binding of a cannabinoid receptor ligand to CB₁ or CB₂ depends on receptor structure, conformation, and tissue-specific expression. They can act as full, partial, or biased agonists depending on how they interact with receptor conformations. 

Therapeutically, agonists are explored for neuropathic pain, epilepsy, and neurodegenerative disorders. Beyond CNS disorders, they hold promise in inflammatory diseases, where activation of CB2 suppresses immune hyperactivity without psychoactive effects.

Figure 1. General structure of the library and diversity elements included in the synthesis and pharmacological characterization (at CB1R and CB2R) of the largest published collection of Synthetic cannabinoid receptor agonists (SCRAs). Source: Gioé-Gallo C, et al. Pharmacological insights emerging from the characterization of a large collection of synthetic cannabinoid receptor agonists designer drugs. Biomed Pharmacother. 2023 Aug;164:114934. doi: 10.1016/j.biopha.2023.114934

Cannabinoid Receptor Antagonists: Mechanisms and Drug Binding Profiles

Cannabinoid receptor antagonists block the activity of the endocannabinoid system by competing with endogenous cannabinoids for binding sites on the receptors. By occupying these sites, they prevent receptor activation and inhibit downstream cellular signaling pathways. These antagonists can act as inverse agonists or neutral antagonists, modulating receptor activity differently. Recent studies using fluorescence-based assays have characterized key kinetic parameters, revealing that binding affinity depends on association and dissociation rates, which vary between CB1 and CB2 receptors.

Antagonists like rimonabant (CB₁ selective) or AM630 (CB₂ selective) are widely used in research to dissect receptor pathways and clarify how different binding affinities translate into clinical profiles.

Examples of well-studied cannabinoid receptor antagonists include:

• Rimonabant: CB₁ inverse agonist with rapid association and moderate dissociation rates.
• SR144528: Highly selective CB₂ antagonist with slow dissociation, promoting sustained receptor blockade. It was also reported to be an inverse agonist
• Taranabant: Peripherally restricted CB₁ antagonist aimed at minimizing central side effects.
• AM6545: CB₁ neutral antagonist with peripheral restriction properties.

Understanding these profiles aids drug development by optimizing efficacy, selectivity, and safety.

Restoring and Modulating the Endocannabinoid System: Assay Strategies

Understanding how different compounds bind to and modulate cannabinoid receptors is a crucial step in the development of new therapeutics targeting the endocannabinoid system. In early drug discovery, the goal is to generate controlled in vitro models that allow precise measurement of ligand–receptor interactions before studying receptor expression directly in patient-derived tissues.

Traditional radioligand binding assays have long served this purpose, but they present several limitations:

• The need to handle radioactive materials
  
• Reliance on membrane fractions instead of intact cellular systems
  
• Lack of dynamic and spatial information about receptor activity

Fluorescence-based high-content screening (HCS) assays now offer a paradigm shift in receptor pharmacology. They provide multiparametric, image-based data from living cells. Unlike traditional radioligand binding assays that use isolated membrane preparations, HCS assays measure ligand-receptor interactions in whole cells expressing high receptor levels, thus closely mimicking physiological conditions. This method not only quantifies binding affinity but also provides visual confirmation of compound engagement, enabling the simultaneous assessment of cellular health.

Figure 2. CELT-331 fluorescent ligand labeling CB2 receptors. The reduction in fluorescence signal observed as the concentration of the compound increases enables the determination of the affinity value of the compound for CB2.

Building on these benefits, at Celtarys, we offer specialized HCS cannabinoid assay kits and screening services for both receptors. These services combine the sensitivity and specificity of HCS technology with rapid, client-oriented turnaround times, delivering comprehensive affinity data alongside high-quality imaging outputs. By working with whole cells rather than membrane fragments, Celtarys’ platform provides more robust and translationally relevant data to support cannabinoid drug discovery efforts.

Boost your cannabinoid drug discovery with our expert screening services. Contact us to learn how to accelerate your pipeline!

References

Borrega-Roman L, Hoare BL, Kosar M, Sarott RC, Patej KJ, Bouma J, Scott-Dennis M, Koers EJ, Gazzi T, Mach L, Barrondo S, Sallés J, Guba W, Kusznir E, Nazaré M, Rufer AC, Grether U, Heitman LH, Carreira EM, Sykes DA, Veprintsev DB. A universal cannabinoid CB1 and CB2 receptor TR-FRET kinetic ligand-binding assay. Front Pharmacol. 2025 Apr 9;16:1469986. doi: 10.3389/fphar.2025.1469986

Howlett AC, Abood ME. CB1 and CB2 Receptor Pharmacology. Adv Pharmacol. 2017;80:169-206. doi: 10.1016/bs.apha.2017.03.007 

Kumari P, Dvorácskó S, Enos MD, Ramesh K, Lim D, Hassan SA, Kunos G, Cinar R, Iyer MR, Rosenbaum DM. Structural mechanism of CB1R binding to peripheral and biased inverse agonists. Nat Commun. 2024 Dec 18;15(1):10694. doi: 10.1038/s41467-024-54206-0

Maldonado R, Cabañero D, Martín-García E. The endocannabinoid system in modulating fear, anxiety, and stress
. Dialogues Clin Neurosci. 2020 Sep;22(3):229-239. doi: 10.31887/DCNS.2020.22.3/rmaldonado

Navarrete F, García-Gutiérrez MS, Jurado-Barba R, Rubio G, Gasparyan A, Austrich-Olivares A, Manzanares J. Endocannabinoid System Components as Potential Biomarkers in Psychiatry. Front Psychiatry. 2020 Apr 27;11:315. doi: 10.3389/fpsyt.2020.00315

Navarro G, Sotelo E, Raïch I, Loza MI, Brea J, Majellaro M. A Robust and Efficient FRET-Based Assay for Cannabinoid Receptor Ligands Discovery. Molecules. 2023 Dec 15;28(24):8107. doi: 10.3390/molecules28248107

Vasincu A, Rusu RN, Ababei DC, Neamțu M, Arcan OD, Macadan I, Beșchea Chiriac S, Bild W, Bild V. Exploring the Therapeutic Potential of Cannabinoid Receptor Antagonists in Inflammation, Diabetes Mellitus, and Obesity. Biomedicines. 2023 Jun 8;11(6):1667. doi: 10.3390/biomedicines11061667