Chemical Probes for Investigating the Endocannabinoid System

Cannabis sativa has been used therapeutically since early civilizations, with key cannabinoids Δ⁹-tetrahydrocannabinol (THC) 3.1 and cannabidiol characterized in the 1960s, leading to the discovery of cannabinoid receptors type 1 (CB₁R) and type 2 (CB₂R) 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. CB₁R and CB₂R, 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 CB₁R and CB₂R probes have enhanced CB receptor studies. CB₂R 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.