Molecular Pharmacology of Synthetic Cannabinoids: Delineating CB1 Receptor-Mediated Cell Signaling

Abstract

Synthetic cannabinoids (SCs) are a class of new psychoactive substances (NPSs) that exhibit high affinity binding to the cannabinoid CB1 and CB2 receptors and display a pharmacological profile similar to the phytocannabinoid (-)-trans-Δ⁹-tetrahydrocannabinol (THC). SCs are marketed under brand names such as K2 and Spice and are popular drugs of abuse among male teenagers and young adults. Since their introduction in the early 2000s, SCs have grown in number and evolved in structural diversity to evade forensic detection and drug scheduling. In addition to their desirable euphoric and antinociceptive effects, SCs can cause severe toxicity including seizures, respiratory depression, cardiac arrhythmias, stroke and psychosis. Binding of SCs to the CB1 receptor, expressed in the central and peripheral nervous systems, stimulates pertussis toxin-sensitive G proteins (G_i/G_o) resulting in the inhibition of adenylyl cyclase, a decreased opening of N-type Ca²⁺ channels and the activation of G protein-gated inward rectifier (GIRK) channels. This combination of signaling effects dampens neuronal activity in both CNS excitatory and inhibitory pathways by decreasing action potential formation and neurotransmitter release. Despite this knowledge, the relationship between the chemical structure of the SCs and their CB1 receptor-mediated molecular actions is not well understood. In addition, the potency and efficacy of newer SC structural groups has not been determined. To address these limitations, various cell-based assay technologies are being utilized to develop structure versus activity relationships (SAR) for the SCs and to explore the effects of these compounds on noncannabinoid receptor targets. This review focuses on describing and evaluating these assays and summarizes our current knowledge of SC molecular pharmacology.

Keywords: CB1 receptors; cell signaling assays; molecular pharmacology; synthetic cannabinoids.

Figure 1

Structural classes of synthetic cannabinoids. Chemical structures of WIN 55,212-2 (aminoalkylindole), JWH-018 (naphtholylindole), XLR-11 (tetramethylcyclopropyl), AB-BICA (indole carboxamide), CP 55,940 (cyclohexylphenol), BB-22 (quinolinyl ester), MDMB-FUBINACA (indazole carboxamide) and AB-FUB7AICA (7-azaindole carboxamide).

Figure 2

Cannabinoid CB1 receptor structure and signaling. (A) Structural model of the CB1 receptor (CB1R)-G_i protein complex obtained from cryoelectron microscopy. The binding site for MDMB-FUBINACA (FUB) is indicated by the magenta SC structure. The CB1-G_i receptor complex structure was obtained from the Protein Data Bank (code 6N4B). (B) Binding of SCs to the CB1 receptor stimulates both neuronal G_i/G_o and β-arrestin signaling pathways (see text for description). In addition, activation of inotropic transient receptor potential (TRP) channels by cannabinoids causes Ca²⁺ influx into the neuron.

Figure 3

Cell-based assay technologies used for delineating SC-mediated signaling. (A) Fragment complementation assays consist of inactive enzyme donor (ED) and enzyme acceptor (EA) components that form an active enzyme when combined. For measuring 3′,5′-adenosine monophosphate (cAMP), the ED consists of an inactive fragment of β-galactosidase that is conjugated to cAMP. When cellular cAMP levels are low or absent, the conjugated cAMP is sequestered by the cAMP antibody and no active enzyme is formed. In the presence of high levels of cAMP (as shown), the ED-cAMP conjugate is free to combine with the EA. β-galactosidase activity is then be detected by adding a substrate that is converted to a fluorescent or luminescent signal. (B) Cyclic nucleotide-gated (CNG) channel cAMP assay. Opening of CNG channels during elevations in intracellular cAMP allows Ca²⁺ to enter the cell and bind to Ca²⁺-sensitive fluorescent dye molecules. (C) Bioluminescence resonance energy transfer (BRET) assays use a biosensor consisting of a BRET donor (D) and acceptor (A) pair. The BRET cAMP sensor consists of a cAMP binding protein coupled to the BRET donor, Renilla luciferase (RLuc) and acceptor, yellow fluorescent protein (YFP). Binding of cAMP to the sensor (as shown) results in a conformational change and a loss of BRET intensity. (D) G protein-gated inward rectifier (GIRK) channel fluorescent membrane potential-sensitive dye (MPSD) assay. Hyperpolarization/depolarization of the cell resting membrane potential (left & right arrows), resulting from G_i protein βγ subunit (G_iβγ) opening/closing of the GIRK channels, alters the distribution of MPSD molecules across the plasma membrane and thus the fluorescent signal. Figure 3A [70] was adapted with permission of Cambridge University Press through PLSclear. Figure 3C,D [71] were reproduced by permission from BMG Labtech and Taylor & Francis Ltd. (www.tandfonline.com), respectively.