Cannabinoid CB 1 and CB 2 receptors are activated by Δ 9-tetrahydrocannabinol, a psychoactive component of marijuana. The cannabinoid CB 1 receptor is primarily located in the brain and is responsible for the psychoactive side effects, whereas the cannabinoid CB 2 receptor is located in immune cells and is an attractive target for immune-related maladies. We identify small molecules that selectively bind to the cannabinoid CB 2 receptor and can be further developed into therapeutics. The affinity of three molecules, ABK5, ABK6, and ABK7, to the cannabinoid CB 2 receptor was determined with radioligand competition binding. The potency of G-protein coupling was determined with GTPγS binding. The three compounds bound selectively to the cannabinoid CB 2 receptor, and no binding to the cannabinoid CB 1 receptor was detected up to 10 μM. Immunoblotting studies show that the amount of ERK1/2 and MEK phosphorylation increased in a G i/o-dependent manner. Furthermore, an immune cell line (Jurkat cells) was treated with ABK5, and as a result, inhibited cell proliferation. These three compounds are novel cannabinoid CB 2 receptor agonists and hold promise to be further developed to treat inflammation and the oftenassociated pain.

Cannabinoid receptor 1 (CB 1) is a G-protein-coupled receptor that is abundant in the central nervous system. It binds several compounds in its orthosteric site, including the endocannabinoids, arachidonoyl ethanolamide (anandamide) and 2-arachidonoyl glycerol, and the plant-derived D 9-tetrahydrocannabinol, one of the main psychoactive components of marijuana. It primarily couples to G i/o proteins to inhibit adenylate cyclase activity and typically induces downstream signaling that is G idependent. Since this receptor is implicated in several maladies, such as obesity, pain, and neurodegenerative disorders, there is interest in developing therapeutics that selectively target this receptor. Allosteric modulators of CB 1 offer one new approach that has tremendous therapeutic potential. Here, we reveal receptor-and cellular-level properties consistent with receptor activation by a series of pyrimidinyl biphenylureas (LDK1285, LDK1288, LDK1305, and PSNCBAM1), including promoting binding of the agonist CP55940 with positive cooperativity and inhibiting binding of the inverse agonist SR141716A with negative cooperativity, demonstrated via radioligand binding studies. Consistent with these findings, the allosteric modulators induced cellular internalization of the receptor and recruitment of b-arrestin 2 in human embryonic kidney cell line 293 cells monitored with confocal and total internal reflective fluorescence microscopy, respectively. These allosteric modulators, however, caused G-protein-independent but b-arrestin 1-dependent phosphorylation of the downstream kinases extracellular signal-regulated kinase 1/2, mitogen-activated protein kinase, and Src, shown by immunoblotting studies. These results are consistent with the involvement of b-arrestin and suggest that these allosteric modulators induce biased signaling.

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Activation of the CB2 receptor has been shown to have anti-inflammatory and antinociceptive effects without causing psychoactive effects. Previously, we reported that the compound ethyl 2(2-(N-(2,3-dimethylphenyl) phenylsulfonamido)acetamido)benzoate (ABK5) is a CB2 subtype selective agonist with anti-inflammatory and antinociceptive effects. In the present study, we tested four ABK5 derivatives, ABK5-1, ABK5-2, ABK5-5, and ABK5-6, to analyze the structure of ABK5 to obtain CB2-selective agonists with higher affinity and efficacy. Affinity, subtype selectivity, and G-protein coupling were determined by radioligand binding assays. Selected compounds were then subjected to evaluation of anti-inflammatory effects using two different cell lines, Jurkat (ABK5-1 and 5-2) and BV-2 cells (ABK5-1), which are models of T cells and microglia, respectively. ABK5-1, ABK5-2, and ABK5-6 had comparable CB2 binding affinity with ABK5 (and stimulated G-protein coupling), while only ABK5-1 and ABK5-2 maintained CB2-subtype selectivity. ABK5-5 did not bind CB2 in the detectable range. RT-PCR and ELISA analysis showed that the two compounds also inhibit IL-2 and TNF-α production, and they were more efficacious than ABK5 in inhibiting TNF-α production. CXCL-12 mediated chemotaxis was also evaluated by the transwell migration assay, and both ABK5-1 and ABK5-2 inhibited chemotaxis with a stronger effect observed in ABK5-1. In the microglia cell line BV-2, ABK5-1 inhibited IL-1β and IL-6 production, which suggests this compound has anti-inflammatory effects through targeting multiple immune cells, and may be a candidate for treatment of inflammation.

Background and objectives: Neuroinflammation is closely associated with various diseases including neuropathic pain. Microglia are immune cells in the central nervous system which are the main players of immunity and inflammation. Since microglia are activated by nerve injury, and they produce proinflammatory mediators to cause neuropathic pain, targeting activated microglia is considered to be a strategy for treating neuropathic pain. Activation of the cannabinoid CB 2 receptor is known to have anti-inflammatory effects in microglia. ABK5-1 is a CB 2 subtype selective agonist which inhibits IL-1β and IL-6 production in the microglia cell line BV-2. The purpose of the current study is to further analyze anti-inflammatory effects of ABK5 in terms of different cytokines and the possible pathway involved in the effect in the BV-2 cell line.Methods: A cytokine array was performed to screen the effect of ABK5-1 on forty inflammatory mediators in BV-2 cells. Changes of the inflammatory mediators was further supported by mRNA analysis, and a possible signaling molecule that involved the observation was evaluated by western blot.Results: Stimulating BV-2 cells by lipopolysaccharide increased expression of eleven inflammatory mediators, and ABK5-1 treatment resulted in more than a 50% decrease of sICAM1, IL-6, and RANTES. Real-time PCR results showed a decrease of G-CSF, ICAM1, MCP-1, MIP-1α, and MIP-1β mRNA levels. Western blot analysis showed that ABK5-1 inhibited LPS-induced ERK phosphorylation, which can be a mechanism of ABK5-1-mediated anti-inflammatory effect. Conclusions:Our current results support the possibility that ABK5-1 is an anti-inflammatory drug for microglia.