The NPXXY Motif Regulates ß-Arrestin Recruitment by the CB1 Cannabinoid Receptor.

Background: Activation of signaling effectors by G-protein coupled receptors (GPCRs) depends on different molecular mechanisms triggered by conserved amino acid residues. Although studies have focused on the G-protein signaling state, the mechanism for ß-arrestin signaling by CB1 is not yet well defined. Studies have indicated that transmembrane helix 7 (TMH7) and the highly conserved NPXXY motif can be subject to different conformational changes in response to biased ligands and could therefore participate in a molecular mechanism to trigger ß-arrestin recruitment. Objective: To investigate the effect of mutations in the NPXXY motif on different signaling pathways activated by the CB1 receptor. Materials and Methods: Point mutations of the NPXXY motif and associated residues were generated in the CB1 receptor using site-directed mutagenesis and transfection into HEK-293 cells. Signaling by wild-type and mutant receptors was analyzed by quantifying inhibition of cAMP, and by ß-arrestin recruitment assays. Results: We found that N7.49 and Y7.53 are essential for ß-arrestin recruitment by CB1. N7.49A and Y7.53F impair ß-arrestin signaling, with no effect on G-protein signaling. We found a regulatory role for residue I2.43; I2.43 interacts with Y7.53, affecting its positioning. Reducing steric bulk at I2.43 (I2.43A) enhances ß-arrestin1 recruitment, while introducing a polar residue (I2.43T) reduces ß-arrestin recruitment. Conclusions: These findings point to a novel mechanism for ß-arrestin recruitment, implicating amino acids in the NPXXY motif as critical for the putative ß-arrestin biased conformational state of Class A GPCRs.

Age-linked suppression of lipoxin A4 associates with cognitive deficits in mice and humans.

Age increases the risk for cognitive impairment and is the single major risk factor for Alzheimer's disease (AD), the most prevalent form of dementia in the elderly. The pathophysiological processes triggered by aging that render the brain vulnerable to dementia involve, at least in part, changes in inflammatory mediators. Here we show that lipoxin A4 (LXA4), a lipid mediator of inflammation resolution known to stimulate endocannabinoid signaling in the brain, is reduced in the aging central nervous system. We demonstrate that genetic suppression of 5-lipoxygenase (5-LOX), the enzyme mediating LXA4 synthesis, promotes learning impairment in mice. Conversely, administration of exogenous LXA4 attenuated cytokine production and memory loss induced by inflammation in mice. We further show that cerebrospinal fluid LXA4 is reduced in patients with dementia and positively associated with cognitive performance, brain-derived neurotrophic factor (BDNF), and AD-linked amyloid-ß. Our findings suggest that reduced LXA4 levels may lead to vulnerability to age-related cognitive disorders and that promoting LXA4 signaling may comprise an effective strategy to prevent early cognitive decline in AD.

CB1 Cannabinoid Receptor Signaling and Biased Signaling.

The CB1 cannabinoid receptor is a G-protein coupled receptor highly expressed throughout the central nervous system that is a promising target for the treatment of various disorders, including anxiety, pain, and neurodegeneration. Despite the wide therapeutic potential of CB1, the development of drug candidates is hindered by adverse effects, rapid tolerance development, and abuse potential. Ligands that produce biased signaling-the preferential activation of a signaling transducer in detriment of another-have been proposed as a strategy to dissociate therapeutic and adverse effects for a variety of G-protein coupled receptors. However, biased signaling at the CB1 receptor is poorly understood due to a lack of strongly biased agonists. Here, we review studies that have investigated the biased signaling profile of classical cannabinoid agonists and allosteric ligands, searching for a potential therapeutic advantage of CB1 biased signaling in different pathological states. Agonist and antagonist bound structures of CB1 and proposed mechanisms of action of biased allosteric modulators are used to discuss a putative molecular mechanism for CB1 receptor activation and biased signaling. Current studies suggest that allosteric binding sites on CB1 can be explored to yield biased ligands that favor or hinder conformational changes important for biased signaling.

Discovery of a Biased Allosteric Modulator for Cannabinoid 1 Receptor: Preclinical Anti-Glaucoma Efficacy.

We apply the magic methyl effect to improve the potency/efficacy of GAT211, the prototypic 2-phenylindole-based cannabinoid type-1 receptor (CB1R) agonist-positive allosteric modulator (ago-PAM). Introducing a methyl group at the α-position of nitro group generated two diastereomers, the greater potency and efficacy of erythro, (±)-9 vs threo, (±)-10 constitutes the first demonstration of diastereoselective CB1R-allosteric modulator interaction. Of the (±)-9 enantiomers, (-)-(S,R)-13 evidenced improved potency over GAT211 as a CB1R ago-PAM, whereas (+)-(R,S)-14 was a CB1R allosteric agonist biased toward G protein- vs ß-arrestin1/2-dependent signaling. (-)-(S,R)-13 and (+)-(R,S)-14 were devoid of undesirable side effects (triad test), and (+)-(R,S)-14 reduced intraocular pressure with an unprecedentedly long duration of action in a murine glaucoma model. (-)-(S,R)-13 docked into both a CB1R extracellular PAM and intracellular allosteric-agonist site(s), whereas (+)-(R,S)-14 preferentially engaged only the latter. Exploiting G-protein biased CB1R-allosteric modulation can offer safer therapeutic candidates for glaucoma and, potentially, other diseases.

Application of Fluorine- and Nitrogen-Walk Approaches: Defining the Structural and Functional Diversity of 2-Phenylindole Class of Cannabinoid 1 Receptor Positive Allosteric Modulators.

Cannabinoid 1 receptor (CB1R) allosteric ligands hold a far-reaching therapeutic promise. We report the application of fluoro- and nitrogen-walk approaches to enhance the drug-like properties of GAT211, a prototype CB1R allosteric agonist-positive allosteric modulator (ago-PAM). Several analogs exhibited improved functional potency (cAMP, ß-arrestin 2), metabolic stability, and aqueous solubility. Two key analogs, GAT591 (6r) and GAT593 (6s), exhibited augmented allosteric-agonist and PAM activities in neuronal cultures, improved metabolic stability, and enhanced orthosteric agonist binding (CP55,940). Both analogs also exhibited good analgesic potency in the CFA inflammatory-pain model with longer duration of action over GAT211 while being devoid of adverse cannabimimetic effects. Another analog, GAT592 (9j), exhibited moderate ago-PAM potency and improved aqueous solubility with therapeutic reduction of intraocular pressure in murine glaucoma models. The SAR findings and the enhanced allosteric activity in this class of allosteric modulators were accounted for in our recently developed computational model for CB1R allosteric activation and positive allosteric modulation.

GPR55-mediated effects on brain microvascular endothelial cells and the blood-brain barrier.

GPR55, an atypical cannabinoid receptor activated by lysophosphatidylinositol (LPI) has been involved in various physiological and pathological processes. We examined the effect of GPR55 activation on rat brain microvascular endothelial cells (RBMVEC), an essential component of the blood-brain barrier (BBB). GPR55 was detected in RBMVEC by western blot and immunocytochemistry. Treatment of RBMVEC with LPI increased cytosolic Ca2+ concentration, [Ca2+]i, in a concentration-dependent manner; the effect was abolished by the GPR55 antagonist, ML-193. Repetitive application of LPI induced tachyphylaxis. LPI-induced increase in [Ca2+]i was not sensitive to U-73122, a phospholipase C inhibitor, but was abolished by the blockade of voltage-gated Ca2+ channels or in Ca2+-free saline, indicating that Ca2+ influx was involved in this response. LPI induced a biphasic change in RBMVEC membrane potential: a fast depolarization followed by a long-lasting hyperpolarization. The hyperpolarization phase was prevented by apamin and charibdotoxin, inhibitors of small- and intermediate-conductance Ca2+-activated K+ channels (KCa). Immunofluorescence studies indicate that LPI produced transient changes in tight and adherens junctions proteins and F-actin stress fibers. LPI decreased the electrical resistance of RBMVEC monolayer assessed with Electric Cell-Substrate Impedance Sensing (ECIS) in a dose-dependent manner. In vivo studies indicate that systemic administration of LPI increased the permeability of the BBB, assessed with Evans Blue method. Taken together, our results indicate that GPR55 activation modulates the function of endothelial cells of brain microvessels, produces a transient reduction in endothelial barrier function and increases BBB permeability.

Age-dependent relevance of endogenous 5-lipoxygenase derivatives in anxiety-like behavior in mice.

When 5-lipoxygenase (5-LO) is inhibited, roughly half of the CNS effect of the prototypic endocannabinoid anandamide (AEA) is lost. Therefore, we decided to investigate whether inhibiting this enzyme would influence physiological functions classically described as being under control of the endocannabinoid system. Although 5-LO inhibition by MK-886 reduced lipoxin A4 levels in the brain, no effect was found in the elevated plus maze (EPM), even at the highest possible doses, via i.p. (10 mg/kg,) or i.c.v. (500 pmol/2 µl) routes. Accordingly, no alterations in anxiety-like behavior in the EPM test were observed in 5-LO KO mice. Interestingly, aged mice, which show reduced circulating lipoxin A4 levels, were sensitive to MK-886, displaying an anxiogenic-like state in response to treatment. Moreover, exogenous lipoxin A4 induced an anxiolytic-like profile in the EPM test. Our findings are in line with other reports showing no difference between FLAP KO or 5-LO KO and their control strains in adult mice, but increased anxiety-like behavior in aged mice. We also show for the first time that lipoxin A4 affects mouse behavior. In conclusion, we propose an age-dependent relevancy of endogenous 5-LO derivatives in the modulation of anxiety-like behavior, in addition to a potential for exogenous lipoxin A4 in producing an anxiolytic-like state.