Cannabidiol (CBD), the main non-euphoric constituent of
Cannabis sativa, exhibits a complex neuropharmacological profile, ranging from anxiolytic, anticonvulsive, and neuroprotective properties to analgesic and anti-inflammatory effects, all potentially useful for the treatment of several neurological and psychiatric disorders
[1][2]. The molecular targets of CBD likely involved in its therapeutic effects have been recently reviewed
[3][4] and include, among others, the transient potential receptor channels (TRPs) TRPV1, TRPA1, and TRPM8
[5], the G-protein coupled receptors GPR55
[6], serotonin 1A receptor
[7], and opioid receptors
[8]. Moreover, even though CBD has a low affinity for the cannabinoid receptors, recent evidence indicated that it may act as a cannabinoid receptor of type 1 (CB1R)-negative allosteric modulator in some circumstances
[9][10][11]. More recently, CBD has been also proposed as a therapeutic tool for addictive disorders such as drug and alcohol abuse
[12][13][14]. In this view, the recent findings that the orexin system is involved in drug addiction
[15], prompted us to investigate the orexin receptors, OX1R and OX2R, as possible targets for CBD. Both receptors are coupled with a Gq protein and their activation induces cell responses mainly via calcium (Ca
2+)- and diacylglycerol (DAG)-mediated pathways
[16][17]. The endogenous ligands of these G-protein coupled receptors are two hypothalamic neuropeptides, orexin-A (OX-A) and orexin-B (OX-B), which regulate different physiological functions in mammals, such as sleep-wake cycles, energy metabolism, and feeding
[18]. Both peptides arise from a common precursor, the prepro-orexin, a 130-amino-acid peptide. Orexin projections extend widely throughout the brain, innervating the neocortex, hippocampus, and forebrain structures implicated in the processing of arousal
[19], wakefulness
[20], feeding
[21], emotion, and motivation
[22]. Defects in the orexin system induce narcolepsy and catalepsy, whereas inhibitors of orexin receptors represent a promising therapy for the treatment of sleep disorders
[23]. Orexin 1 and Orexin 2 receptors are differently distributed in the central nervous system (CNS), thus reflecting different biological profiles—while OX2R is mainly involved in sleep and arousal, OX1R is implicated in compulsive behaviour related to drug addiction and anxiety
[24]. OX1R has a higher affinity for OX-A than OX-B, whereas OX2R binds both OX-A and OX-B with the same affinity
[23]. In the present study, by performing binding experiments on both orexin receptors, we found that CBD binds OX1R selectively over OX2R. Then, by exploiting the recently-released X-ray structures of both orexin receptors
[25][26], the putative binding modes of CBD to OX1R were investigated by molecular docking and molecular dynamics simulations. Computational data, in addition to providing a possible structural basis for the observed binding, also allowed a rationalization of CBD selectivity toward the OX1R subtype. Finally, functional studies based on the measurement of intracellular calcium imaging and mobilization in OX1R-transfected Chinese hamster ovary (CHO) cells allowed the characterization of CBD as an OX1R antagonist.