Molecular pharmacology of cannabinoids: beyond THC

Cannabinoids from plants are some of the oldest human medicine, while synthetic cannabinoid receptor agonists (SCRAs) new psychoactive substances have been responsible for hundreds of deaths all over the world. The studies presented in this thesis were undertaken to further the understanding of the molecular pharmacology of cannabinoids, and how structurally diverse cannabinoids have greatly varied outcomes when acting via the same target. The characterisation of cannabinoids at activating cannabinoid receptors (CB1 and CB2) combined with operational model, functional selectivity and allosteric modulation together form the basis of the current thesis to better understand the molecular contributions to the toxic effects of SCRAs and therapeutic effects of phytocannabinoids. The first major finding of this thesis was the quantitative determination of the efficacy of SCRAs downstream of CB1 using the operational model of pharmacological agonism. A panel of 17 cannabinoids were compared for their ability to induce response after the pharmacological knockdown of CB1 receptor using AM6544 (irreversible CB1 antagonist). The operational efficacy of cannabinoids ranged from 233 (5F-MDMB-PICA) to 0.9 (THC), with CP55940 in the middle of the efficacy rank order. SCRAs generally demonstrated substantially higher efficacy at activating CB1 receptors than THC. This work is the first of its kind to provide a framework to quantify the efficacy of chemically diverse cannabinoids and help identify some of the potential underlying molecular mechanisms regarding the SCRAs-related adverse effect on CB1 activation. The functional activity of the same panel of cannabinoids was also characterised in two signalling endpoints - Gαi/o (inhibition) and Gαs (stimulation) of cAMP signalling. The rank order of potency of the cannabinoids to stimulate Gαs-like signalling compared to Gαi/o signalling was significantly different. This suggests the differing ability of cannabinoids to preferentially induce CB1-dependent cAMP inhibition over stimulation of cAMP (functional selectivity). Cannabinoids showed diverse signalling profile (wide range of EMAX) at Gαs-like pathway than their activity at canonical Gi-mediated signalling pathway. Evaluating the differences in G protein preference among SCRAs may contribute to unravelling their complex effects in humans. vi Another important concept in molecular pharmacology is the allosteric modulation of receptor signalling. The fourth chapter of this thesis was a natural continuation of the original investigation of the SCRA-associated toxicity, where we utilised the concept of allosteric modulation to understand mechanisms underlying the adverse effects related to the mixing of brodifacoum (superwarfarin) with SCRAs. Results revealed that cannabinoid signalling was not affected by brodifacoum, indicating that combining SCRA with brodifacoum is not likely to enhance user experience through interactions with cannabinoid receptors. Allosteric modulation of cannabinoid receptors was also studied in the context of the 'entourage effect' - an emerging idea amongst users of cannabis is that the whole plant must be used in order to achieve the maximal therapeutic effect. However, Chapter 5 determined that aromatic compounds called terpenoids that are commonly found in cannabis had no additive effect on cannabinoid receptor function in the presence of a high efficacy agonist (CPP5940) or a low efficacy agonist (THC), suggesting the absence of 'entourage effect'. While the earlier chapters are focused on the pathways involved in the on-targets effects associated with SCRA toxicity, Chapter 6 featured an exploratory study primarily concerned with the pharmacological effects underlying the potential therapeutic effects of cannabidiol (CBD) signalling on multiple receptors. We demonstrate the specific inhibitory effects of CBD on CB1 responses at physiologically relevant temperature, however, the effect of CBD on CB2, or mu-opioid receptor appears relatively non-specific in nature. The outcome of this chapter indicates that more work is required to understand whether the promiscuity of CBD interaction with multiple receptors is due to the membrane interaction. The finding of this thesis provides new mechanistic insight into cannabinoid receptor function, and would hopefully direct future investigations into design of cannabinoids with high specificity and improved pharmaceutical properties -- abstract.