Abdulmaqsood, Yousra
(2021)
Molecular characterization of endocannabinoid signalling.
PhD thesis, University of Nottingham.
Abstract
The endocannabinoid system has gained an increased focus in exploiting its therapeutic potential for the management of multiple diseases. This research aimed to investigate two components of the EC system, the EC-hydrolysing enzyme ABHD6 and signalling through CB2 receptor. Selective inhibition of ABHD6 was reported to possess anti-inflammatory and neuroprotective effects and was implicated in diverse pathological processes. Likewise, selective modulation of CB2 receptors could have a therapeutic potential in the management of inflammatory, neuropathic pain and degenerative diseases without inducing the psychoactive effects of CB1 receptor activation. This research initially aimed to develop a rapid, cost-effective, reproducible and sensitive HTS assay for screening human recombinant ABHD6. The investigated substrate 4-MUH was a promising tool for screening novel ABHD6 inhibitors. The assay also identified the potential for ABHD6 to recognise multiple endogenous lipids, which may be natural substrates of the enzyme. Nonetheless, the assay in its current configuration employed to ‘real’ tissues, of rat intestine and hippocampus, generated data that were complex and difficult to interpret. It is possible that further adapting this assay may resolve these issues.
Subsequent sections of the thesis provide comprehensive investigation of CB2 receptor signalling, the effects of ligands derived from multiple pharmacophores and the dominant amino acids involved in the receptor’s signalling and function. The signalling pathways investigated in this study focused on rapid (typically <15 min) timepoints including Ca2+ mobilization, ERK phosphorylation, and cAMP accumulation, in addition to exploring changes in [35S]-GTPγS, [3H]-CP55940 and fluorescent ligand binding assays. A series of compounds synthesised at the University of Nottingham based on fenofibrate namely FD24, FD43, FD44 and FD46 were initially investigated along with two commercially available alternatives reported to have agonist and inverse agonist/antagonist properties namely SER601 and COR170. This led to the identification of a promising novel CB2 receptor agonist, FD24 with a ‘balanced’ agonist profile across the investigated signalling pathways. Further investigations pertaining to the selectivity of FD24 at CB2 receptors compared to CB1 receptors could identify beneficial therapeutic effects devoid of psychotropic effects. Furthermore, SER601 was identified as an ERK-phosphorylation biased agonist, without agonist activity in the other signalling pathways. Subsequent to further investigations, this compound could be a beneficial starting point for managing inflammatory or neuropathic pain through selective targeting of CB2 receptors (and possibly PPARα). Additional range of commercially available, structurally diverse agents reported to be CB2 receptor-selective agonists, CB65, GP2a, GW405833, HU308, L759656 and MDA19, were investigated to assess their potential for bias within the same rapid responding assays. HU308 and MDA19 were efficacious CB2 receptor agonists ‘balanced’ at the investigated pathways. HU308 is suggested to be useful as a ‘balanced’/unbiased agonist for characterization of CB2 receptors and the current study confirms this profile. GW405833 acted as a low efficacy agonist/antagonist, dependent on the signalling pathway. GP2a displayed an inverse agonist profile in the cAMP pathway and was ineffective in all of the investigated pathways, while L759656 evoked inhibition of cAMP accumulation with no effects on the other pathways. The profile of CB65 was consistent with a low efficacy agonist/antagonist and not consistent with the literature. For investigation of in vivo profiles of synthetic CB2 receptor-selective agents, it would be interesting to compare the profiles of L759656, SER601 and GW405833 or HU308 to investigate whether signalling bias through the pathways investigated here align with potentially beneficial therapeutic properties.
Following investigations of CB2 receptor signalling, the concluding part of the project aimed to investigate the role of specific residues contributing towards CB2 receptor signalling. The hydrophobic amino acids F117, F200 and W258 were mutated to their alanine counterparts and assessed in the rapid signalling pathways ERK phosphorylation and [3H]-cAMP accumulation. Mutations of F117A, F200A and W258A led to complete loss of functional activity via both signalling pathways. It was not possible to confirm expression of the mutant receptors through immuno- and/or molecular tagging techniques. Thus, the lack of coupling of the mutated receptors could be attributed to either the vital nature of these amino acids or that the receptor was not expressed subsequent to mutation. In order to gain solid conclusions regarding the prominence of these amino acids, future investigations could aim to utilise alternative approaches of transfection of the mutated receptor to gain more confidence that the receptor was expressed. Moreover, mutating an amino acid that was not involved in the receptor signalling or ligand binding could be used as a comparison that the receptor was functional subsequent to site-directed mutagenesis. Overall these investigations enhanced our understanding of the EC system.
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