Exploring Cannabinoid Receptors in Humans and Zebrafish Through the Lens of Molecular Pharmacology

<p dir="ltr">Cannabinoid receptors (CBRs) play a vital role in regulating various physiological functions and are responsible for the psychoactive effects in humans. The exploration of cannabinoid therapeutics has led to the development of Synthetic Cannabinoid Receptor Agonists (SCRAs) and other treatments for neurological disorders like Multiple Sclerosis. However, some therapeutics that work well in disease models, such as Zebrafish (<i>Danio rerio</i>), often fail in clinical trials. The distinct interactions between therapeutics in the zebrafish model and the human body may explain these failures. Although zebrafish share 70% of their genetic makeup with humans, understanding how cannabinoids interact with CBRs in both species is crucial for translating findings from zebrafish models into human disease treatments. This thesis aims to uncover the molecular mechanisms governing cannabinoid interactions with human and zebrafish CBRs. This thesis investigates the structure-activity relationship of SCRAs related to SDB-006, 5F-SDB-006, CUMYL-PICA, and 5F-CUMYL-PICA at hCB1 and hCB2 receptors. Cellular hyperpolarisation through membrane potential assay in FlexStation 3 revealed the high selectivity of these SCRAs towards CB1 receptor activation. The potency ranking of SCRAs was cumyl > (S)-α-methylbenzyl > (R)-α-methylbenzyl ≈ benzyl, with enantiomeric effects favouring (S)-α-methylbenzyl-derived SCRAs at hCB1. Furthermore, this thesis explores the pharmacological activities of cannabinoids on human and zebrafish CBRs. CBRs expressed in mammalian AtT20 cells activate GIRK channels, leading to cannabinoid-induced hyperpolarisation, assessed via membrane potential assay. Responses varied between zebrafish and human CBRs. For instance, CP 55,940 and THC hyperpolarised hCB1 and zfCB1 cells similarly, while WIN 55,212-2 affected zfCB1 more potently. AEA and 2-AG were more potent at zfCB1 than at hCB1. CP 55,940 had reduced potency at zfCB2A compared to hCB2, whereas AEA and 2-AG showed similar potency. Observing the amino acid sequence similarities between human and zebrafish CBRs, it becomes evident that there are subtle differences that contribute to distinct responses. Additionally, with the differences in cannabinoid response observed in zebrafish CBRs, we determined that these distinctions could potentially result in modified responses when assessing cannabinoids designed for human diseases in zebrafish. With the hypothesis that bisphenols induce obesity in zebrafish through the activation of CBRs, we further investigated the effect of bisphenol and analogues for the CBRs activity in two G protein-dependent signalling pathways - Gβγ mediated GIRK channel activation in AtT20 cells and Gαi mediated adenylyl cyclase inhibition in HEK293 cells. Our results indicate that none of the tested bisphenols activate CBRs through the tested signalling pathways. Molecular docking analysis showed that bisphenol analogues have the propensity to activate the receptor. However, the precise mode of action of how bisphenols activate zebrafish CBRs and induce obesity needs to be studied further. In summary, cannabinoids, both natural and synthetic, hold promise for treating various medical conditions. Zebrafish models offer a valuable tool for studying these compounds despite challenges stemming from genetic differences between humans and zebrafish. By comparing cannabinoid interactions at the receptor level, researchers can enhance the translation of research findings into clinical practice.</p>