Off-target activity of NBOMes and NBOMe analogs at the μ opioid receptor

Abstract

New psychoactive substances (NPS) are introduced on the illicit drug market at a rapid pace. They are usually poorly pharmacologically characterized, leaving users unaware of the potential harms they could be exposed to. Apart from the unknown effects and potencies of these new substances, their exact molecular targets are often inadequately elucidated. Guided by recent literature, we investigated the μ opioid receptor (MOR) activation potential of a large set of psychedelics, which typically activate their target receptor, the serotonin (5-HT2A) receptor. Screening for MOR activity of a panel of psychedelics was done using a cell-based NanoBiT® bioassay. The NanoBiT® assay monitors the recruitment of the intracellular signaling protein β-arrestin2 to the ligand activated MOR receptor. The principle exploits functional complementation of a split nanoluciferase enzyme of which the inactive subunits are either fused to the receptor and βarr2. Receptor activation causes the 2 subunits to come in close proximity, resulting in restoration of the nanoluciferase activity. Upon addition of the substrate, a luminescent signal can be detected using a luminometer. Based on the results of this initial screening, MOR activity of a selected panel of NBOMes and NBOMe analogs was evaluated using the both the NanoBiT® and AequoScreen® assay, the latter measuring the rapid intracellular calcium flux caused by receptor activation, a more downstream effect caused by G protein signaling. Using the NanoBiT® assay, 5 NBOMes showed MOR activation potential. Based on these findings, a panel of NBOMe analogs was selected and evaluated using 2 distinct assays. Comparing both assays, the same overall trends regarding MOR activity could be determined. The iodo analog 25I-NBOMe was more potent than the bromo substituted 25B-NBOMe, which was more potent than the chloro analog 25C-NBOMe, signifying that MOR activity increased with an increased size of the halogen atom. Similarly, MOR activity increased with increasing size of the alkyl side chain, with the ethylated 25E-NBOMe being more active than the methylated 25D-NBOMe. However, a propyl or isopropyl side chain might be too long or bulky, as the 25P-NBOMe and 25iP-NBOMe showed a substantial decrease in activity. NBOMe compounds were more active at MOR than their NBOH counterparts, whereas earlier data showed that they had a similar activity at their designated target receptor 5-HT2A. Moving the methoxy group from position 2 to position 3 or 4 resulted in an important decrease in MOR activity. Furthermore, the effects at MOR of the analyzed compounds could be blocked by the opioid antagonist naloxone, suggesting that these NBOMes occupy the same common opioid binding pocket as conventional opioids. Using the AequoScreen® assay, aspecific, receptor independent effects were found for the alkylated 25iP-NBOMe, 25E-NBOMe and 25D-NBOMe, indicating that these may directly interact with other targets in the downstream signaling pathway. A limited potential to activate the MOR was found for 10 NBOMes and NBOMe analogs, which was confirmed using 2 distinct cell-based assays. Structure-activity relationships could be determined, and additional experiments revealed that the effects of these psychedelics at MOR could be antagonized by naloxone, signifying that these substances occupy the same binding pocket as common opioids. Some compounds showed an aspecific effect in the AequoScreen® assay, which was not observed using the NanoBiT® assay. As MOR activity of these psychedelics was only noticed at high concentrations, it is unlikely that these will contribute to pronounced opioid toxicity at physiologically relevant concentrations. However, small modifications to the original NBOMe structure may result in a panel of more efficacious and potent MOR agonists.