Structure-activity relationship of prevalent synthetic cannabinoid metabolites on hCB1 in vitro and in silico dynamics.

Åstrand A, Laudadio E, Gameli PS, Martin L, Carlier J, Busardò FP, Dahlén J, Wu X, Konradsson P, Vikingsson S, Kronstrand R, Gréen H

Acta Pharmacol Sin 47 (3) 776-789 [2026-03-00; online 2025-11-03]

Synthetic cannabinoids (SC) target the human cannabinoid receptor 1 (hCB1) and are extensively metabolized, but the metabolite activity on the hCB1 receptor after a SC intake is largely unknown. In this study we compared the in vitro hCB1 receptor activity of 26 metabolites of the synthetic cannabinoid receptor agonists (SCRA) JWH-018, AM-2201, THJ-018 and THJ-2201 as a model system for SC metabolite activity to elucidate their structure-activity relationships. The efficacy and potency of metabolites were assessed using an AequoScreen hCB1 receptor assay in triplicates and 7-8 concentration points (20 µg/mL-9.5 ng/mL) were used to construct dose-response curves and to determine EC50 and Emax. In silico docking and molecular dynamics were performed using a model of the active form of the hCB1 receptor with all the metabolites. Final poses were simulated to assess stability under physiological conditions. We showed that carboxylic acid metabolites and 2-hydroxyindole biotransformational products were inactive, while 5-hydroxypentyl SCRA metabolites decreased efficacy to <70%, qualifying them as partial agonists. Eighteen metabolites retained >70% efficacy of their parent compound. Metabolite potencies ranged from 13-3500 nM where the most potent were the 4-hydroxypentyl derivatives of THJ-2201 and THJ-018 and the 4-hydroxyindole derivatives of AM-2201 and JWH-018, also known to be prevalent in vivo metabolites. The efficacy data from in silico experiments were correlated with the in vitro results demonstrating a linear trend (R2 = 0.9457), significant (P < 0.0001) at the 95% confident interval between the binding energies and efficacies of the compounds investigated. In silico analysis with docking and molecular dynamics simulations showed that active metabolites maintained a minimum of six amino acid interactions involving all substructures. The in silico molecular dynamics simulations revealed that the efficacy and potency seemed to be driven by a complex network of hydrophobic weak amino acid-ligand interactions. Most prevalent were CH-π interactions and π-π stackings. This study demonstrates the clear structure-activity relationships well correlated to the molecular dynamics simulations, suggesting that metabolites, especially the 4-hydroxy pentyl metabolites, may contribute to the overall effect of SCs in vivo.

PubMed 41184621

DOI 10.1038/s41401-025-01678-5

Crossref 10.1038/s41401-025-01678-5

pmc: PMC12932713
pii: 10.1038/s41401-025-01678-5