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<p>Synthetic cannabinoids (SC) target the human cannabinoid receptor 1 (hCB<sub>1</sub>) and are extensively metabolized, but the metabolite activity on the hCB<sub>1</sub> receptor after a SC intake is largely unknown. In this study we compared the in vitro hCB<sub>1</sub> 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 hCB<sub>1</sub> 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 EC<sub>50</sub> and <i>E</i><sub>max</sub>. In silico docking and molecular dynamics were performed using a model of the active form of the hCB<sub>1</sub> 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 &lt;70%, qualifying them as partial agonists. Eighteen metabolites retained &gt;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 (<i>R</i><sup>2</sup> = 0.9457), significant (<i>P</i> &lt; 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.</p><p></p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年11月3日

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  • Anna Åstrand,
  • Emiliano Laudadio,
  • Prince S. Gameli,
  • Laura Martin,
  • Jeremy Carlier,
  • Francesco P. Busardò,
  • Johan Dahlén,
  • Xiongyu Wu,
  • Peter Konradsson,
  • Svante Vikingsson,
  • Robert Kronstrand,
  • Henrik Gréen

摘要

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.