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

## TL;DR

This study explores how synthetic cannabinoid metabolites interact with a human receptor, finding that some metabolites remain highly active and contribute to the drug's effects.

## Contribution

The study identifies specific metabolites that retain high efficacy and correlates their activity with in silico molecular dynamics simulations.

## Key findings

- 4-hydroxypentyl metabolites of THJ-2201 and THJ-018 are the most potent, with EC50 values as low as 13 nM.
- In silico simulations showed a strong correlation (R² = 0.9457) between binding energy and in vitro efficacy.
- Active metabolites maintain at least six amino acid interactions, primarily through CH-π and π-π stacking interactions.

## Abstract

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.