Identification of the Putative Binding Site of a Benzimidazole Opioid (Etazene) and Its Metabolites at µ-Opioid Receptor: A Human Liver Microsomal Assay and Systematic Computational Study

Abstract

The synthetic benzimidazole opioid etazene (which has a 70-times higher analgesic activity than morphine), a recreational drug, has gained popularity as a novel psychoactive substance (NPS) on the illegal/darknet market; however, no experimental information is available at the molecular level on the binding mechanism and putative binding site of etazene and its metabolites at the µ-opioid receptor (MOR). In the present study, we investigated the metabolism of etazene in human liver microsomes using ultra-high-performance liquid chromatography–mass spectrometry (UHPLC–MS). We also explored the possibilities of MOR activation by etazene and its metabolites by studying their binding mechanisms and interaction profiles at an active-state MOR model via molecular docking, binding free energy calculations, and all-atom molecular dynamics (MD) simulations. The putative metabolites of etazene were also predicted using the ADMET Predictor 10.1. The molecular docking studies and free energy calculations showed that etazene and its metabolites (M1, M2, and M5–M7) exhibited strong predicted binding affinity at MOR and showed overlapped binding orientation with MOR-bound agonist BU72, which was co-crystallized in the MOR X-ray crystal structure (PDB ID: 5C1M). MD also confirmed the stability of the MOR–etazene and MOR–M6 complexes. These results suggest that etazene and its metabolites may act as strong MOR agonists, highlighting the necessity of experimental validation. The insights from this study, such as key interactions between etazene and its metabolites and the MOR, will allow authorities to predict potential analogs and clarify the target–protein interactions associated with this illicit substance, granting advanced or rapid reactions to confiscating or banning potential emerging drugs.

In vitro structure–activity relationships and forensic case series of emerging 2-benzylbenzimidazole ‘nitazene’ opioids

“…In addition, it has been hypothesized that nitazenes have a long MOR residence time, maintaining the receptor in an active state for a longer period (Malcolm et al 2023). In agreement with this, recent in silico studies confirm that nitazenes may have a MOR binding pose that is distinct from other opioids (De Luca et al 2022;Chaturvedi et al 2023). Future structural studies of MOR bound to 2-benzylbenzimidazoles may provide an explanation for the nitazene SAR trends observed by us and others.…”

Cannabinoid Receptor Type II Ligands from Sandalwood Oil and Synthetic α-Santalol Derivatives

“…2.3, 2019.1 (Schrodinger) relaxation protocol with slight modifications, as mentioned in our previous publication. 42 In brief, the protocol involved an initial minimization of the solvent while keeping restraints on the solute, followed by short MD simulations, including the following steps: (1) simulation (1 ns) using Brownian dynamics in the NVT ensemble at 10 K with heavy solute atoms restrained; (2) simulation (100 ps) in the NVT ensemble using a Berendsen thermostat (10 K) with heavy solute atoms restrained; (3) simulation (100 ps) in the NPT ensemble using a Berendsen thermostat (10 K) and a Berendsen barostat (1 atm) with nonhydrogen solute atoms restrained; (4) simulation (100 ps) in the NPT ensemble using a Berendsen thermostat (300 K) and a Berendsen barostat (1 atm) with non-hydrogen solute atoms restrained; (5) simulation (200 ps) in the NPT ensemble using a Berendsen thermostat (300 K) and a Berendsen barostat (1 atm) with no restraints; (6) simulation (5000 ps) in the NPT ensemble using a Langevin thermostat (300 K) and a Langevin barostat (1 atm) with no restraints. The 200 ns MD simulations were run in the canonical ensemble (NPT) using a Langevin thermostat at 300 K. The trajectory analysis was performed using Event Analysis and the Simulation Interaction Diagram (SID) implemented in the Desmond software.…”