A putative binding model of nitazene derivatives at the μ-opioid receptor

Nitazenes are a class of novel synthetic opioids with exceptionally high potency. Currently, an experimental structure of μOR-opioid receptor (μOR) in complex with a nitazene is lacking. Here we used a suite of computational tools, including consensus docking, conventional molecular dynamics (MD) and metadynamics simulations, to investigate the μOR binding modes of nitro-containing meto-, eto-, proto-, buto-, and isotonitazenes and nitro-less analogs, metodes-, etodes-, and protodesnitazenes. Docking generated three binding modes, whereby the nitro-substituted or unsubstituted benzimidazole group extends into SP1 (subpocket 1 between transmembrane helix or TM 2 and 3), SP2 (subpocket 2 between TM1, TM2, and TM7) or SP3 (subpocket 3 between TM5 and TM6). Simulations suggest that etonitazene and likely also other nitazenes favor the SP2-binding mode. Comparison to the experimental structures of μOR in complex with BU72, fentanyl, and mitragynine pseudoindoxyl (MP) allows us to propose a putative model for μOR-ligand recognition in which ligand can access hydrophobic SP1 or hydrophilic SP2, mediated by the conformational change of Gln124^2.60. Interestingly, in addition to water-mediated hydrogen bonds, the nitro group in nitazenes forms a π-hole interaction with the conserved Tyr75^1.39. Our computational analysis provides new insights into the mechanism of μOR-opioid recognition, paving the way for investigations of the structure-activity relationships of nitazenes.