Computational Analysis of the Binding Poses of Nitazene Derivatives at the μ -Opioid Receptor

Nitazenes, a novel class of synthetic opioids with exceptionally high potency, has recently emerged on illicit markets, resulting in many overdose deaths. Currently, an experimental structure of nitazene bound to the molecular target, the µ -opioid receptor ( µ OR), is lacking, hindering the understanding of their structure-activity relationships. Here we employed a suite of computational approaches, encompassing consensus docking, metadynamics, and conventional molecular dynamics (cMD) refinement, to determine the putative µ OR binding poses of eight nitazenes, including the nitro-containing meto-, eto-, proto-, buto-, and isotonitazene and the nitro-less metodes-, etodes-, and protodesnitazene. Docking generated three possible binding modes, whereby the nitro-substituted or unsubstituted benzimidazole group extends into subpocket 1 (SP1) between transmembrane helix (TM) 2 and 3, subpocket 2 (SP2) between TM2, TM7, and TM1 or subpocket 3 (SP3) between between TM5 and TM6. Metadynamics and cMD simulations suggested that the SP2-binding mode is most stable for etonitazene and analogs as well as etodesnitazene. Based on the comparison to the experimental structures of BU72-, fentanyl- and mitragynine pseudoindoxyl (MP)-bound µ OR, we proposed a general model for receptor-opioid recognition, which involves interactions with SP1, SP2 and SP3. The hydrophobic SP1 prefers an aromatic moiety, such as the phenyl ring of BU72 and fentanyl. In contrast, the hydrophilic SP2 and SP3 favor a polar group which can form water-mediated hydrogen bond interactions with the conserved Tyr75 ^1.39 and His297 ^6.52 . Our findings provide molecular insights into the mechanism of receptor-opioid recognition, paving the way for investigations of the structure-activity relationships of nitazenes.