Coordinated subpocket engagement underlies nitazene potency at the μ-opioid receptor.

Nitazenes are emerging synthetic opioids that exhibit exceptionally high potency at the {micro}-opioid receptor (MOR) and contribute to rising overdose fatalities worldwide. Despite extensive in vitro profiling, the structural determinants underlying their structure-activity relationships (SARs) remain unresolved. Here, we combine functional profiling with quantum mechanical calculations and molecular dynamics (MD) simulations to establish a MOR structure-based SAR for nitazenes. Across functional assays, systematic variation at the R1, R2, and R3 positions revealed non-additive effects on potency and identified optimal R1 chain length, R2 N-desethylation, and retention of the 5-nitro group as key determinants of high MOR potency. Consistent with this framework, N-desethyl isotonitazene emerged as the most potent analogue. Structural analysis of the cryo-EM MOR-Gi-fluornitrazene complex, together with MD simulations of multiple nitazene analogues, revealed a conserved trivalent binding architecture in which each substituent engages distinct subpockets. N-desethylation at R2 increases the positive electrostatic surface at the protonated amine, reduces steric constraints near transmembrane helix (TM) 7, strengthens R3 interactions, and allosterically modulates R1 engagement in a substituent-dependent manner. Additionally, optimal R1 chain length and shape stabilize the TM5-TM6 interface and influence activation-relevant TM6 dynamics, defining a unified SAR at R1 across nitazene and fentanyl scaffolds. Together, these findings indicate that nitazene potency reflects substituent-dependent coupling among R1, R2, and R3 within the MOR binding pocket, with R3 engagement distinguishing nitazenes from fentanyl. This framework establishes a coherent structural model of nitazene-MOR recognition that accounts for their unusually high potency and efficacy.