Membrane Permeability Drives the Extreme Potency of Fentanyl but not Isotonitazene

Fentanyl is a leading cause of drug overdose deaths in the United States, yet the mechanisms underlying its extreme in vivo potency remain poorly understood. Recently, new synthetic opioids nitazene derivatives have emerged, among which isotonitazene is 50 times more potent than fentanyl. Here we used state-of-the-art molecular dynamics (MD) simulations and experiments to investigate the membrane-dependent pharmacology of fentanyl, isotonitazene, morphine, and naloxone. Using the weighted-ensemble continuous constant pH MD, we estimated the effective permeability of fentanyl at pH 7.5 to be on the order of 10^-7 cm/s, which is about two orders of magnitude faster than the simulation estimate for morphine. In contrast, isotonitazene and naloxone effectively do not partition into the membrane under the same conditions. The simulations captured the proton-coupled permeation processes, challenging as well as refining the long-standing pH-partition hypothesis. Subsequent BRET reporter cell experiments demonstrated that cells exposed to fentanyl, but not morphine, reactivated the receptor after washout and in competition with naloxone. Immobilized affinity membrane chromatography confirmed fentanyl's high affinity for phospholipids. Our findings strongly support the hypothesis that fentanyl's extreme in vivo potency may be driven by its accumulation within the plasma membrane or intracellularly, enabling it to repartition into the extracellular space to rebind the receptor, or potentially access it via a lipid-mediated route. This highlights the importance of membrane-dependent pharmacology for understanding opioid toxicity and guiding the design of more effective antagonists. Our simulation methodology enables accurate prediction and analysis of membrane permeation of ionizable molecules, providing a valuable tool for ADME optimization in drug development.