Fentanyl and its derivatives are potent mu-opioid receptor (mOR) agonists, and their illicit use has exacerbated the ongoing opioid crisis. Despite the rise of overdose related deaths, very little is known about their molecular action at mOR. Using the state-of-the-art molecular dynamics (MD) techniques, including the weighted ensemble (WE), metadynamics, and replica-exchange continuous constant-pH MD, we studied the detailed fentanyl-mOR binding mechanism and retrospectively predicted the unbinding rates of fentanyl and analogs. Consistent with the morphinan compound BU72, fentanyl binding is enabled through a salt bridge with D1473.32, although its position is lower than BU72. Surprisingly, we found a secondary binding mode, in which fentanyl is further inserted in mOR and forms a hydrogen bond with a conserved H2976.52 in the neutral ND-tautomer state. Furthermore, the H297-binding mode significantly prolongs the ligand-mOR dissociation time. The predicted ranking of fentanyl analog dissociation times is consistent with experimental measurements. Our data may offer a molecular basis to explain the significant pharmacological differences between fentanyl and morphinan derivatives. Traditional protein simulations employ standard protonation states; our work underscores the need to move beyond this practice, in order to further advance the knowledge and quantitative prediction of protein-ligand binding and unbinding. The developed protocol may be used to prospectively predict the relative dissociation rates of illicit opioids emerging from the dark market to aid in the regulatory decision making. The insights from the work may also inform the design of safer analgesics to combat the opioid crisis.

Mechanism of binding and unbinding of fentanyl and analogs at the mu-opioid receptor

ACS Fall 2021

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