Kinetics and Mechanism of Fentanyl Dissociation from the mu-Opioid Receptor

Driven by illicit fentanyl, opioid related deaths have reached the highest level in 2020. Currently, an opioid overdose is resuscitated by the use of naloxone, which competitively binds and antagonizes the mu-opioid receptor (mOR). Thus, knowledge of the residence times of opioids at mOR and the unbinding mechanisms is valuable for assessing the effectiveness of naloxone. In the present study, we calculate the fentanyl-mOR dissociation time and elucidate the mechanism by applying an enhanced sampling molecular dynamics (MD) technique. Two sets of metadynamics simulations with different initial structures were performed while accounting for the protonation state of the conserved H297(6.52), which has been suggested to modulate the ligand-mOR affinity and binding mode. Surprisingly, with the N delta-protonated H297(6.52), fentanyl can descend as much as 10 angstrom below the level of the conserved D147(3.32) before escaping the receptor and has a calculated residence time tau of 38 s. In contrast, with the N epsilon- and doubly protonated H297(6.52), the calculated tau are 2.6 and 0.9 s, respectively. Analysis suggests that formation of the piperidine-Hid297 hydrogen bond strengthens the hydrophobic contacts with the transmembrane helix (TM) 6, allowing fentanyl to explore a deep pocket. Considering the experimental tau of similar to 4 min for fentanyl and the role of TM6 in mOR activation, the deep insertion mechanism may be biologically relevant. The work paves the way for large-scale computational predictions of opioid dissociation rates to inform evaluation of strategies for opioid overdose reversal. The profound role of the histidine protonation state found here may shift the paradigm in computational studies of ligand-receptor kinetics.