How mu-Opioid Receptor Recognizes Fentanyl

Abstract

The opioid crisis has escalated during the COVID-19 pandemic. More than half of the overdose-related deaths are related to synthetic opioids represented by fentanyl which is a potent agonist of mu-opioid receptor (mOR). In recent years, crystal structures of mOR complexed with morphine derivatives have been determined; however, structural basis of mOR activation by fentanyl-like synthetic opioids remains lacking. Exploiting the X-ray structure of mOR bound to a morphinan ligand and several state-of-the-art simulation techniques, including weighted ensemble and continuous constant pH molecular dynamics, we elucidated the detailed binding mechanism of fentanyl with mOR. Surprisingly, in addition to the orthosteric site common to morphinan opiates, fentanyl can move deeper and bind mOR through hydrogen bonding with a conserved histidine H297, which has been shown to modulate mOR's ligand affinity and pH dependence in mutagenesis experiments, but its precise role remains unclear. Intriguingly, the secondary binding mode is only accessible when H297 adopts a neutral HID tautomer. Alternative binding modes and involvement of tautomer states may represent general mechanisms in G protein-coupled receptor (GPCR)-ligand recognition. Our work provides a starting point for understanding mOR activation by fentanyl analogs that are emerging at a rapid pace and assisting the design of safer analgesics to combat the opioid crisis. Current protein simulation studies employ standard protonation and tautomer states; our work demonstrates the need to move beyond the practice to advance our understanding of protein-ligand recognition.

A comprehensive evaluation of the potential binding poses of fentanyl and its analogs at the µ-opioid receptor

“…Previous MD simulation studies showed that the amide moiety of fentanyl could be stable in either the trans or cis conformation (Fig. 1D) in the MOR binding pocket, without any reported transition between these two conformations (de Waal et al, 2020;Lipinski et al, 2019;Podlewska et al, 2020;Ricarte et al, 2021;Vo et al, 2021). These results suggest the transition between the trans and cis conformations may have a relatively high energy barrier to overcome in the MD simulations, but these studies did not evaluate which conformation has lower energy.…”

“…In addition, the conformations of the bound fentanyl are also different in these studies. The dihedral angle between amide carbonyl and aniline was in the trans configuration in the Ricarte, de Waal, and Lipinski poses (de Waal et al, 2020;Lipinski et al, 2019;Ricarte et al, 2021), and cis in the Vo and Podleska poses (Podlewska et al, 2020;Vo et al, 2021) (see the trans and cis definitions in Fig. 1D).…”

“…By selecting and then manually adjusting the docked poses, we established fentanyl poses in both the APF and FPA orientations (see the definitions in Fig. 1B,C) and in both trans and cis conformations, as proposed by previous studies (de Waal et al, 2020;Lipinski et al, 2019;Podlewska et al, 2020;Ricarte et al, 2021;Vo et al, 2021) (see the definition in Fig. 1D).…”

“…By taking advantage of these structures, several groups have computationally investigated the binding mechanism of fentanyl, and its analogs, at the MOR, but they have not reached a consensus (de Waal et al, 2020;Ellis et al, 2018;Lipinski et al, 2019;Podlewska et al, 2020;Ricarte et al, 2021;Vo et al, 2021). While these studies commonly found that the positively charged nitrogen on the piperidine ring forms a salt bridge with Asp 3.32 (superscripts denote Ballesteros-Weinstein numbering (Ballesteros and Weinstein, 1995)) in the binding site of the MOR, they proposed different binding modes of fentanyl and its analogs.…”

“…While these studies commonly found that the positively charged nitrogen on the piperidine ring forms a salt bridge with Asp 3.32 (superscripts denote Ballesteros-Weinstein numbering (Ballesteros and Weinstein, 1995)) in the binding site of the MOR, they proposed different binding modes of fentanyl and its analogs. Specifically, based on the crystal structure of the MOR in an active conformation, Vo et al carried out extensive molecular dynamics (MD) simulations recently and proposed that, in addition to forming a saltbridge with Asp147 3.32 in the orthosteric binding site (OBS), fentanyl can move deeper toward the intracellular side with its positively charged nitrogen forming a hydrogen bond to His297 6.52 (Vo et al, 2021). They found that the amide and aniline groups of fentanyl point towards the extracellular portions of transmembrane segments (TMs) 2 and 7, an orientation of which is similar to that proposed by Lipinski et al (Lipinski et al, 2019;Vo et al, 2021) (defined as the phenyl-piperidine-amide (FPA) orientation in Fig.…”