Entry from the Lipid Bilayer: A Possible Pathway for Inhibition of a Peptide G Protein-Coupled Receptor by a Lipophilic Small Molecule

Biochemistry. 2018 Oct 2;57(39):5748-5758. doi: 10.1021/acs.biochem.8b00577. Epub 2018 Aug 27.


The pathways that G protein-coupled receptor (GPCR) ligands follow as they bind to or dissociate from their receptors are largely unknown. Protease-activated receptor-1 (PAR1) is a GPCR activated by intramolecular binding of a tethered agonist peptide that is exposed by thrombin cleavage. By contrast, the PAR1 antagonist vorapaxar is a lipophilic drug that binds in a pocket almost entirely occluded from the extracellular solvent. The binding and dissociation pathway of vorapaxar is unknown. Starting with the crystal structure of vorapaxar bound to PAR1, we performed temperature-accelerated molecular dynamics simulations of ligand dissociation. In the majority of simulations, vorapaxar exited the receptor laterally into the lipid bilayer through openings in the transmembrane helix (TM) bundle. Prior to full dissociation, vorapaxar paused in metastable intermediates stabilized by interactions with the receptor and lipid headgroups. Derivatives of vorapaxar with alkyl chains predicted to extend between TM6 and TM7 into the lipid bilayer inhibited PAR1 with apparent on rates similar to that of the parent compound in cell signaling assays. These data are consistent with vorapaxar binding to PAR1 via a pathway that passes between TM6 and TM7 from the lipid bilayer, in agreement with the most consistent pathway observed by molecular dynamics. While there is some evidence of entry of the ligand into rhodopsin and lipid-activated GPCRs from the cell membrane, our study provides the first such evidence for a peptide-activated GPCR and suggests that metastable intermediates along drug binding and dissociation pathways can be stabilized by specific interactions between lipids and the ligand.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Video-Audio Media

MeSH terms

  • Animals
  • Binding Sites
  • Fibroblasts
  • Humans
  • Lactones / chemistry
  • Lactones / metabolism*
  • Ligands
  • Lipid Bilayers / metabolism*
  • Molecular Dynamics Simulation
  • Molecular Structure
  • Phosphatidylcholines / metabolism
  • Protein Binding
  • Protein Conformation
  • Pyridines / chemistry
  • Pyridines / metabolism*
  • Rats
  • Receptor, PAR-1 / antagonists & inhibitors*
  • Receptor, PAR-1 / chemistry
  • Receptor, PAR-1 / metabolism*


  • Lactones
  • Ligands
  • Lipid Bilayers
  • Phosphatidylcholines
  • Pyridines
  • Receptor, PAR-1
  • 1-palmitoyl-2-oleoylphosphatidylcholine
  • vorapaxar