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

The mechanisms by which G protein-coupled receptor (GPCR) ligands bind to and dissociate from their receptors remain poorly understood. Protease-activated receptor-1 (PAR1), a GPCR, is activated through intramolecular binding of a tethered agonist peptide revealed by thrombin cleavage. In contrast, the antagonist vorapaxar is a lipophilic compound that binds within a receptor pocket largely shielded from the extracellular environment. However, the precise binding and dissociation pathway of vorapaxar is unknown.
Using the crystal structure of the PAR1–vorapaxar complex, we conducted temperature-accelerated molecular dynamics simulations to explore ligand dissociation. Most simulations revealed that vorapaxar exited the receptor laterally into the lipid bilayer through openings between transmembrane (TM) helices. Before complete dissociation, the ligand paused at metastable intermediate states, stabilized by interactions with both the receptor and lipid headgroups.
Vorapaxar derivatives modified with alkyl chains—designed to extend between TM6 and TM7 into the membrane—exhibited inhibitory activity and apparent association rates similar to the parent compound in cell-based signaling assays. These findings support a model in which vorapaxar accesses its binding site from the lipid bilayer via a path between TM6 and TM7, consistent with the most frequently observed route in simulations.
While membrane entry has been suggested for ligands targeting rhodopsin and lipid-activated GPCRs, our study offers the first evidence of this mechanism for a peptide-activated GPCR. Moreover, it highlights how metastable intermediates along ligand binding and unbinding pathways can be stabilized through specific lipid–ligand interactions.