Binding pose depth modulates photoswitchable ligands’ efficacy at the 5-HT2A receptor

The efficacy of photoswitchable ligands for the human 5-HT2A receptor depends on the vertical depth of ligand insertion into the orthosteric binding pocket, determined by the position of a methoxy substituent. Molecular dynamics simulations show that a para-methoxy ligand (compound 1) acts as a weak antagonist in its trans form because it forms hydrogen bonds with Asp231 and Thr160, anchoring it deeper and preventing engagement with activation-critical residues. A meta-methoxy ligand (compound 2) lacks these anchoring interactions, adopts a shallower pose, and maintains agonist activity in both forms. The authors propose that ligand insertion depth is a critical determinant of efficacy, offering a framework for designing light-sensitive GPCR ligands with tunable signaling.

Study at a glance

Abstract

Abstract Photoswitchable ligands enable reversible control of receptor signaling through light-induced cis – trans isomerization, yet predicting how subtle structural modifications affect efficacy remains challenging. Here, we use molecular dynamics simulations to investigate two azobenzene-based human 5–HT 2A receptor ligands differing only by a methoxy substituent position ( para – vs meta –methoxy). Compound 1 ( para –methoxy) switches from acting as a weak antagonist ( trans ) to a moderate agonist ( cis ), whereas compound 2 ( meta -methoxy) maintains agonist activity in both forms, with cis - 2 exhibiting the highest efficacy. Our simulations reveal that the key determinant of these efficacy differences lies in the vertical depth of ligand insertion into the orthosteric binding pocket. The para –methoxy moiety of trans – 1 forms hydrogen bonds with Asp231 5.35 and Thr160 3.37 , anchoring the ligand deeper than typical tryptamine agonists and preventing engagement with activation-critical residues, thereby stabilizing the inactive receptor. Conversely, trans – 2 lacks these anchoring interactions and adopts a shallower, agonist-compatible pose. In the active receptor, cis – 2 forms a persistent Thr160 3.37 hydrogen bond that allows deeper penetration between TM4 and TM5, whereas cis – 1 ’s para –methoxy causes steric hindrances limiting this interaction. Based on these findings, we suggest that ligand insertion depth is a critical determinant of efficacy. This provides a framework for designing light-sensitive GPCR ligands with tunable signaling properties.