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

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 5–HT2A 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 indicate 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 Asp2315.35 and Thr1603.37, 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 Thr1603.37 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.