Exploring a Druggable Hydrophobic Tunnel in the 5-HT2A Receptor with Potent Phenethylamines

The serotonin 2A receptor (5-HT2AR) is the most abundant excitatory serotonin receptor in the brain and the primary target for serotonergic psychedelics. Despite its importance, the receptor's mechanism of action and structure-activity relationships are not fully understood. The experimental 5-HT2AR structures have identified a hydrophobic tunnel lateral to the orthosteric site, situated in between the transmembrane helices 4 and 5. Here, we have examined this tunnel and its implications for 5-HT2AR agonist pharmacology. The tunnel characteristics and the key roles of Phe2345x39 and Gly2385x43 were delineated by molecular modeling and the synthesis and functional characterization of ten phenethylamine analogs. Molecular modeling, structure-activity relationship determination, and mutagenesis experiments demonstrate that the agonist potencies at the 5-HT2AR exhibited by analogs with 4-substituents with at least four heavy atoms are rooted in their ability to protrude into and form interactions within this tunnel. Thus, these findings provide new insights into the molecular basis for phenethylamine-induced 5-HT2AR activation and identify this tunnel, for the first time, as a druggable region to employ in designing potent 5-HT2AR agonists.