Mechanistic Basis for 5-HT_2AR Over 5-HT_2BR Activation

Selective 5-HT_2AR agonism without 5-HT_2BR activation is critical for safe psychedelic-inspired therapeutics. Here, we systematically probe the ligand-binding pockets of 5-HT_2AR and 5-HT_2BR and identify steric and conformational constraints at the side-extended pocket (SEP) and extended binding pocket (EBP) that dictate ligand orientation and receptor signaling. Guided by these insights, we design derivatives across tryptamine and phenethylamine scaffolds that integrate SEP and EBP engagement with scaffold modification to reinforce 5-HT_2AR activation while limiting 5-HT_2BR activity. Cryo-EM structures of 5-HT_2AR and 5-HT_2BR bound to IHCH-2330, a selective 5-HT_2AR agonist and 5-HT_2BR antagonist, confirm the design principle. These findings show differential activation mechanisms between 5-HT_2AR and 5-HT_2BR and provide a rational framework for engineering safer, functionally selective serotonergic compounds, mitigating 5-HT_2BR-mediated side effects while preserving therapeutic efficacy.