Molecular Interaction of Serotonin 5-HT2A Receptor Residues Phe339(6.51) and Phe340(6.52) with Superpotent N-Benzyl Phenethylamine Agonists

Experiments were conducted to examine the molecular basis for the high affinity and potency of a new class of 5-HT_2A receptor agonists, N-benzyl phenethylamines. Competition binding assays at several serotonin receptors confirmed that an N-arylmethyl substitution was necessary for affinity increases up to 300-fold over simple N-alkyl homologs, as well as enhanced selectivity for 5-HT_2A versus 5-HT_2C and 5-HT_1A receptors. PI hydrolysis functional assays confirmed that these N-benzyl phenethylamines are potent and highly efficacious agonists at the rat 5-HT_2A receptor. Virtual docking of these compounds into a human 5-HT_2A receptor homology model indicated that the N-benzyl moiety might be interacting with Phe339^(6.51), whereas the phenethylamine portion was likely to be interacting with Phe340^(6.52). Experiments in h5-HT_2A receptors with Phe339^(6.51)L and Phe340^(6.52)L mutations seem to support this hypothesis. Dramatic detrimental effects on affinity, potency, and intrinsic activity were observed with the Phe339^(6.51)L mutation for all N-benzyl analogs, whereas most N-unsubstituted phenethylamines and traditional agonists were only weakly affected, if at all. Consistent with other published studies, the Phe340^(6.52)L mutation detrimentally affected affinity, potency, and intrinsic activity of nearly all compounds tested, although a strong change in intrinsic activity was not seen with most N-aryl analogs. These data further validate the topology of our h5-HT_2A receptor homology model. It is noteworthy that this study is the first to identify a hitherto unrecognized role for residue 6.51 in agonist activation of a serotonin G protein-coupled receptor (GPCR), whereas most previous reports have suggested a varied and sometimes contradictory role in homologous GPCRs.