Received for publication July 10, 2006.
Revised September 25, 2006.
Accepted for publication September 25, 2006.

Molecular interaction of serotonin 5 HT_2A receptor residues Phe339^(6.51) and Phe340^(6.52) with super-potent N-benzyl phenethylamine agonists

Abstract

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 homologues, as well as enhanced selectivity for 5 HT_2A vs. 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 F339^(6.51), whereas the phenethylamine portion was likely interacting with F340^(6.52). Experiments in h5 HT_2A receptors with F339^(6.51)L and F340^(6.52)L mutations appear to support this hypothesis. Dramatic detrimental effects on affinity, potency, and intrinsic activity were observed with the F339^(6.51)L mutation for all N-benzyl analogues, whereas most N-unsubstituted phenethylamines and traditional agonists were only weakly affected, if at all. Consistent with other published studies, the F340^(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 analogues. These data further validate the topology of our h5 HT_2A receptor homology model. Importantly, this study is the first to identify a hitherto unrecognized role for residue 6.51 in agonist activation of a serotonin GPCR, whereas most previous reports have suggested a varied and sometimes contradictory role in homologous GPCRs.

Key words: Serotonin, Gq/11 family, Structure-activity relationships and modeling, Mutagenesis/Chimeric approaches, Receptor binding studies

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