Abstract
Experiments were conducted to examine the molecular basis for the high affinity and potency of a new class of 5-HT2A 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-HT2A versus 5-HT2C and 5-HT1A receptors. PI hydrolysis functional assays confirmed that these N-benzyl phenethylamines are potent and highly efficacious agonists at the rat 5-HT2A receptor. Virtual docking of these compounds into a human 5-HT2A 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-HT2A 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-HT2A 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.
Footnotes
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This research was supported by National Institutes of Health grant DA02189 from the National Institute on Drug Abuse, and a grant from the Heffter Research Institute. The work was conducted in a facility constructed with support from Research Facilities Improvement Program Grant Number C06-14499 from the National Center for Research Resources of the National Institutes of Health.
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ABBREVIATIONS: LSD, d-lysergic acid diethylamide; 5-HT, 5-hydroxytryptamine, serotonin; CHO, Chinese hamster ovary; HEK, human embryonic kidney; psilocin, 4-hydroxy-N,N-dimethyltryptamine; 5-MeO-DMT, 5-methoxy-N,N-dimethyltryptamine; mescaline, 3,4,5-trimethoxyphenethylamine; DOI, 4-iodo-2,5-dimethoxyphenylisopropylamine; DOI-NBOMe, N-(2-methoxybenzyl)-4-iodo-2,5-dimethoxyphenylisopropylamine; 25H, 2,5-dimethoxyphenethylamine; 25H-NMe, N-methyl-2,5-dimethoxyphenethylamine; 25H-NPr, N-propyl-2,5-dimethoxyphenethylamine; 25H-NB, N-benzyl-2,5-dimethoxyphenethylamine; 25H-NBOMe, N-(2-methoxybenzyl)-2,5-dimethoxyphenethylamine; 25H-NBOH, N-(2-hydroxybenzyl)-2,5-dimethoxyphenethylamine; 24, 2,4-dimethoxyphenethylamine; 24-NB, N-benzyl-2,4-dimethoxyphenethylamine; 24-NBOMe, N-(2-methoxybenzyl)-2,4-dimethoxyphenethylamine; 24-NBOH, N-(2-hydroxybenzyl)-2,4-dimethoxyphenethylamine; 25I, 4-iodo-2,5-dimethoxyphenethylamine; 25I-NB, N-benzyl-4-iodo-2,5-dimethoxyphenethylamine; 25I-NNap, N-methylnapthyl-4-iodo-2,5-dimethoxyphenethylamine; 25I-NBOMe, N-(2-methoxybenzyl)-4-iodo-2,5-dimethoxyphenethylamine; 25I-NBOH, N-(2-hydroxybenzyl)-4-iodo-2,5-dimethoxyphenethylamine; 25I-NBF, N-(2-fluorobenzyl)-4-iodo-2,5-dimethoxyphenethylamine; 25I-NBMD, N-(2,3-methylenedioxybenzyl)-4-iodo-2,5-dimethoxyphenethylamine; PI, phosphatidylinositide(s); TM, transmembrane.
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The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material. - Received July 10, 2006.
- Accepted September 25, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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