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 Nbenzyl 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.Agonist activity at the serotonin 2A (5-HT 2A ) receptor is essential for the psychopharmacology of serotonergic psychedelics such as LSD, DOI, psilocin, and 5-MeO-DMT, compounds with unique and dramatic effects on certain aspects of consciousness (Nichols 2004). Moreover, we have recently identified a functionally selective 5-HT 2A receptor agonist that selectively activates phosphoinositide turnover over production of eicosanoids (McLean et al., 2006a). A key aspect to understanding the effects on consciousness of psychedelics is the study of the receptor-ligand interaction at the molecular level and how it modulates second messenger generation subsequent to receptor activation.We have been particularly interested in experimental val-

A series of conformationally restricted analogues of the hallucinogenic phenethylamine 1 (2,5-dimethoxy-4-bromophenethylamine, 2C-B) was synthesized to test several hypotheses concerning the bioactive conformation of phenethylamine ligands upon binding to the 5-HT(2A) receptor. These benzocycloalkane analogues were assayed for their receptor binding affinity and ability to activate downstream signaling pathways, and one exceptional compound was selected for testing in an in vivo drug discrimination model of hallucinogenesis. All compounds were examined in silico by virtual docking into a homology model of the 5-HT(2A) receptor. On the basis of these docking experiments, it was predicted that the R enantiomer of benzocyclobutene analogue 2 would be the most potent. Subsequent chemical resolution and X-ray crystallography confirmed this prediction, as (R)-2 proved to be equipotent to LSD in rats trained to discriminate LSD from saline. Thus, we propose that the conformation of 2 mimics the active binding conformation of the more flexible phenethylamine type hallucinogens. In addition, (R)-2 is one of the most potent and selective compounds yet discovered in the in vivo drug discrimination assay. Further, 2 was found to be a functionally selective agonist at the 5-HT(2A) receptor, having 65-fold greater potency in stimulating phosphoinositide turnover than in producing arachidonic acid release. If hallucinogenic effects are correlated with arachidonic acid production, such functionally selective 5-HT(2A) receptor agonists may lack the intoxicating properties of hallucinogens such as LSD.

Previous studies in our laboratory have shown that in NIH3T3-5HT 2A cells, 5-HT-induced AA release is PLA 2 -coupled and independent of 5-HT 2A receptor-mediated PLC activation. Although 5-HT 2A receptor-mediated PLC activation is known to be Ga q -coupled, much less is understood about 5-HT 2A receptor-mediated PLA 2 activation. Therefore, the studies presented here were aimed at elucidating the signal transduction pathway linking stimulation of the 5-HT 2A receptor to PLA 2 activation. By employing various selective inhibitors, toxins, and antagonistic peptide constructs, we propose that the 5-HT 2A receptor can couple to PLA 2 activation through two parallel signaling cascades. Initial experiments were designed to examine the role of pertussis toxin-sensitive G proteins, namely Ga i/o , as well as pertussis toxin-insensitive G proteins, namely Ga 12/13 , in 5-HT-induced AA release. Furthermore, inactivation of both Gbc heterodimers and Rho proteins resulted in decreased agonist-induced AA release, without having any effect on PLC-IP accumulation. We also demonstrated 5-HT 2A receptor-mediated phosphorylation of ERK1,2 and p38. Moreover, pretreatment with selective ERK1,2 and p38 inhibitors resulted in decreased 5-HT-induced AA release. Taken together, these results suggest that the 5-HT 2A receptor expressed in NIH3T3 cells can couple to PLA 2 activation though a complex signaling mechanism involving both Ga i/o -associated Gbc-mediated ERK1,2 activation and Ga 12/13 -coupled, Rho-mediated p38 activation.

Experiments compared a series of phenethylamine hallucinogens with their phenylisopropylamine analogues for binding affinity and ability to stimulate serotonin 5-HT 2A receptor-mediated hydrolysis of phosphatidyl inositol in cells expressing cloned rat and human 5-HT 2A receptors. The (±) phenylisopropylamine analogues had significantly higher intrinsic activities for 5-HT 2A receptor-mediated hydrolysis of phosphatidyl inositol compared to their phenethylamine analogues. With respect to the effects of the stereochemistry of the phenylisopropylamines, those with the (R) absolute configuration at the alpha carbon had higher intrinsic activities for hydrolysis of phosphatidyl inositol in a cell line expressing the human 5-HT 2A receptor compared to those with the (S) absolute configuration. In virtual docking studies comparing the (R)-and (S)-phenylisopropylamines with their phenethylamine analogues, there were distinct differences in the orientations of key ligand binding domain residues that have been identified as important by previous mutagenesis studies.In conclusion, our data support the hypothesis that phenylisopropylamines have higher hallucinogenic potency than their phenethylamine analogues primarily because they have higher intrinsic activities at 5-HT 2A receptors. Although virtual ligand binding led to significant perturbations of certain key residues, our results emphasize the conclusion reached by others that overall three-dimensional structural microdomains within the receptor must be considered.

To date, several studies have demonstrated that phospholipase C-coupled receptors stimulate the production of endocannabinoids, particularly 2-arachidonoylglycerol. There is now evidence that endocannabinoids are involved in phospholipase C-coupled serotonin 5-HT 2A receptor-mediated behavioral effects in both rats and mice. The main objective of this study was to determine whether activation of the 5-HT 2A receptor leads to the production and release of the endocannabinoid 2-arachidonoylglycerol. NIH3T3 cells stably expressing the rat 5-HT 2A receptor were first incubated with [ 3 H]-arachidonic acid for 24 h. Following stimulation with 10 lM serotonin, lipids were extracted from the assay medium, separated by thin layer chromatography, and analyzed by liquid scintillation counting. Our results indicate that 5-HT 2A receptor activation stimulates the formation and release of 2-arachidonoylglycerol. The 5-HT 2A receptor-dependent release of 2-arachidonoylglycerol was partially dependent on phosphatidylinositol-specific phospholipase C activation. Diacylglycerol produced downstream of 5-HT 2A receptormediated phospholipase D or phosphatidylcholine-specific phospholipase C activation did not appear to contribute to 2-arachidonoylglycerol formation in NIH3T3-5HT 2A cells. In conclusion, our results support a functional model where neuromodulatory neurotransmitters such as serotonin may act as regulators of endocannabinoid tone at excitatory synapses through the activation of phospholipase C-coupled G-protein coupled receptors.