No abstract

Phenylalkylamines such as 1-(4-bromo-2, 5-dimethoxyphenyl)-2-aminopropane (DOB; 1a) and its corresponding iodo derivative DOI (2) are commonly used 5-HT(2) serotonin agonists. Previous studies have established that the 2,5-dimethoxy substitution pattern found in these compounds is optimal for high affinity at 5-HT(2A) receptors and that substituents at the 4-position can modulate affinity over a wide range. We have previously shown, however, that when the 4-position is substituted with a 3-phenylpropyl substituent (i.e., 3), the compound binds with an affinity comparable to that of 1a but that it possesses 5-HT(2A) antagonist character. The present study examined the structure-affinity relationships of 3, and the results were very much unexpected. That is, the 2,5-dimethoxy substitution pattern of 3 is not required for high affinity. Either of the two methoxy groups can be removed without untoward effect on affinity, and relocation of the methoxy substituents actually enhances affinity by as much as an order of magnitude. None of the compounds displayed more than 20-fold selectivity for 5-HT(2A) over 5-HT(2C) receptors. In addition, several were demonstrated to act as 5-HT(2A) partial agonists. As such, the results of this study suggest that the structure-affinity relationships of phenylalkylamines as 5-HT(2A) ligands now be reinvestigated in greater detail.

Risperidone displays a novel mechanism of antagonism of the h5-HT 7 receptor. Pretreatment of the cells with 5 or 20 nM risperidone, followed by removal of the drug from the media, renders the 5-HT 7 receptors unresponsive to 10 M 5-HT for at least 24 h. Thus, risperidone seems to be producing a rapid, long-lasting inactivation of the h5-HT 7 receptor. Whole-cell radioligand binding studies indicate that risperidone interacts in an irreversible or pseudo-irreversible manner with the h5-HT 7 receptor, thus producing the inactivation. Internalization of the h5-HT 7 receptor was not detected by monitoring green fluorescent protein-labeled fluorescent forms of the h5-HT 7 receptor exposed to 20 nM risperidone. Ten other antagonists were tested for h5-HT 7 -inactivating properties, and only 9-OH-risperidone and methiothepin were found to demonstrate the same anomalous properties as risperidone. These results indicate that the h5-HT 7 receptor may possess unique structural features that allow certain drugs to induce a conformation resulting in an irreversible interaction in the intact membrane environment. This may indicate that the h5-HT 7 receptor is part of a subfamily of G-protein-coupled receptors (GPCRs) possessing this property or that many GPCRs have the potential to be irreversibly blocked, but only select drugs can induce this effect. At the very least, the possibility that highly prescribed drugs, such as risperidone, are irreversibly antagonizing GPCR function in vivo is noteworthy.

In a previous publication, using human 5-hydroxytryptamine 7 (h5-HT 7 ) receptor-expressing human embryonic kidney (HEK) 293 cells, we reported the rapid, potent inactivation of the h5-HT 7 receptor stimulation of cAMP production by three antagonists: risperidone, 9-OH-risperidone, and methiothepin (Smith et al., 2006). To better understand the drug-receptor interaction producing the inactivation, we 1) expanded the list of inactivating drugs, 2) determined the inactivating potencies and efficacies by performing concentration-response experiments, and 3) determined the potencies and efficacies of the inactivators as irreversible binding site inhibitors. Three new drugs were found to fully inactivate the h5-HT 7 receptor: lisuride, bromocryptine, and metergoline. As inactivators, these drugs displayed potencies of 1, 80, and 321 nM, respectively. Pretreatment of 5-HT 7 -expressing HEK cells with increasing concentrations of the inactivating drugs risperidone, 9-OH-risperidone, methiothepin, lisuride, bromocriptine, and metergoline potently inhibited radiolabeling of the h5-HT 7 receptor, with IC 50 values of 9, 5.5, 152, 3, 73, and 10 nM, respectively. We were surprised to find that maximal concentrations of risperidone and 9-OH-risperidone inhibited only 50% of the radiolabeling of h5-HT 7 receptors. These results indicate that risperidone and 9-OH risperidone may be producing 5-HT 7 receptor inactivation by different mechanisms than lisuride, bromocryptine, metergoline, and methiothepin. These results are not interpretable using the conventional model of G-protein-coupled receptor function. The complex seems capable of assuming a stable inactive conformation as a result of the interaction of certain antagonists. The rapid, potent inactivation of the receptor-G-protein complex by antagonists implies a constitutive, pre-existing complex between the h5-HT 7 receptor and a G-protein.