Pharmacological Analysis of the Novel, Rapid, and Potent Inactivation of the Human 5-Hydroxytryptamine₇Receptor by Risperidone, 9-OH-Risperidone, and Other Inactivating Antagonists

Abstract

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.

Human 5-HT7 Receptor-Induced Inactivation of Forskolin-Stimulated Adenylate Cyclase by Risperidone, 9-OH-Risperidone and Other “Inactivating Antagonists”

“…at ASPET Journals on May 12, 2018 molpharm.aspetjournals.org previous observations, indicating that they display somewhat different properties as inactivators (Knight et al, 2009). As shown in Fig.…”

“…These interactions have been shown to be classic competitive antagonist interactions (Roth et al, 1994;Smith et al, 2006). In previous publications, using h5-HT 7 receptor-expressing HEK293 cells, we reported the rapid, potent inactivation of h5-HT 7 receptor stimulation of cAMP production by six antagonists: risperidone, 9-OH-risperidone, methiothepin, bromocriptine, metergoline, and lisuride (Smith et al, 2006;Knight et al, 2009). The mechanism seems to involve the pseudo-irreversible interaction of the drugs with the h5-HT 7 receptor, thus occluding the orthosteric binding site and preventing stimulation by 5-HT.…”

“…Six of the drugs tested displayed this property: risperidone, 9-OH-risperidone, methiothepin, bromocriptine, metergoline, and lisuride. These six drugs had been previously characterized as h5-HT 7 receptor inactivators (Smith et al, 2006;Knight et al, 2009). The six drugs tested that had inactivating ability exhibited h5-HT 7 receptor affinities that ranged 5-HT 7 Inactivation of Forskolin-Stimulated Adenylate Cyclase from 0.4 to 143 nM.…”

“…Risperidone and 9-OHrisperidone irreversibly inhibited only 50% of the h5-HT 7 orthosteric binding sites, whereas the other four inactivators irreversibly inhibited all the h5-HT 7 receptor binding sites (Knight et al, 2009). In addition, metergoline's potency as an inactivator was significantly lower than predicted from its affinity for the h5-HT 7 receptor, whereas the other five inactivators' potencies matched their affinities for the h5-HT 7 receptor.…”

“…There seem to be no reports of acute effects of antagonists on forskolin-stimulated adenylate cyclase activity. During the previously reported investigation of h5-HT 7 receptor inactivation by risperidone and other inactivating antagonists (Knight et al, 2009), forskolin-stimulated adenylate cyclase activity was monitored as a control for cellular capacity to produce cAMP. It was anticipated that forskolin-stimulated adenylate cyclase activity would not be affected by the inactivating drugs.…”