Milt Teitler

Binding of β-carbolines and related agents at serotonin (5-HT2 and 5-HT1A), dopamine (D2) and benzodiazepine receptors

A large series of β-carbolines was examined for their ability to bind at [3H]agonist-labeled 5-HT2A serotonin receptors. Selected β-carbolines were also examined at 5-HT2C serotonin receptors, 5-HT1A serotonin receptors, dopamine D2 receptors, and benzodiazepine receptors. Indolealkylamines and phenylisopropylamines were also evaluated in some of these binding assays. The β-carbolines were found to bind with modest affinity at 5-HT2A receptors, and affinity was highly dependent upon the presence of ring substituents and ring saturation. The β-carbolines displayed little to no affinity for 5-HT1A serotonin receptors, dopamine D2 receptors and, with the exception of β-CCM, for benzodiazepine receptors. Examples of β-carbolines, indolealkylamines (i.e. N,N-dimethyltryptamine analogs), and phenylisopropylamines have been previously shown to produce common stimulus effects in animals trained to discriminate the phenylisopropylamine hallucinogen DOM (i.e. 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane) from vehicle. Although the only common receptor population that might account for this action is 5-HT2A, on the basis of a lack of enhanced affinity for agonist-labeled 5-HT2A receptors, as well as on their lack of agonist action in the PI hydrolysis assay, it is difficult to conclude that the β-carbolines behave in a manner consistent with that of other classical hallucinogens.

Beta-Blocker Selectivity at Cloned Human Beta1- and Beta2-Adrenergic Receptors

Summary. The ratio between the affinities of beta-blockers for the beta2- and beta1-receptors is often used to predict the cardioselectivity and the potential consequences of blocking beta2-receptor–mediated effects of adrenergic receptor blockers. These ratios have been traditionally determined using various in vitro models of beta2 and beta1-receptor antagonist activity, including isolated organ preparations and redioligand binding in tissues from various species. The data from these studies, while useful, are complicated by the use of different preparations, techniques, and nonhuman models. Recombinant cell lines expressing human beta2 and beta1 receptors have been developed, allowing for the direct comparison of the affinities of the beta-blockers for the beta2 and beta1 receptors under identical conditions, and allowing a precise determination of the beta1-receptor selectivity of the beta-blockers. Bisoprolol, atenolol, propranolol, betaxolol, metoprolol, carvedilol, and ICI 118, 551 were compared for their beta-receptor selectivity using membranes prepared from recombinant cells selectively expressing human beta2 and beta1 receptors. Bisoprolol was found to have the highest selectivity for the beta1 receptor, displaying a beta2//beta1 ratio of 19 (a 19-fold higher affinity for the beta receptor than for the beta2 receptor). Atenolol, metoprolol, and betaxolol displayed lower, although significant, selectivity for the beta1 receptor, whereas propranolol and carvedilol displayed no signiicant beta-adrenergic selectivity. ICI 118,55 was selective for the beta2 receptor. The equilibrium dissociation constants of the beta-blockers for the beta1 and beta2 receptors were generally similar to previously reported values. The affinity ratios were also generally similar to previously reported values.

Agonist activity of LSD and lisuride at cloned 5HT2A and 5HT2C receptors.

Abstract Evidence from studies with phenylisopropylamine hallucinogens indicates that the 5HT2A receptor is the likely target for the initiation of events leading to hallucinogenic activity associated with LSD and related drugs. Recently, lisuride (a purported non-hallucinogenic congener of LSD) was reported to be a potent antagonist at the 5HT2C receptor and an agonist at the 5HT2A receptor. LSD exhibited agonist activity at both receptors. These data were interpreted as indicating that the 5HT2C receptor might be the initiating site of action for hallucinogens. To test this hypothesis, recombinant cells expressing 5HT2A and 5HT2C receptors were used to determine the actions of LSD and lisuride. LSD and lisuride were potent partial agonists at 5HT2A receptors with EC50 values of 7.2 nM and 17 nM, respectively. Also, LSD and lisuride were partial agonists at 5HT2C receptors with EC50 values of 27 nM and 94 nM, respectively. We conclude that lisuride and LSD have similar actions at 5HT2A and 5HT2C receptors in recombinant cells. As agonist activity at brain 5HT2A receptors has been associated with hallucinogenic acitivity, these results indicate that lisuride may possess hallucinogenic activity, although the psychopharmacological effects of lisuride appear to be different from the hallucinogenic effects of LSD.

Activating Mutations of the Serotonin 5‐HT2C Receptor

Abstract: Site‐directed mutagenesis was performed to create a mutant serotonin 5‐HT2C receptor that would mimic the active conformation of the native receptor. Structural alteration of receptor conformation was achieved by changing amino acid no. 312 from serine to phenylalanine (S312F) or lysine (S312K). After expression in COS‐7 cells, the binding affinity of 5‐HT for [3H]‐mesulergine‐labeled 5‐HT2C receptors increased from 203 nM (native) to 76 nM for S312F and 6.6 nM for S312K mutant receptors. 5‐HT potency for stimulation of phosphatidylinositol (PI) hydrolysis increased from 70 nM (native) to 28 nM for S312F and 2.7 nM for S312K mutant receptors. The mutant receptors were constitutively active, stimulating PI hydrolysis in the absence of agonist. S312F and S312K mutations resulted in twofold and five‐fold increases, respectively, in basal levels of PI hydrolysis. Mianserin and mesulergine displayed inverse agonist activity by decreasing basal levels of PI hydrolysis stimulated by S312K mutant receptors. [3H]5‐HT and [3H]‐mesulergine labeled the same number of S312K mutant receptors and 5′‐guanylylimidodiphosphate had no effect on [3H]5‐HT binding. These results indicate that serine → lysine mutation at amino acid no. 312 produces an agonist high‐affinity state of the 5‐HT2C receptor that spontaneously couples to G proteins and stimulates PI hydrolysis in the absence of agonist.

Investigation of hallucinogenic and related β-carbolines

Abstract Certain β-carbolines are known to be hallucinogenic in humans, and several produce stimulus effects in animals similar to those of the classical hallucinogen 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM). Classical hallucinogens bind at 5-HT2 serotonin receptors and these receptors are thought to play a role in their mechanism of action. In the present study, we examined the binding of 15 β-carbolines at rat 5-HT2A and 5-HT2C receptors. Affinities (Ki values) of the β-carbolines ranged from about 100 nM to greater than 10 000 nM depending upon the degree of saturation of the pyridyl ring, and upon the presence and location of methoxy substituents in the benzenoid ring. In a further study, six rats were trained to discriminate the hallucinogenic β-carboline harmaline (3.0 mg/kg, i.p.) from vehicle using a VI-15s schedule of reinforcement. This represents the first time a hallucinogenic β-carboline has been used as a training drug in a drug discrimination study. Administration of DOM to the harmaline-trained animals resulted in 76% harmaline-appropriate responding at 1.25 mg/kg DOM and disruption of behavior at a higher dose. Taken together, the results of the present investigation demonstrate that: (a) certain β-carbolines bind at 5-HT2 receptors; (b) that harmaline serves as a training drug at 3.0 mg/kg in drug discrimination studies with rats as subjects; and that (c) there is some similarity between the stimulus effects produced by harmaline and DOM.

Agonist high and low affinity state ratios predict drug intrinsic activity and a revised Ternary complex mechanism at serotonin 5-HT2A and 5-HT2C receptors

The ternary complex model as applied to G‐protein coupled receptors (GPCR) predicts that an agonist binds with low affinity (KL) to the free receptor (R), leading to an agonist/receptor/G‐protein complex. This ternary complex displays high agonist affinity (KH), resulting in signal transduction. Classical dogma states that the ratio KL/KH predicts intrinsic activity of drugs: the higher the ratio the higher the intrinsic activity. This model was based on studies in which KL and KH were indirectly determined by computer analyses of antagonist radioligand binding data. In order to investigate the relationship of KL, KH, and intrinsic activity for agonists at 5‐HT2A and 5‐HT2C receptors, we utilized 3H‐agonist and 3H‐antagonist radioligands to directly determine KH and KL. Comparisons of the log KL/KH ratios and intrinsic activities of drugs for stimulating intracellular phosphatidylinositol (PI) hydrolysis revealed a strong correlation for 5‐HT2A (r2 = 0.92) and 5‐HT2C (r2 = 0.96) receptors. The data were fit to computer simulations based on the original ternary complex model and the revised ternary complex model in which an activated state of the receptor (R*) exists in equilibrium with the resting state of the receptor (R). Data produced for both 5‐HT2A and 5‐HT2C receptors were better‐fitted to a revised ternary complex model, rather than the classical ternary complex model. These data support a revised model for the molecular events coupling GPCR to activation of G‐proteins and indicate that a strong correlation between the KL/KH ratio and intrinsic activity for agonist action at GPCR is consistent with the existence of R*. Synapse 35:144–150, 2000.

Risperidone Irreversibly Binds to and Inactivates the h5-HT7 Serotonin Receptor

Risperidone displays a novel mechanism of antagonism of the h5-HT7 receptor. Pretreatment of the cells with 5 or 20 nM risperidone, followed by removal of the drug from the media, renders the 5-HT7 receptors unresponsive to 10 μM 5-HT for at least 24 h. Thus, risperidone seems to be producing a rapid, long-lasting inactivation of the h5-HT7 receptor. Whole-cell radioligand binding studies indicate that risperidone interacts in an irreversible or pseudo-irreversible manner with the h5-HT7 receptor, thus producing the inactivation. Internalization of the h5-HT7 receptor was not detected by monitoring green fluorescent protein-labeled fluorescent forms of the h5-HT7 receptor exposed to 20 nM risperidone. Ten other antagonists were tested for h5-HT7-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-HT7 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-HT7 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.

Ketanserin and Spiperone as Templates for Novel Serotonin 5-HT2A Antagonists

The structures of ketanserin (1) and spiperone (2) were examined in detail to determine the role of various substituent groups on 5-HT(2A) receptor affinity and selectivity. It was found that the presence of the quinazoline ring of ketanserin detracts from selectivity and that various ring-opened analogs displayed ketanserin-like affinity and up to 30-fold enhanced selectivity. The triazaspirodecanone portion of spiperone is a major determinant of its 5-HT affinity and selectivity. The conformational rigidity imposed by the ring, as well as the nature of the N(1)-substituent, are important factors in controlling binding at 5-HT(2A), 5-HT(2C), 5-HT(1A), and dopamine D2 receptors. Replacement of the N(1)-phenyl ring of spiperone with a methyl group (KML-010; 48) resulted in a compound that binds at 5-HT(2A) receptors with slightly lower affinity than spiperone, but that lacked affinity (Ki >10,000 nM) for 5-HT(2C) and 5-HT(1A) receptors and binds with 400-fold reduced affinity at D2 receptors.

Constitutive Activity of G-Protein Coupled Receptors: Emphasis on Serotonin Receptors

Several lines of evidence indicate that G-protein coupled receptors (GPCR) may exist in a state that allows a tonic level of stimulation in vivo (constitutive activity). Several native forms of GPCR, when expressed in recombinant cell lines, display significant signal transduction stimulation in the absence of activating ligand. Many GPCR, including three serotonin receptors, display robust constitutive activation upon the mutation of a single amino acid, indicating mutations producing inappropriate constitutive activation may be etiological factors in diseases. If constitutive activity of GPCR is as common a phenomenon as some researchers suspect, this would suggest significant alterations in the classical model of ligand-receptor interactions. One of the most significant implications of constitutive activity for pharmacologists and medicinal chemists, is the possibility of developing drugs that lower the level of constitutive activity. Such compounds have been termed inverse agonists . These drugs, in theory, would have different physiological effects, and therefore possibly different therapeutic potential, than classical competitive receptor antagonists ( neutral antagonists ). Theoretical issues concerning constitutive activity in the GPCR family and some of the evidence supporting the existence of constitutive activity in the GPCR family is reviewed. Studies are presented demonstrating the procedures for producing and characterizing constitutive activated forms of serotonin receptors, including the demonstration of inverse agonist activity of drugs on these receptors.

Agonist activity of sumatriptan and metergoline at the human 5-HT1Dβ receptor: further evidence for a role of the 5-HT1D receptor in the action of sumatriptan

We have recently cloned a novel human 5-HT1D receptor subtype termed 5-HT1D beta. CHO K1 cells expressing the human serotonin 5-HT1D beta receptor were assayed to determine the second messenger system of this receptor. Cyclic AMP radioimmunoassays revealed that the 5-HT1D beta receptor is negatively coupled to adenylate cyclase in this cell system. A maximum of 50% inhibition of forskolin stimulated cAMP production was obtained with 5-HT1 receptor agonists which was blocked by the non-selective 5-HT receptor antagonist methiothepin (pKB = 100 nM). The novel anti-migraine drug sumatriptan, a putative 5-HT1D selective compound, acted as an agonist at the 5-HT1D beta receptor. Most notably metergoline, a putative 5-HT1 receptor antagonist, did not block the effects of 5-HT and was found to be acting as a full agonist at the 5-HT1D beta receptor. The ability of metergoline to act as an agonist at the 5-HT1D beta receptor may explain why it does not inhibit 5-HT and sumatriptan induced contraction of dog saphenous vein and other large conducting arteries. These results suggest that the 5-HT1D beta receptor may be the site of action of sumatriptan in preventing migraine, and that metergolines actions on the dog saphenous vein are not contradictory to that hypothesis, as previously reported.