Ligand-specific roles for transmembrane 5 serine residues in the binding and efficacy of dopamine D(1) receptor catechol agonists.

To refine further the structure-activity relationships of D(1) dopamine receptor agonists, we investigated the roles of three conserved serine residues [Ser198(5.42), Ser199(5.43), and Ser202(5.46)] in agonist binding and receptor activation. These transmembrane domain 5 (TM5) residues are believed to engage catechol ligands through polar interactions. We stably expressed wild-type or mutant (S198A, S199A, and S202A) D(1) receptors in human embryonic kidney cells. These receptors were expressed at similar levels (approximately 2000 fmol/mg) and bound the radioligand [(3)H]R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH 23390), although S198A and S199A displayed significant losses of affinity compared with that for wild-type receptors. The endogenous agonist, dopamine, had losses of potency at each of the mutant receptors. We tested cyclohexyl-substituted isochroman, carbocyclic, and chroman bicyclic dopamine analogs and found that the mutations affected the chroman to a lesser extent than the other compounds. These results support our hypothesis that the decreased D(1) activity of chroman analogs results from a ligand intramolecular hydrogen bond that impairs the ability of the catechol to engage the receptor. Sensitivities of these rigid catechol agonists to the effects of the serine mutations were dependent on ligand geometry, particularly with respect to the rotameric conformation of the ethylamine side chain and the distance between the amino group and each catechol hydroxyl. Functional experiments in striatal tissue suggest that the ability to engage TM5 serines is largely correlated with agonist efficacy for cAMP stimulation. These results provide a new understanding of the complexities of D(1) ligand recognition and agonist activation and have implications for the design of rigid catechol ligands.

Facile synthesis of octahydrobenzo[h]isoquinolines: novel and highly potent D1 dopamine agonists.

The octahydrobenzo[h]isoquinoline scaffold is of interest as a conformationally-restricted phenethylamine that may be useful for constructing biologically active products. Surprisingly, however, no tractable synthesis of this ring system has been reported. We now describe a facile method for obtaining this framework, and illustrate that our approach is easily amenable to substitutions at the 5-position. Importantly, we demonstrate that the 7,8-dihydroxy-5-phenyl-substituted ligand is an extremely potent, high-affinity, full D1 dopamine receptor-selective agonist.

Ligand-dependent oligomerization of dopamine D(2) and adenosine A(2A) receptors in living neuronal cells.

Adenosine A(2A) and dopamine D(2) receptors (A(2A) and D(2)) associate in homo- and heteromeric complexes in the striatum, providing a structural basis for their mutual antagonism. At the cellular level, the portion of receptors engaging in homo- and heteromers, as well as the effect of persistent receptor activation or antagonism on the cell oligomer repertoire, are largely unknown. We have used bimolecular fluorescence complementation (BiFC) to visualize A(2A) and D(2) oligomerization in the Cath.a differentiated neuronal cell model. Receptor fusions to BiFC fluorescent protein fragments retained their function when expressed alone or in A(2A)/A(2A), D(2)/D(2), and A(2A)/D(2) BiFC pairs. Robust fluorescence complementation reflecting A(2A)/D(2) heteromers was detected at the cell membrane as well as in endosomes. In contrast, weaker BiFC signals, largely confined to intracellular domains, were detected with A(2A)/dopamine D(1) BiFC pairs. Multicolor BiFC was used to simultaneously visualize A(2A) and D(2) homo- and heteromers in living cells and to examine drug-induced changes in receptor oligomers. Prolonged D(2) stimulation with quinpirole lead to the internalization of D(2)/D(2) and A(2A)/D(2) oligomers and resulted in decreased A(2A)/D(2) relative to A(2A)/A(2A) oligomer formation. Opposing effects were observed in cells treated with D(2) antagonists or with the A(2A) agonist 5'-N-methylcarboxamidoadenosine (MECA). Subsequent radioreceptor binding analysis indicated that the drug-induced changes in oligomer formation were not readily explained by alterations in receptor density. These observations support the hypothesis that long-term drug exposure differentially alters A(2A)/D(2) receptor oligomerization and provide the first demonstration for the use of BiFC to monitor drug-modulated GPCR oligomerization.

High specific activity tritium-labeled N-(2-methoxybenzyl)-2,5-dimethoxy-4-iodophenethylamine (INBMeO): a high-affinity 5-HT2A receptor-selective agonist radioligand.

The title compound ([3H]INBMeO) was prepared by an O,O-dimethylation reaction of a t-BOC protected diphenolic precursor using no carrier added tritiated iodomethane in DMF with K(2)CO(3). Removal of the t-BOC protecting group and purification by HPLC afforded an overall yield of 43%, with a radiochemical purity of 99% and specific activity of 164Ci/mmol. The new radioligand was suitable for labeling human 5-HT(2A) receptors in two heterologous cell lines and had about 20-fold higher affinity than [(3)H]ketanserin.

trans-2,3-dihydroxy-6a,7,8,12b-tetrahydro-6H-chromeno[3,4-c]isoquinoline: synthesis, resolution, and preliminary pharmacological characterization of a new dopamine D1 receptor full agonist.

We report the synthesis of trans-2,3-dihydroxy-6a,7,8,12b-tetrahydro-6H-chromeno[3,4-c]isoquinoline hydrochloride 6 and the resolution of its enantiomers. This new compound is an oxygen bioisostere of the potent dopamine D1-selective full agonist dihydrexidine. The initial synthetic approach involved, as a key step, a Suzuki coupling between a chromene triflate and a boronate ester, followed by isoquinoline formation and reduction of the resulting isoquinoline. Subsequently, a more efficient route was developed that involved conjugate addition of an aryl Grignard reagent to a 2-nitrochromene. The title compound possessed high affinity (Ki=20-30 nM) for porcine D1-like receptors in native striatal tissue and full intrinsic activity at cloned human dopamine D1 receptors but had much lower affinity at dopamine D2-like receptors (Ki=3000 nM). The binding and functional properties of this compound illustrate again the utility of constructing dopamine D1 agonist ligands around the beta-phenyldopamine pharmacophore template.

WAY-100635 is a potent dopamine D4 receptor agonist.

RATIONALE AND OBJECTIVES: WAY-100635 is a prototypical 5-HT1A receptor antagonist and has been used widely as a pharmacological probe to investigate the distribution and function of 5-HT1A receptors. Results from our studies suggested that WAY-100635 was potently inducing effects unrelated to its 5-HT1A receptor affinity. In the present work, we evaluated the in vitro pharmacology of this compound at two D2-like receptor subtypes. METHOD: The functional properties and binding affinities of WAY-100635 were evaluated in HEK 293 cells stably expressing dopamine D2L or D4.4 receptors. RESULTS: Initial screens performed by the NIMH Psychoactive Drug Screening Program indicated that WAY-100635 displayed 940, 370, and 16 nM binding affinities at D2L, D3, and D4.2 receptors, respectively. Subsequent saturation analyses demonstrated that the Kd of [3H]WAY-100635 at D4.2 receptors was 2.4 nM, only tenfold higher than 5-HT1A. WAY-100635 and its major metabolite, WAY-100634, were potent agonists in HEK-D4.4 cells (EC50=9.7+/-2.2 and 0.65+/-0.2 nM, respectively). WAY-100635 behaved as a full agonist, and WAY-100634 was a nearly full agonist. In HEK-D2L cells, WAY-100635 weakly antagonized the effects of 300 nM quinpirole. Subsequent radioligand binding studies confirmed that WAY-100635 possesses high affinity for D4.4 receptors but binds weakly to D2L receptors (3.3+/-0.6 and 420+/-11 nM, respectively). CONCLUSIONS: This study demonstrates that WAY-100635 is not a "selective" 5-HT1A receptor antagonist, as previously reported, and conclusions drawn from studies that employed WAY-100635 as a selective 5-HT1A antagonist may need to be reevaluated.

Cell-type specific increases in female hamster nucleus accumbens spine density following female sexual experience.

Female sexual behavior is an established model of a naturally motivated behavior which is regulated by activity within the mesolimbic dopamine system. Repeated activation of the mesolimbic circuit by female sexual behavior elevates dopamine release and produces persistent postsynaptic alterations to dopamine D1 receptor signaling within the nucleus accumbens. Here we demonstrate that sexual experience in female Syrian hamsters significantly increases spine density and alters morphology selectively in D1 receptor-expressing medium spiny neurons within the nucleus accumbens core, with no corresponding change in dopamine receptor binding or protein expression. Our findings demonstrate that previous life experience with a naturally motivated behavior has the capacity to induce persistent structural alterations to the mesolimbic circuit that can increase reproductive success and are analogous to the persistent structural changes following repeated exposure to many drugs of abuse.

Analogues of doxanthrine reveal differences between the dopamine D1 receptor binding properties of chromanoisoquinolines and hexahydrobenzo[a]phenanthridines.

Efforts to develop selective agonists for dopamine D(1)-like receptors led to the discovery of dihydrexidine and doxanthrine, two bioisosteric ß-phenyldopamine-type full agonist ligands that display selectivity and potency at D(1)-like receptors. We report herein an improved methodology for the synthesis of substituted chromanoisoquinolines (doxanthrine derivatives) and the evaluation of several new compounds for their ability to bind to D(1)- and D(2)-like receptors. Identical pendant phenyl ring substitutions on the dihydrexidine and doxanthrine templates surprisingly led to different effects on D(1)-like receptor binding, suggesting important differences between the interactions of these ligands with the D(1) receptor. We propose, based on the biological results and molecular modeling studies, that slight conformational differences between the tetralin and chroman-based compounds lead to a shift in the location of the pendant ring substituents within the receptor.

Mapping the catechol binding site in dopamine D1 receptors: synthesis and evaluation of two parallel series of bicyclic dopamine analogues.

A novel class of isochroman dopamine analogues, originally reported by Abbott Laboratories, have >100-fold selectivity for D1-like over D2-like receptors. We synthesized a parallel series of chroman compounds and showed that repositioning the oxygen atom in the heterocyclic ring decreases potency and confers D2-like receptor selectivity to these compounds. In silico modeling supports the hypothesis that the altered pharmacology for the chroman series is due to potential intramolecular hydrogen bonding between the oxygen in the chroman ring and the meta-hydroxy group of the catechol moiety. This interaction realigns the catechol hydroxy groups and disrupts key interactions between these ligands and critical serine residues in TM5 of the D1-like receptors. This hypothesis was tested by the synthesis and pharmacological evaluation of a parallel series of carbocyclic compounds. Our results suggest that if the potential for intramolecular hydrogen bonding is removed, D1-like receptor potency and selectivity are restored.

Dopamine D4 receptor involvement in the discriminative stimulus effects in rats of LSD, but not the phenethylamine hallucinogen DOI.

RATIONALE: Lysergic acid diethylamide (LSD) differs from other types of hallucinogens in that it possesses direct dopaminergic effects. The exact nature of this component has not been elucidated. OBJECTIVE: The present study sought to characterize the effects of several dopamine D(4) agonists and antagonists on the discriminative stimulus effect of LSD at two pretreatment times and 2,5-dimethoxy-4-iodoamphetamine (DOI), a selective 5-HT(2A/2C) agonist. MATERIALS AND METHODS: Male Sprague-Dawley rats were trained in a two-lever, fixed ratio (FR) 50, food-reinforced task with LSD-30 (0.08 mg/kg, i.p., 30-min pretreatment time), LSD-90 (0.16 mg/kg, i.p., 90-min pretreatment time), and DOI (0.4 mg/kg, i.p., 30-min pretreatment time) as discriminative stimuli. Substitution and combination tests with the dopamine D(4) agonists, ABT-724 and WAY 100635, were performed in all groups. Combination tests were run using the dopamine D(4) antagonists A-381393 and L-745,870 and two antipsychotic drugs, clozapine and olanzapine. RESULTS: WAY 100635 produced full substitution in LSD-90 rats, partial substitution in LSD-30 rats, and saline appropriate responding in DOI-trained rats. ABT-724 partially mimicked the LSD-90 and LSD-30 cues, but produced no substitution in DOI-trained rats. In combination tests, both agonists shifted the dose-response curve of LSD leftward, most potently for the LSD-90 cue. The D(4) antagonists significantly attenuated both the LSD-90 and LSD-30 cue, but had no effect on the DOI cue. CONCLUSION: Dopamine D(4) receptor activation plays a significant modulatory role in the discriminative stimulus effects in LSD-90-trained rats, most markedly for the later temporal phase of LSD, but has no effect on the cue produced by DOI.

Comparison of the enantiomers of (+/-)-doxanthrine, a high efficacy full dopamine D(1) receptor agonist, and a reversal of enantioselectivity at D(1) versus alpha(2C) adrenergic receptors.

Parkinson's disease is a neurodegenerative condition involving the death of dopaminergic neurons in the substantia nigra. Dopamine D(1) receptor agonists are potential alternative treatments to current therapies that employ L-DOPA, a dopamine precursor. We evaluated the pharmacological profiles of the enantiomers of a novel dopamine D(1) receptor full agonist, doxanthrine (DOX) at D(1) and alpha(2C) adrenergic receptors. (+)-DOX displayed greater potency and intrinsic activity than (-)-DOX in porcine striatal tissue and in a heterologous D(1) receptor expression system. Studies in MCF7 cells, which express an endogenous human dopamine D(1)-like receptor, revealed that (-)-DOX was a weak partial agonist/antagonist that reduced the functional activity of (+)-DOX and dopamine. (-)-DOX had 10-fold greater potency than (+)-DOX at alpha(2C) adrenergic receptors, with an EC50 value of 4 nM. These findings demonstrate a reversed stereoselectivity for the enantiomers of DOX at D(1) and alpha(2C) receptors and have implications for the therapeutic utility of doxanthrine.