Chapter Seven - Beyond Small-Molecule SAR: Using the Dopamine D3 Receptor Crystal Structure to Guide Drug Design

A large number of ligand binding studies have shown that clozapine has a number of receptor affinities, including those of the dopamine (DA) D1 and D2 receptor families. The study of intrinsic efficacy at these receptors is less straight-forward. In the experiments summarised here, evidence is presented that clozapine behaves as an agonist at DA D1 receptors. Thus, the hypothermia produced by clozapine (2.5 mg kg(-1)) in the rat is fully antagonised by either of the selective DA D1 receptor antagonists SCH-23390 (0.1 mg kg(-1)) or NNC-687 (4 mg kg(-1)). These results provide an intriguing explanation for the clinical profile of clozapine as an atypical antipsychotic drug. Thus, there are supporting clinical and laboratory observations implicating DA D1 receptors in the prefrontal cortex in cognitive functions. Finally, clozapine displays features with regard to extrapyramidal motor mechanisms, and seizure thresholds, that could be explained by its properties as a DA D1 receptor agonist.

In addition to its role as a neurotransmitter, dopamine can stimulate neurite outgrowth and morphological effects upon primary neurons. To investigate the signal transduction mechanisms used by dopamine in developing striatal neurons, we focused upon the effects of activating the dopamine D1 receptor. Using the D1 receptor agonist SKF38393, we found that Trk neurotrophin receptors were activated in embryonic day 18 striatal neurons. K-252a, a Trk tyrosine kinase inhibitor, and a dopamine D1 receptor antagonist could block the effects of SKF38393. The increase in TrkB phosphorylation was not the result of increased neurotrophin production. Induction of TrkB activity by SKF38393 was accompanied by the phosphorylation of several Trk signaling proteins, including phospholipase Cgamma, Akt, and MAPK. Biotinylation experiments followed by immunostaining by phospho-TrkB-specific antibodies indicated that the mechanism involved increased TrkB surface expression by dopamine D1 receptor activation. This increase in cell surface TrkB expression was dependent upon an increase in intracellular Ca(2+). These results indicate that stimulation of dopamine D1 receptors can be coupled to the neurotrophin receptor signaling to mediate the effects of dopamine upon striatal neurons.

Decoding Dopamine Signaling

We investigated the role of G protein-coupled receptor kinase (GRK)-mediated phosphorylation in agonist-induced desensitization, arrestin association, endocytosis, and intracellular trafficking of the D(2) dopamine receptor (DAR). Agonist activation of D(2) DARs results in rapid and sustained receptor phosphorylation that is solely mediated by GRKs. A survey of GRKs revealed that only GRK2 or GRK3 promotes D(2) DAR phosphorylation. Mutational analyses resulted in the identification of eight serine/threonine residues within the third cytoplasmic loop of the receptor that are phosphorylated by GRK2/3. Simultaneous mutation of these eight residues results in a receptor construct, GRK(-), that is completely devoid of agonist-promoted GRK-mediated receptor phosphorylation. We found that both wild-type (WT) and GRK(-) receptors underwent a similar degree of agonist-induced desensitization as assessed using [(35)S]GTPgammaS binding assays. Similarly, both receptor constructs internalized to the same extent in response to agonist treatment. Furthermore, using bioluminescence resonance energy transfer assays to directly assess receptor association with arrestin3, we found no differences between the WT and GRK(-) receptors. Thus, phosphorylation is not required for arrestin-receptor association or agonist-induced desensitization or internalization. In contrast, when we examined recycling of the D(2) DARs to the cell surface, subsequent to agonist-induced endocytosis, the GRK(-) construct exhibited less recycling in comparison with the WT receptor. This impairment appears to be due to a greater propensity of the GRK(-) receptors to down-regulate once internalized. In contrast, if the receptor is highly phosphorylated, then receptor recycling is promoted. These results reveal a novel role for GRK-mediated phosphorylation in regulating the post-endocytic trafficking of a G protein-coupled receptor.

Eight Cebus apella monkeys previously treated with dopamine D1 and D2 receptor antagonists were used to elucidate the pathophysiology of acute oral dyskinesia. Five monkeys had mild oral tardive dyskinesia because of previous antagonist treatment. Interactions between the dopamine (DA) D1 receptor agonist SKF 81297 and the DA D2/D3 receptor agonist quinpirole, DA D1 and D2 receptor antagonists, and the anticholinergic biperiden were investigated. SKF 81297, 0.3 mg/kg, induced acute oral dyskinesia and grooming, which were each inhibited by the DA D1 receptor antagonist NNC 756 in a dose-dependent manner. The DA D2 receptor antagonist raclopride enhanced SKF 81297-induced acute oral dyskinesia and suppressed SKF 81297-induced grooming, each with bell-shaped dose-effect curves. Quinpirole, 0.1 mg/kg, induced a hyperarousal syndrome (i.e., increased arousal, stereotypy, and locomotion). Concomitant treatment with SKF 81297 and quinpirole caused an extreme hyperarousal syndrome but antagonized acute oral dyskinesia and grooming, suggesting a synergistic effect of high-efficacy DA D1 and D2/D3 receptor agonists regarding the induction of the hyperarousal syndrome and the antagonism of acute oral dyskinesia and grooming. Biperiden, 0.25 mg/kg, antagonized both the SKF 81297-induced and raclopride-induced acute oral dyskinesia. The results suggest that oral dyskinesia and grooming are independent but most often simultaneously occurring behaviors. Grooming is induced by DA D1 receptor agonists and antagonized by D1 and D2 antagonists and D2/D3 agonists. Acute oral dyskinesia is induced by D1 agonists and lower doses of D2 antagonists, but antagonized by D1 antagonists, D2/D3 agonists, and anticholinergics. These results suggest varying interactions between dopaminergic receptor subtypes in different types of dopaminergic behaviors.

The affinities of D3 dopamine receptors for antagonists are similar to those of D2 receptors. D3 receptors have been reported, however, to have affinities nearly 100-fold higher than those of D2 receptors for some agonists, including (+/-)-7-hydroxy-n,n-dipropyl-aminotetralin (7-OH-DPAT) and quinpirole. This has led to the use of these agonists to try to identify functional responses mediated by D3 receptors in vivo. However, D2 receptors exist in multiple states having high and low affinities for agonists. The G protein-coupled state of D2 receptors is believed to be the functional state of these receptors. When receptors were labeled with the D2 receptor antagonist [125I]-(S)-3-iodo-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5,6- dimethoxysalicylamide ([125I]-NCQ-298) under conditions that promote uncoupling of receptors from G proteins, the affinities of D3 receptors were approximately 130-fold higher than those of D2 receptors for 7-OH-DPAT and quinpirole. When receptors were labeled with the D2 receptor agonist [125I]-(R)trans-7-hydroxy-2-[N-propyl-N-(3'-iodo-2'- propenyl)-amino]tetralin ([125I]-7-OH-PIPAT) under conditions that favor interactions of receptors with G proteins, the affinities of D3 receptors were less than sevenfold higher than the affinities of D2 receptors for the same drugs. Similarly, small differences in the affinities of D2 and D3 receptors for other agonists were seen when receptors were labeled with [125I]-7-OH-PIPAT. These data demonstrate that putative D3 receptor-selective agonists also interact with a high-affinity, G protein-coupled state of D2 receptors. The similarities in affinities of the agonist-preferring state of D2 and D3 receptors means that currently available agonists cannot be used to discriminate between behavioral effects mediated by D2 and D3 receptors.

The brain reward circuitry located in the mesolimbic system of the brain regulates our emotional behaviors. It is well know that certain neurotransmitter pathways are involved in maintain homeostatic conditions which dictate normal physiology of reward and associated behaviors (motivation for food, drink and sex). Our laboratory and others have provided? preclinical and clinical evidence that a number of genes? associate with impulsive, compulsive and addictive behaviors.? We have defined these behaviors as having common genetic antecedents? and coined the? term Reward Deficiency Syndrome (RDS). Numerous peer-reviewed studies have now provided evidence that impairment of dopaminergic among other neurotransmitter pathways induce RDFS behaviors. The treatment, however, has been confounded by certain myths and has resulted in failure. We are suggesting a newer and more appropriate approach? as discussed herein. Based on neurochemical and genetic evidence, we suggest that both prevention and treatment of multiple addictions, such as dependence to alcohol, nicotine and glucose, should involve a biphasic approach. Thus, acute treatment should consist of preferential blocking of postsynaptic Nucleus Accumbens (NAc) dopamine receptors (D1-D5), whereas long term activation of the mesolimbic dopaminergic system should involve activation and/or release of Dopamine (DA) at the NAc site.? Failure to do so will result in abnormal mood, behavior and potential suicide ideation. Individuals possessing a paucity of serotonergic and/or dopaminergic receptors, and an increased rate of synaptic DA catabolism due to high catabolic genotype of the COMT gene, are predisposed to self-medicating any substance or behavior that will activate DA release, including alcohol, opiates, psychostimulants, nicotine, gambling, sex, and even excessive internet gaming. Acute utilization of these substances and/or stimulatory behaviors induces a feeling of well being. Unfortunately, sustained and prolonged abuse leads to a toxic pseudo feeling of well being resulting in tolerance and disease or discomfort. Thus, a reduced number of DA receptors, due to carrying the DRD2 A1 allelic genotype, results in excessive craving behavior; whereas a normal or abundant amount of DA receptors results in low craving behavior. In terms of preventing substance abuse, one goal would be to induce a proliferation of DA D2 receptors in genetically prone individuals.? Experiments in vitro have shown that constant stimulation of the DA receptor system via a known D2 agonist results in significant proliferation of D2 receptors in spite of genetic antecedents. In essence, D2 receptor stimulation signals negative feedback mechanisms in the mesolimbic system to induce mRNA expression causing proliferation of D2 receptors. The authors propose that D2 receptor stimulation can be accomplished via Haveos augmentation, a natural but therapeutic nutraceutical formulation that induces DA release, causing the same induction of D2-directed mRNA and thus proliferation of D2 receptors in the human. This proliferation of D2 receptors in turn will induce the attenuation of craving behavior.? In fact as mentioned earlier, this model has been proven in research showing DNA-directed compensatory overexpression (a form of gene therapy) of the DRD2 receptors, resulting in a significant reduction in alcohol craving behavior in alcohol preferring rodents. Utilizing natural dopaminergic repletion therapy to promote long term dopaminergic activation will ultimately lead to a common, safe and effective modality to treat RDS behaviors including Substance Use Disorders (SUD), Attention Deficit Hyperactivity Disorder (ADHD), Obesity and other reward deficient aberrant behaviors. While we are cognizant that there are potentially over 395 genes that associate with addictive behavior, we are confident that activation of dopaminergic? pathways provide the best approach as we know it to date.

Although amphetamine induces hyperactivity by releasing dopamine (DA), mice that lack alpha1b-adrenoceptors do not release DA in response to amphetamine and do not, therefore, exhibit locomotor supersensitivity to amphetamine. However, such mice reveal hyperlocomotion to p-chloroamphetamine (PCA). Because these alpha1b-adrenoceptor knockout mice have no alterations in the striatal densities of DA D1 or D2 receptors, the basis for any possible dopaminergic contribution to the PCA-induced hyperlocomotion to PCA is unclear. Therefore, because supersensitive animals are generally known to have a higher proportion of DA D2 receptors in the high-affinity state for DA D2(High), we investigated whether there was any change in the alpha1b-adrenoceptor knockout striata in the proportion of DA D2(High) receptors to determine whether there could be a DA-based contribution to the PCA-induced hyperlocomotion. We found that the proportion of D2(High) in the wild type striata was 23 +/- 3.3%, whereas that in the alpha1b-adrenoceptor knockout striata was 52 +/- 2.9%, an increase of 2.3-fold. This elevation agrees with other types of DA-supersensitive animal striata and could assist in eliciting a supersensitive response in these alpha1b-adrenoceptor knockout mice.

Autoradiographic and pharmacological studies on the role of dopamine D3 receptors in genetically dystonic ( dtsz) hamsters

Neonatal, bilateral lesion of the ventral hippocampus (VH) in rats recently has been proposed as a model of schizophrenia because these animals show postpubertal hypersensitivity to stress and to dopamine (DA) agonists that can be reversed by neuroleptic treatment. In search of the mechanisms of postpubertal emergence of hyperdopaminergic behavior in this model, we investigated developmental expressions of DA D1, D2, and D3 receptors in various striatal and limbic subregions of rats that had received bilateral ibotenic acid lesion of the VH at postnatal day 7 (PD7). D-Amphetamine-, apomorphine-, and stress-induced changes in locomotor activity were measured and, in accordance with previous reports, we observed an increased locomotor activity at PD56 in the hippocampal-lesioned group. The expression of DA D1, D2, and D3 receptors was then estimated in these rats by ligand autoradiography at PD41 and PD62. We observed that the levels of DA D3 receptors, as measured by tritiated 7-hydroxy-N,N-di-n-propyl-2-amino-tetralin ([3H]7-OH-DPAT) binding, are markedly reduced at PD62 in the limbic areas of lesioned rats compared with sham controls particularly in the nucleus accumbens, olfactory tubercles, and islands of Calleja. A small but significant increase in D1 receptors was also seen in the caudate-putamen of the lesioned animals at PD62, whereas no significant change in the overall expression of D2 receptors ([3H]spiperone binding) was noted. In view of the inhibitory role of D3 receptors on locomotion and, presumably, other DA-mediated behaviors, it is suggested that behavioral changes in the neonatally hippocampal-lesioned rats may be mediated by altered D3 receptor levels.

SUMMARY Overeating in obese individuals shares similarities with the loss of control and compulsive drug taking behavior observed in drug-addicted subjects. The mechanism of these behaviors is not well understood. Our prior studies with positron emission tomography (PET) in drug-addicted subjects documented reductions in striatal dopamine (DA) D2 receptors. In pathologically obese subjects, we found reductions in striatal DA D2 receptors similar to that in drug-addicted subjects. Moreover, DA D2 receptor levels were found to have an inverse relationship to the body mass index of the obese subjects. We postulated that decreased levels of DA D2 receptors predisposed subjects to search for reinforcers; in the case of drug-addicted subjects for the drug and in the case of the obese subjects for food as a means to temporarily compensate for a decreased sensitivity of DA D2 regulated reward circuits. Understanding the mechanism in food intake will help to suggest strategies for the treatment of obesity.

To explore the possible differential trafficking properties of the dopamine D1-like receptor subtypes, D1 receptor and D5 receptor. To visualize distributions of dopamine D1-like receptor subtypes at subcellular level, the yellow and cyan variants of green fluorescent protein (GFP) were used to tag D1 and D5 receptors. After transfection with the tagged dopamine receptors, the neuroblastoma cells NG108-15 were treated with D1 agonist SKF38393 or acetylcholine (ACh). Then we observed the subcellular distributions of the tagged receptors under the confocal microscopy and tried to determine trafficking properties by comparing their distribution patterns before and after the drug treatment. In resting conditions, D1 receptors located in the plasma membrane of NG108-15 cells, while D5 receptors located in both plasma membrane and cytosol. With the pre-treatment of SKF38393, the subcellular distribution of D1 receptors was changed. The yellow particle-like fluorescence of tagged D1 receptors appeared in the cytosol, indicating that D1 receptors were internalized into cytosol from the cell surface. Same situation also occurred in ACh pre-treatment. In contrast, the subcellular distribution of D5 receptors was not changed after SKF38393 or ACh treatment, indicating that D5R was not translocated to cell surface. Interestingly, when D1 and D5 receptors were co-expressed in the same cell, both kept their distinct subcellular distribution patterns and the trafficking properties. Our present study reveals that in NG108-15 nerve cells, dopamine D1 and D5 receptors exhibit differential subcellular distribution patterns, and only D1 receptor has a marked trafficking response to the drug stimulation. We further discuss the potential role of the differential trafficking properties of D1-like receptors in complex modulation of DA signaling.

The discovery of PD 89211 and related compounds: Selective dopamine D4 receptor antagonists

Changes in striatal dopamine D2 receptor mRNA levels provoked by unilateral 6-hydroxydopamine-induced lesion of the nigrostriatal dopamine pathway were studied by in situ hybridization. The influence of embryonic dopaminergic neurons implanted into the dopamine-depleted striatum on the lesion-induced changes was also examined. Changes in D2 mRNA levels were compared with changes in D2 receptor densities measured in the same animals by receptor autoradiography using [3H]spiperone or [3H]SDZ 205-501 as ligands. The distribution of D2 mRNA in the striatum of control animals closely paralleled that of the D2 receptor itself, as assessed by autoradiography, and the highest density of D2 mRNA occurred in the lateral part of the striatum. One month after lesion, levels of D2 mRNA were 34% higher in the dorsolateral part of the dopamine-depleted striatum than in the corresponding region of the contralateral control striatum. D2 receptor density in this region was increased by 40% relative to the control level. No significant increases could be measured in the medial part of the striatum. The increases in the lateral part were similar at 7 months post-lesion; however, at this time the increase in both D2 mRNA and receptor levels had spread to the medial part of the striatum as well. In the graft-bearing striatum levels of both D2 mRNA and D2 receptors reverted to control levels. This study shows that the post-lesion increase in striatal dopamine receptor and mRNA level is a biphasic phenomenon with a late-occurring component in the medial striatum. It also shows that once the increase in striatal D2 receptor gene expression is accomplished, it is maintained unchanged for long periods, similar to that of D2 receptor levels themselves. Moreover, grafts of embryonic dopaminergic neurons are able to modulate the expression of the dopamine D2 receptor gene.

Activation of dopamine D2 receptors in the shell of nucleus accumbens triggers conditioned avoidance responses in rats