Development of Novel Tavapadon Analogs as Dual-targeted Partial Agonists Based on the Dopamine D1/D5 Receptors

Rotigotine acts as a dopamine receptor agonist with high affinity for the dopamine D2, D3, D4 and D5 receptors but with a low affinity for the dopamine D1 receptor. We have investigated this further in radioligand binding and functional studies and compared the profile of rotigotine with that of other drugs used in the treatment of Parkinson's disease (PD). The binding of rotigotine to human dopamine D1, D2, D3, D4 and D5 receptors was determined in radioligand binding studies using [(3)H]rotigotine and compared with that of standard antagonist radioligands. Functional interactions of rotigotine with human dopamine receptors was also determined. [(3)H]rotigotine can be used as an agonist radioligand to label all dopamine receptor subtypes and this can be important to derive agonist affinity estimates. Rotigotine maintains this high affinity in functional studies at all dopamine receptors especially D1, D2 and D3 receptors and, to a lesser extent, D4 and D5 receptors. Rotigotine, like apomorphine but unlike ropinirole and pramipexole, was a potent agonist at all dopamine receptors. Rotigotine is a high-potency agonist at human dopamine D1, D2 and D3 receptors with a lower potency at D4 and D5 receptors. These studies differentiate rotigotine from conventional dopamine D2 agonists, used in the treatment of PD, such as ropinirole and pramipexole which lack activity at the D1 and D5 receptors, but resembles that of apomorphine which has greater efficacy in PD than other dopamine agonists but has suboptimal pharmacokinetic properties.

Dopamine signaling through D1 receptors in the prefrontal cortex (PFC) plays a critical role in the maintenance of higher cognitive functions, such as working memory. At the cellular level, these functions are predicated to involve alterations in neuronal calcium levels. The dendrites of PFC neurons express D1 receptors and N-type calcium channels, yet little information exists regarding their coupling. Here, we show that D1 receptors potently inhibit N-type channels in dendrites of rat PFC neurons. Using coimmunoprecipitation, we demonstrate the existence of a D1 receptor-N-type channel signaling complex in this region, and we provide evidence for a direct receptor-channel interaction. Finally, we demonstrate the importance of this complex to receptor-channel colocalization in heterologous systems and in PFC neurons. Our data indicate that the N-type calcium channel is an important physiological target of D1 receptors and reveal a mechanism for D1 receptor-mediated regulation of cognitive function in the PFC.

We have investigated the anatomic localization of dopamine D1 and D2 receptors in the human brain using selective high affinity ligands for both types of dopamine receptors and the technique of receptor autoradiography. Dopamine D1 receptors were labeled in postmortem human brain tissue sections using the antagonist [3H]SCH 23390. Dopamine D2 receptors were labeled in consecutive tissue sections using the agonist [3H]205-502 and the antagonist [3H]spiroperidol. D1 and D2 dopamine receptors presented a heterogeneous distribution in the human brain. The highest concentrations of both D1 and D2 receptors were found in parts of the basal ganglia, particularly the nucleus caudatus and putamen. Lower concentrations were seen in other areas for example, the lateral globus pallidus was enriched in D2 receptors and the medial globus pallidus in D1 receptors. The substantia nigra contained intermediate densities of both D1 and D2, D1 receptors being present in higher concentrations. Dopamine D1 receptors were also localized in areas outside of the basal ganglia, particularly in the neocortex, amygdala and hippocampal formation. Dopamine D2 receptors were also present in areas outside of the basal ganglia, the most significant densities being found in the hippocampal formation. We observed a marked age-dependent decline in the density of D1 receptors during the first decades of life. In contrast, D2 receptor concentrations appeared to be unaltered with age. The distribution and densities of dopamine receptors were examined in 12 cases of Parkinson's disease and compared to a control adult population. No significant differences in density and distribution were seen for either D1 nor D2 receptors.

ObjectiveDopamine D2‐like receptors – mainly dopamine D2 receptors (D2R) and dopamine D3 receptors (D3R) – are believed to be greatly involved in the pathology of Parkinson disease (PD) progression. However, these receptors have not been precisely examined in PD patients. Our aim was to quantitatively calculate the exact densities of dopamine D1 receptors (D1R), D2R, and D3R in control, Alzheimer disease (AD), and Lewy body disease (LBD) patients (including PD, Dementia with Lewy bodies, and Parkinson disease dementia); and analyze the relationship between dopamine receptors and clinical PD manifestations.MethodsWe analyzed the densities of D1R, D2R, and D3R in the striatum and substantia nigra (SN) using a novel quantitative autoradiography procedure previously developed by our group. We also examined the expression of D2R and D3R mRNA in the striatum by in situ hybridization.ResultsThe results showed that although no differences of striatal D1R were found among all groups; D2R was significantly decreased in the striatum of PD patients when compared with control and AD patients. Some clinical manifestations: age of onset, PD stage, dopamine responsiveness, and survival time after onset; showed a better correlation with striatal D1R + D3R densities combined compared to D1R or D3R alone.InterpretationThere is a possibility that we may infer the results in diagnosis, treatment, and prognosis of PD by detecting D1R + D3R as opposed to using dopamine D1 or D3 receptors alone. This is especially true for elderly patients with low D2R expression as is common in this disease.

As for all proteins, G protein-coupled receptors (GPCRs) undergo synthesis and maturation within the endoplasmic reticulum (ER). The mechanisms involved in the biogenesis and trafficking of GPCRs from the ER to the cell surface are poorly understood, but they may involve interactions with other proteins. We have now identified the ER chaperone protein calnexin as an interacting protein for both D(1) and D(2) dopamine receptors. These protein-protein interactions were confirmed using Western blot analysis and co-immunoprecipitation experiments. To determine the influence of calnexin on receptor expression, we conducted assays in HEK293T cells using a variety of calnexin-modifying conditions. Inhibition of glycosylation either through receptor mutations or treatments with glycosylation inhibitors partially blocks the interactions with calnexin with a resulting decrease in cell surface receptor expression. Confocal fluorescence microscopy reveals the accumulation of D(1)-green fluorescent protein and D(2)-yellow fluorescent protein receptors within internal stores following treatment with calnexin inhibitors. Overexpression of calnexin also results in a marked decrease in both D(1) and D(2) receptor expression. This is likely because of an increase in ER retention because confocal microscopy revealed intracellular clustering of dopamine receptors that were co-localized with an ER marker protein. Additionally, we show that calnexin interacts with the receptors via two distinct mechanisms, glycan-dependent and glycan-independent, which may underlie the multiple effects (ER retention and surface trafficking) of calnexin on receptor expression. Our data suggest that optimal receptor-calnexin interactions critically regulate D(1) and D(2) receptor trafficking and expression at the cell surface, a mechanism likely to be of importance for many GPCRs.

Evidence for antagonist activity of the dopamine D3 receptor partial agonist, BP 897, at human dopamine D3 receptor

The clinical utility of dopamine (DA) D1 receptor agonists in the treatment of Parkinson's disease (PD) is still unclear. The therapeutic use of selective DA D1 receptor agonists such as SKF-82958 (6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzaze pine hydrobromide) and A-77636 ([1R, 3S] 3-[1'-admantyl]-1-aminomethyl-3,4-dihydro-5,6-dihydroxy-1H-2-benzo pyran hydrochloride) seems limited because of their duration of action, which is too short for SKF-82958 (< 1 hr) and too long for A-77636 (> 20 hr, leading to behavioral tolerance). We therefore conducted the present acute dose-response study in four 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-exposed cynomolgus monkeys primed to exhibit levodopa-induced dyskinesias to evaluate the locomotor and dyskinetic effects on challenge with four doses (from 0.03 to 1.0 mg/kg) of A-86929 ([-]-[5aR,11bS]-4,5,5a,6,7,11b-hexahydro-2-propyl-3-thia-5-+ ++azacyclopent-1- ena[c]phenathrene-9-10-diol), a selective and full DA D1-like receptor agonist with an intermediate duration of action. Levodopa and the DA D2-like receptor agonist, LY-171555 ([4aR-trans]-4,4a,5,6,7,8,8a,9-o-dihydro-5n-propyl-2H-pyrazo lo-3-4-quinoline hydrochloride) were also used for comparison. Acute administration of A-86929 was as efficacious in alleviating MPTP-induced parkinsonism as levodopa and LY-171555, but was less likely to reproduce the levodopa-induced dyskinesias in these animals than with either LY-171555 or subsequent challenge of levodopa. Selective stimulation of the DA D1 receptor may provide better integration of neural inputs transmitted to the internal segment of the globus pallidus (referred to as the basal ganglia output) compared with levodopa and selective DA D2 receptor agonist. Potent DA D1 receptor agents with an intermediate duration of efficacy such as A-86929 (approximately 4 hr at higher doses tested) are potential therapeutic tools in PD and merit further attention.

Atypical Antipsychotics: Mechanism of Action

G protein-coupled receptors occur as dimers within arrays of oligomers. We visualized ensembles of dopamine receptor oligomers in living cells and evaluated the contributions of receptor conformation to the dynamics of oligomer association and dissociation, using a strategy of trafficking a receptor to another cellular compartment. We incorporated a nuclear localization sequence into the D1 dopamine receptor, which translocated from the cell surface to the nucleus. Receptor inverse agonists blocked this translocation, retaining the modified receptor, D1-nuclear localization signal (NLS), at the cell surface. D1 co-translocated with D1-NLS to the nucleus, indicating formation of homooligomers. (+)-Butaclamol retained both receptors at the cell surface, and removal of the drug allowed translocation of both receptors to the nucleus. Agonist-nonbinding D1(S198A/S199A)-NLS, containing two substituted serine residues in transmembrane 5 also oligomerized with D1, and both were retained on the cell surface by (+)-butaclamol. Drug removal disrupted these oligomerized receptors so that D1 remained at the cell surface while D1(S198A/S199A)-NLS trafficked to the nucleus. Thus, receptor conformational differences permitted oligomer disruption and showed that ligand-binding pocket occupancy by the inverse agonist induced a conformational change. We demonstrated robust heterooligomerization between the D2 dopamine receptor and the D1 receptor. The heterooligomers could not be disrupted by inverse agonists targeting either one of the receptor constituents. However, D2 did not heterooligomerize with the structurally modified D1(S198A/S199A), indicating an impaired interface for their interaction. Thus, we describe a novel method showing that a homogeneous receptor conformation maintains the structural integrity of oligomers, whereas conformational heterogeneity disrupts it.

The focal distribution of the dopamine (DA) D(3) receptor in brain regions implicated in emotional and cognitive functions has made this target a main focus of drug discovery efforts. This paper will review the most recent lines of research in support of the use of selective DA D(3) receptor antagonists for the pharmacotherapeutic management of drug addiction: (1) expression of the DA D(3) receptor in the rodent and human brain; (2) changes in expression of the DA D(3) receptor following exposure to drugs of abuse, and (3) efficacy of selective DA D(3) receptor antagonists in preclinical paradigms assessing the behavioral effects of drugs such as cocaine, nicotine, alcohol, methamphetamine, and heroin. This manuscript, however, will not review the effects of nonselective DA D(2)/D(3) receptor antagonists or partial D(3) receptor agonists. Growing evidence suggests that selective DA D(3) receptor antagonists do not affect the primary reinforcing effects of drugs of abuse, but rather seem to regulate the motivation to self-administer drugs under schedules of reinforcement that require an increase in work demand. In addition, selective antagonism at DA D(3) receptors appears to disrupt significantly the responsiveness to drug-associated stimuli that play a key role in reinstatement of drug-seeking behavior. These preclinical findings will be discussed in the context of translational research relevant to the design of early clinical trials and hypothesis testing in humans.

In treating Parkinson's disease with dopaminergic agonists, such as pramipexole, ropinirole, pergolide, rotigotine, apomorphine, or bromocriptine, it has been observed that a significant number of patients develop impulse-control disorders, such as compulsive shopping, pathological gambling, or hypersexuality. Because the dopamine agonists have high affinities for the dopamine D2 and D3 receptors, the drug dissociation constants of these drugs at the functional high-affinity states of these receptors, namely D2High and D3High, were compared. The data show that, compared to the other dopamine agonist drugs, pramipexole has a relatively high selectivity for the dopamine D3 receptor, as compared to D2, suggesting that the D3 receptor may be a primary target for pramipexole. There is a trend showing that the proportion of impulse-control disorders is related to the selectivity for D3 receptors over D2 receptors, with pramipexole having the highest association with, or frequency of, impulse-control disorders. While the number of studies are limited, the proportion of patients with impulse-control disorder in Parkinson patients treated with an add-on agonist were 32% for pramipexole, 25% for ropinirole, 16% for pergolide, 22% for rotigotine, 10% for apomorphine, and 6.8% for bromocriptine. Clinically, temporary replacement of pramipexole by bromocriptine may provide relief or reversal of the impulsive behavior associated with selective D3 stimulation by either pramipexole or ropinirole, while maintaining D2 stimulation needed for the anti-Parkinson action.

Datasets of antimanic potency ratings and receptor-binding affinities [inhibition constants (K(i))] at dopamine D2 and serotonin 5-HT2A brain receptors were accessed from published literature for a large series (n = 24) of typical neuroleptic drugs, many of which are now obsolete and unobtainable. There was a strong positive association between antimanic potency and affinity for D2 receptors, in support of a 'dopamine-blockade hypothesis' of antimanic drug action. Taking the series of neuroleptics as a whole, there was no association between antimanic potency and affinity for 5-HT2A receptors. Despite this, within a subsample of typical neuroleptics with low affinity for D2 receptors resembling new generation atypical antipsychotics, a positive association between antimanic potency and affinity for 5-HT2A receptors emerged. This suggests that blockade of brain 5-HT2A receptors plays at least a subsidiary role in the antimanic effects of some typical neuroleptics. Other considerations also suggest that combining drugs to achieve high affinity for and blockade of both dopamine D2 receptors and serotonin 5-HT2A receptors, possibly with additional direct or indirect stimulation of postsynaptic 5-HT1A receptors, might maximize antimanic efficacy.

SKF-83959 is not a highly-biased functionally selective D1 dopamine receptor ligand with activity at phospholipase C

The phenomenon of “biased agonism” presents an attractive avenue for drug development as it allows the separation of therapeutic effects from side effects mediated by the same target. A prototypical G protein-coupled receptor at which biased agonism has been extensively studied is the dopamine D₂ receptor, an important therapeutic target for current treatments of Parkinson’s disease and schizophrenia. There is increasing evidence that biased agonism is important for the antipsychotic efficacy of dopamine D₂ receptor partial agonists, such as aripiprazole and cariprazine. However, a clear relationship between biased agonism at the dopamine D₂ receptor and antipsychotic efficacy remains elusive, not least due to discrepancies in literature describing aripiprazole as ‘biased’ or ‘unbiased’, despite the same signalling endpoints being studied using the same cell background. To clarify such conflicts, and to aid the drug discovery efforts aimed at identifying novel dopamine D₂ receptor biased agonists, the focus of this thesis is to gain greater insight into the mechanisms that mediate biased agonism at the dopamine D₂ receptor. Through the utilization of both mutagenesis-based and structure-activity-based approaches, a secondary binding pocket was identified for being crucial in the affinity, efficacy, and bias of different ligands at the dopamine D₂ receptor. A structure-activity relationship study indicated that both efficacy and biased agonism can be finely tuned by minor structural modifications to the head group, the tail group, and the orientation and length of a spacer region of cariprazine. In particular, it was demonstrated that modifications to the tail region, and thus the interaction with a potential secondary binding site, alter the orientation of the head group within the orthosteric binding site regulating both efficacy and biased agonism. These results were corroborated with a mutagenesis study, in which mutations within a putative secondary binding site significantly impacted the affinity and efficacy of a number of dopamine D₂ receptor agonists. Finally, it was demonstrated that “kinetic context”, as determined by both ligand-binding kinetics and the kinetics intrinsic to different cellular signalling processes, can dramatically impact observations of biased agonism. Such findings illustrate, for the first time, the importance of incorporating kinetic profiling in future studies focussed on biased agonism to allow a more informed selection of preclinical candidates and thus an improved foundation for drug discovery of biased agonists.

Many effects resulting from D2 dopamine (DA) receptor stimulation are manifest only when D1 DA receptors are stimulated by endogenous DA. When D1 receptor stimulation is enhanced by administration of selective D1 receptor agonists, the functional effects of selective D2 agonists are markedly increased. These qualitative and quantitative forms of D1/D2 DA receptor synergism are abolished by chronic DA depletion when both D1 and D2 DA receptors are supersensitive. Using both electrophysiological and behavioral methods, the present study examined the effects of selective D1 and D2 receptor supersensitivity, induced by repeated administration of selective D1 or D2 receptor antagonists, on the synergistic relationships between D1 and D2 receptors. Daily administration of the selective D2 antagonist eticlopride (0.5 mg/kg, s.c.) for 3 weeks produced a selective supersensitivity of both dorsal (caudate-putamen) and ventral (nucleus accumbens) striatal neurons to the inhibitory effects of the D2 agonist quinpirole (applied by microiontophoresis). This treatment also abolished the normal ability of the D1 agonist SKF 38393 to potentiate quinpirole-induced inhibition, and relieved D2 receptors from the necessity of D1 receptor stimulation by endogenous DA (enabling), as indicated by significant electrophysiological and behavioral (stereotypy) effects of quinpirole in eticlopride-pretreated, but not saline-pretreated, rats that were also acutely depleted of DA. Daily administration of the selective D1 receptor antagonist SCH 23390 (0.5 mg/kg, s.c.) caused supersensitivity of striatal neurons to the inhibitory effects of SKF 38393 and also abolished both the ability of SKF 38393 to potentiate quinpirole-induced inhibition and the necessity of D1 receptor stimulation for such inhibition. However, both quinpirole-induced inhibition of striatal cells and stereotyped responses were also somewhat enhanced in SCH 23390-pretreated rats. When such D1-sensitized rats were acutely depleted of DA, the behavioral effects of quinpirole were intermediate between saline-pretreated rats with acute DA depletion and SCH 23390-pretreated rats without acute DA depletion. Based upon these and related results, it is argued that the enhanced effects of quinpirole in D1-sensitized rats are due to a heterologous sensitization of D2 receptors rather than to enhanced enabling resulting from supersensitive D1 receptors. It is suggested that supersensitivity of either D1 or D2 receptors can lead to an uncoupling of normal qualitative and quantitative D1/D2 synergisms and that the heterologous regulation of D2 receptor sensitivity by D1 receptors may be related to uncoupling of functional D1/D2 synergisms.