The High Affinity Dopamine D2 Receptor Agonist MCL-536: A New Tool for Studying Dopaminergic Contribution to Neurological Disorders.

The dopamine D2 receptor exists in two different states, D2high and D2low; the former is the functional form of the D2 receptor and associates with intracellular G-proteins. The D2 agonist [3H]MCL-536 has high affinity for the D2 receptor (Kd 0.8 nM) and potently displaces the binding of (R-(-)-N-n-propylnorapomorphine (NPA; Ki 0.16 nM) and raclopride (Ki 0.9 nM) in competition binding assays. Here, we further characterize [3H]MCL-536. [3H]MCL-536 was metabolically stable, with about 75% of the compound remaining intact after 1 h incubation with human liver microsomes. Blood-brain barrier penetration in rats was good, attaining at 15 min a % injected dose per gram of wet tissue (%ID/g) of 0.28 in males versus 0.42 in females in the striatum. Specific uptake ratios ([%ID/g striatum]/[%ID/g cerebellum]) were stable in males during the first 60 min and in females up to 15-30 min. The D2-rich striatum exhibited the highest uptake and slowest washout compared to D2-poor cortex or cerebellum. In peripheral organs, uptake peaked at 15 min but declined to baseline at 60 min, indicating good clearance from the body. In vitro autoradiography on transaxial and coronal brain sections showed specific binding of [3H]MCL-536, which was abolished by preincubation with D2/D3 ligands sulpiride, NPA, and raclopride and in the presence of the stable GTP analogue guanylylimidodiphosphate. In amphetamine-sensitized animals, striatal binding was higher than in controls, indicating specificity for the D2high receptor state. [3H]MCL-536's unique properties make it a valuable tool for research on neurological disorders involving the dopaminergic system like Parkinson's disease or schizophrenia.

New Dopamine D2 Receptor Agonist, [3H]MCL-536, for Detecting Dopamine D2high Receptors in Vivo.

Increases in the D2 receptor high affinity state are associated with certain neurological disorders. We synthesized and characterized the high-affinity D2high ligand [3H]MCL-536 in competition binding against the D2/3 agonist R-(-)- N- n-propylnorapomorphine (NPA) and the D2/3 antagonist raclopride. The total binding of [3H]MCL-536 (minus that in the presence of 100 nM NPA) was measured by saturation binding in CHO cells expressing human D2long; the data yielded separable, nonsaturable nonspecific, and saturable specific components. The former represents an aporphine site common to NPA and [3H]MCL-536. The latter indicated specific binding to the total D2 receptors (both high and low-affinity states). [3H]MCL-536 had a Kd of 0.8 nM. In competition binding, NPA had a Ki of 0.16 nM, and raclopride had a Ki of 0.9 nM. Co-incubation with guanylylimidodiphosphate abolished binding to D2high. This unique profile makes radiolabeled MCL-536 a versatile tool for diagnostics and therapeutics, and may quantify D2high sites in schizophrenia and PD patients in vivo.

Structural Investigation of the Dopamine-2 Receptor Agonist Bromocriptine Binding to Dimeric D2HighR and D2LowR States.

The active (D2HighR) and inactive (D2LowR) states of dimeric dopamine D2 receptor (D2R) models were investigated to clarify the binding mechanisms of the dopamine agonist bromocriptine, using Molecular Dynamics (MD) simulation. The aim of this comprehensive study was to investigate the critical effects of bromocriptine binding on each distinct receptor conformation. The different binding modes of the bromocriptine ligand in the active and inactive states have a significant effect on the conformational changes of the receptor. Based on the MM/GBSA approach, the calculated binding enthalpies of bromocriptine demonstrated selectivity toward the D2HighR active state. There is good agreement between the calculated and experimentally measured D2HighR selectivity. In the ligand-binding site, the key amino acids identified for D2HighR were Asp114(3.32) and Glu95(2.65), and for D2LowR, it was Ser193(5.42). Moreover, analysis of replicate MD trajectories demonstrated that the bromocriptine structure was more rigid at the D2HighR state and more flexible at the D2LowR state. However, the side chains of the ligand-receptor complex of D2HighR showed larger variations relative to the corresponding regions of D2LowR. The present study is part of an ongoing research program to study D2R conformational changes during ligand activation and to evaluate the conformational state selectivity for ligand binding.

Analysis of the Glutamate Agonist LY404,039 Binding to Nonstatic Dopamine Receptor D2 Dimer Structures and Consensus Docking.

Dopamine receptor D2 (D2R) plays an important role in the human central nervous system and is a focal target of antipsychotic agents. The D2HighR and D2LowR dimeric models previously developed by our group are used to investigate the prediction of binding affinity of the LY404,039 ligand and its binding mechanism within the catalytic domain. The computational data obtained using molecular dynamics simulations fit well with the experimental results. The calculated binding affinities of LY404,039 using MM/PBSA for the D2HighR and D2LowR targets were -12.04 and -9.11 kcal/mol, respectively. The experimental results suggest that LY404,039 binds to D2HighR and D2LowR with binding affinities (Ki) of 8.2 and 1640 nM, respectively. The high binding affinity of LY404,039 in terms of binding to [3H]domperidone was inhibited by the presence of a guanine nucleotide, indicating an agonist action of the drug at D2HighR. The interaction analysis demonstrated that while Asp114 was among the most critical amino acids for D2HighR binding, residues Ser193 and Ser197 were significantly more important within the binding cavity of D2LowR. Molecular modeling analyses are extended to ensemble docking as well as structure-based pharmacophore model (E-pharmacophore) development using the bioactive conformation of LY404,039 at the binding pocket as a template and screening of small-molecule databases with derived pharmacophore models.

The signaling pathway of dopamine D2 receptor (D2R) activation using normal mode analysis (NMA) and the construction of pharmacophore models for D2R ligands.

G-protein-coupled receptors (GPCRs) are targets of more than 30% of marketed drugs. Investigation on the GPCRs may shed light on upcoming drug design studies. In the present study, we performed a combination of receptor- and ligand-based analysis targeting the dopamine D2 receptor (D2R). The signaling pathway of D2R activation and the construction of universal pharmacophore models for D2R ligands were also studied. The key amino acids, which contributed to the regular activation of the D2R, were in detail investigated by means of normal mode analysis (NMA). A derived cross-correlation matrix provided us an understanding of the degree of pair residue correlations. Although negative correlations were not observed in the case of the inactive D2R state, a high degree of correlation appeared between the residues in the active state. NMA results showed that the cytoplasmic side of the TM5 plays a significant role in promoting of residue-residue correlations in the active state of D2R. Tracing motions of the amino acids Arg219, Arg220, Val223, Asn224, Lys226, and Ser228 in the position of the TM5 are found to be critical in signal transduction. Complementing the receptor-based modeling, ligand-based modeling was also performed using known D2R ligands. The top-scored pharmacophore models were found as 5-sited (AADPR.671, AADRR.1398, AAPRR.3900, and ADHRR.2864) hypotheses from PHASE modeling from a pool consisting of more than 100 initial candidates. The constructed models using 38 D2R ligands (in the training set) were validated with 15 additional test set compounds. The resulting model correctly predicted the pIC50 values of an additional test set compounds as true unknowns.

Biological Insights of the Dopaminergic Stabilizer ACR16 at the Binding Pocket of Dopamine D2 Receptor.

The dopamine D2 receptor (D2R) plays an important part in the human central nervous system and it is considered to be a focal target of antipsychotic agents. It is structurally modeled in active and inactive states, in which homodimerization reaction of the D2R monomers is also applied. The ASP2314 (also known as ACR16) ligand, a D2R stabilizer, is used in tests to evaluate how dimerization and conformational changes may alter the ligand binding space and to provide information on alterations in inhibitory mechanisms upon activation. The administration of the D2R agonist ligand ACR16 [3H](+)-4-propyl-3,4,4a,5,6,10b-hexahydro-2H-naphtho[1,2-b][1,4]oxazin-9-ol ((+)PHNO) revealed Ki values of 32 nM for the D2highR and 52 µM for the D2lowR. The calculated binding affinities of ACR16 with post processing molecular dynamics (MD) simulations analyses using MM/PBSA for the monomeric and homodimeric forms of the D2highR were -9.46 and -8.39 kcal/mol, respectively. The data suggests that the dimerization of the D2R leads negative cooperativity for ACR16 binding. The dimerization reaction of the D2highR is energetically favorable by -22.95 kcal/mol. The dimerization reaction structurally and thermodynamically stabilizes the D2highR conformation, which may be due to the intermolecular forces formed between the TM4 of each monomer, and the result strongly demonstrates dimerization essential for activation of the D2R.

Binding Interactions of Dopamine and Apomorphine in D2High and D2Low States of Human Dopamine D2 Receptor Using Computational and Experimental Techniques.

We have recently reported G-protein coupled receptor (GPCR) model structures for the active and inactive states of the human dopamine D2 receptor (D2R) using adrenergic crystal structures as templates. Since the therapeutic concentrations of dopamine agonists that suppress the release of prolactin are the same as those that act at the high-affinity state of the D2 receptor (D2High), D2High in the anterior pituitary gland is considered to be the functional state of the receptor. In addition, the therapeutic concentrations of anti-Parkinson drugs are also related to the dissociation constants in the D2High form of the receptor. The discrimination between the high- and low-affinity (D2Low) components of the D2R is not obvious and requires advanced computer-assisted structural biology investigations. Therefore, in this work, the derived D2High and D2Low receptor models (GPCR monomer and dimer three-dimensional structures) are used as drug-binding targets to investigate binding interactions of dopamine and apomorphine. The study reveals a match between the experimental dissociation constants of dopamine and apomorphine at their high- and low-affinity sites of the D2 receptor in monomer and dimer and their calculated dissociation constants. The allosteric receptor-receptor interaction for dopamine D2R dimer is associated with the accessibility of adjacent residues of transmembrane region 4. The measured negative cooperativity between agonist ligand at dopamine D2 receptor is also correctly predicted using the D2R homodimerization model.

Parkinson's disease treatment may cause impulse-control disorder via dopamine D3 receptors.

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.

Parkinson's disease: low-dose haloperidol increases dopamine receptor sensitivity and clinical response.

Background. It is known that ultra-low doses of haloperidol can cause dopamine supersensitivity of dopamine D2 receptors and related behaviour in animals. Objective. The objective was to determine whether a daily ultra-low dose of 40 micrograms of haloperidol could enhance the clinical action of levodopa in Parkinson's disease patients. Method. While continuing their daily treatment with levodopa, 16 patients with Parkinson's disease were followed weekly for six weeks. They received an add-on daily dose of 40 micrograms of haloperidol for the first two weeks only. The SPES/SCOPA scale (short scale for assessment of motor impairments and disabilities in Parkinson's disease) was administered before treatment and weekly throughout the trial. Results. The results showed a mean decrease in SPES/SCOPA scores after one week of the add-on treatment. Conclusion. SCOPA scores decreased after the addition of low-dose haloperidol to the standard daily levodopa dose. This finding is consistent with an increase in sensitivity of dopamine D2 receptors induced by haloperidol. Such treatment for Parkinson's disease may possibly permit the levodopa dose to be reduced and, thus, delay the onset of levodopa side effects.

Depression treatment by withdrawal of short-term low-dose antipsychotic, a proof-of-concept randomized double-blind study.

BACKGROUND AND OBJECTIVE: Because increased dopamine neurotransmission occurs with most antidepressants, and because antipsychotics cause behavioural supersensitivity to dopamine, short-term low-dose antipsychotic treatment was tested on depressed patients with an expectation of clinical improvement in the supersensitive phase following drug withdrawal. METHOD: This was a randomized, double-blind, placebo-controlled study of 48 patients who met criteria for DSM-IV(®) Major Depressive Disorder, were in a Major Depressive Episode, and had a Hamilton Depression Rating Scale (HAMD) rating of ≥14. Half the participants received 0.25mg oral haloperidol each day for 7 days, after which they received placebo daily for 4 weeks. The other half received placebo throughout the trial. RESULTS: One week after stopping the medication, the HAMD ratings of the drug-treated patients fell by 9.96 points, as compared to a reduction of 8.73 points in the placebo-treated patients, when comparing visits 1 and 4. There was no such difference when comparing visits 2 and 4. The differences were not significant, but indicated a trend. One week after the medication was stopped, the Clinical Global Index fell 1.64±0.18 units for the medication-treated patients, compared to 1.12±0.26 units for the placebo group (P=0.05). The regimen was well tolerated. CONCLUSIONS: Seven days of an ultra-low dose of 0.25mg haloperidol, followed by withdrawal of haloperidol, resulted in clinical depression improvement greater than placebo and significantly decreased psychomotor retardation, consistent with haloperidol-induced behavioural supersensitivity to dopamine. LIMITATIONS: The sample was small. More patients are needed in a future study.

(18)F-MCL-524, an (18)F-Labeled Dopamine D2 and D3 Receptor Agonist Sensitive to Dopamine: A Preliminary PET Study.

UNLABELLED: PET has been used to examine changes in neurotransmitter concentrations in the living brain. Pioneering PET studies on the dopamine system have used D2 and D3 receptor (D2/D3) antagonists such as (11)C-raclopride. However, more recently developed agonist radioligands have shown enhanced sensitivity to endogenous dopamine. A limitation of available agonist radioligands is that they incorporate the short-lived radioisotope (11)C. In the current study, we developed the (18)F-labeled D2/D3 receptor agonist (R)-(-)-2-(18)F-fluoroethoxy-N-n-propylnorapomorphine ((18)F-MCL-524). METHODS: In total, 10 PET measurements were conducted on 5 cynomolgus monkeys. Initially, the binding of (18)F-MCL-524 was compared with that of (11)C-MNPA in 3 monkeys. Second, the specificity of (18)F-MCL-524 binding was examined in pretreatment studies using raclopride (1.0 mg/kg) and d-amphetamine (1.0 mg/kg). Third, a preliminary kinetic analysis was performed using the radiometabolite-corrected arterial input function of the baseline studies. Finally, 2 whole-body PET measurements were conducted to evaluate biodistribution and radiation dosimetry after intravenous injection of (18)F-MCL-524. RESULTS: (18)F-MCL-524 entered the brain and provided striatum-to-cerebellum ratios suitable for reliable quantification of receptor binding using the multilinear reference tissue model. Mean striatal nondisplaceable binding potential (BPND) values were 2.0 after injection of (18)F-MCL-524 and 1.4 after (11)C-MNPA. The ratio of the BPND values of (18)F-MCL-524 and (11)C-MNPA was 1.5 across striatal subregions. After administration of raclopride and d-amphetamine, the (18)F-MCL-524 BPND values were reduced by 89% and 56%, respectively. Preliminary kinetic analysis demonstrated that BPND values obtained with the 1-tissue- and 2-tissue-compartment models were similar to values obtained with the multilinear reference tissue model. Estimated radiation doses were highest for gallbladder (0.27 mSv/MBq), upper large intestine (0.19 mSv/MBq), and small intestine (0.17 mSv/MBq). The estimated effective dose was 0.035 mSv/MBq. CONCLUSION: The (18)F-labeled agonist (18)F-MCL-524 appears suitable for quantification of D2/D3 receptor binding in vivo, and the results encourage extension to human studies. The longer half-life of (18)F makes (18)F-MCL-524 attractive for studies on modulation of the dopamine concentration-for example, in combination with simultaneous measurement of changes in blood-oxygen-level-dependent signal using bimodal PET/functional MRI.

Discovery of why acute lymphoblastic leukaemia cells are killed by asparaginase: Adventures of a young post-doctoral student, Bertha K Madras.

A surprising finding was made by JG Kidd (1909-1991) that guinea pig serum could make tumours disappear in mice. A later finding made by JD Broome (1939-) showed that asparaginase could suppress or kill tumour cells. However, the major mystery was why were only tumour cells but not normal cells affected by the asparaginase? The biology underlying this mechanism was unravelled by a young post-doctoral student, Bertha K Madras (1942-) who hypothesized that cells with low asparagine synthetase are those that die following treatment with asparaginase. To test her theory, Madras developed an assay for asparagine synthetase. The hypothesis was supported by the results that cells with normal asparagine synthetase were protected, while cells with low levels of this enzyme were killed by asparaginase. The findings provide a clinical guide for the use of asparaginase in acute lymphoblastic leukaemia in children and adults.

Clozapine, a fast-off-D2 antipsychotic.

Ever since clozapine was first synthesized and tested, it showed the unique property of having antipsychotic action but no Parkinson-like motor side effects. The antipsychotic basis of clozapine is to transiently occupy dopamine D2 receptors in the human striatum, in contrast to haloperidol and chlorpromazine, which have a prolonged occupation of D2 receptors. The chemical structure of clozapine facilitates a relatively rapid dissociation from D2 receptors. After short-term occupation of D2 receptors, peak neural activity raises synaptic dopamine, which then displaces clozapine. While clozapine also occupies other types of receptors, they may not have a significant role in preventing parkinsonism. Clozapine's transient occupation of D2 receptors permits patients to move easily and comfortably.

Is schizophrenia a dopamine supersensitivity psychotic reaction?

Adolf Meyer (1866-1950) did not see schizophrenia as a discrete disorder with a specific etiology but, rather, as a reaction to a wide variety of biopsychosocial factors. He may have been right. Today, we have evidence that gene mutations, brain injury, drug use (cocaine, amphetamine, marijuana, phencyclidine, and steroids), prenatal infection and malnutrition, social isolation and marginalization, can all result in the signs and symptoms of schizophrenia. This clinical picture is generally associated with supersensitivity to dopamine, and activates dopamine neurotransmission that is usually alleviated or blocked by drugs that block dopamine D2 receptors. While the dopamine neural pathway may be a final common route to many of the clinical symptoms, the components of this pathway, such as dopamine release and number of D2 receptors, are approximately normal in schizophrenia patients who are in remission. Postmortem findings, however, reveal more dimers of D1D2 and D2D2 receptors in both human schizophrenia brains and in animal models of schizophrenia. Another finding in animal models is an elevation of high-affinity state D2High receptors, but no radioactive ligand is yet available to selectively label D2High receptors in humans. It is suggested that synaptic dopamine supersensitivity in schizophrenia is an attempt at compensation for the original damage by heightening dopamine neurotransmission pathways (preparing the organism for fight or flight). The dopamine overactivity is experienced subjectively as overstimulation, which accounts for some of the clinical symptoms, with attempts at dampening down the stimulation leading to still other symptoms. Reaction and counter-reaction may explain the symptoms of schizophrenia.

Schizophrenia thalamus imaging: low benzamide binding to dopamine D2 receptors suggests fewer D2Short receptors and fewer presynaptic terminals.

The dopamine D2 receptor continues to be the major target for the treatment of schizophrenia and is one of many genes genetically associated with this disease. Recent data show that fewer short forms of the D2 receptor protein are synthesized if there is a genetic variant in the D2 receptor (with a T in rs 1076560 in intron 6). At the same time, at least six publications report that the binding of radioactive benzamides is reduced in the schizophrenia thalamus. A review of the benzamide pharmacology of the short and long forms of the D2 receptor shows that benzamides have a 2.4-fold higher affinity for the D2Short receptor relative to the D2Long form. Hence, the reduced amount of benzamide binding to the D2 receptors in the schizophrenia thalamus suggests that there is a reduced amount of D2Short receptors in this diseased region, and may possibly also mean fewer presynaptic terminals because that is where D2Short receptors mostly reside. If so, fewer presynaptic dopamine terminals in various brain regions may be the basis of the known behavioural dopamine supersensitivity in schizophrenia.

Schizophrenia and dopamine receptors.

Schizophrenia patients are behaviorally supersensitive to dopamine-like drugs such as amphetamine or methylphenidate, meaning that patients respond to such drugs with increased psychotic symptoms, as compared to control subjects. A basis of such supersensitivity may be an increased pre-synaptic release of dopamine or a post-synaptic elevation of D2 receptors or of D2High receptors in active stages of schizophrenia. While the pre-synaptic release of dopamine is normal in stable patients with schizophrenia, brain imaging studies find that D2 receptors are increased by an average of 5.8% in antipsychotic-free schizophrenia patients. It is possible that the behavioral supersensitivity may stem from more D2High receptors in schizophrenia. Although the antipsychotic/dopamine D2 receptor can exist in vitro in a state of high affinity for dopamine (as D2High), or in a state of low affinity for dopamine (as D2Low), there is no clear evidence that D2High states can be selectively labeled or stably exist in vivo. Nevertheless, two studies revealed an 80% increase in apparent D2High receptors in schizophrenia patients after reducing endogenous dopamine. The elevation in apparent D2High receptors in vivo in schizophrenia matches the elevation in D2High receptors in vitro in animal models of psychosis, including dopamine-supersensitive animals pretreated with amphetamine, marijuana, or phencyclidine, or animals with gene knockouts in various neurotransmitter pathways, including those for glutamate receptors. The elevation of D2High receptors in vitro and the increased apparent D2High receptors in vivo is consistent with behavioral dopamine supersensitivity in schizophrenia patients.

Are dopamine D2 receptors out of control in psychosis?

It is known that schizophrenia patients are behaviorally supersensitive to dopamine-like drugs (amphetamine, methylphenidate). There is evidence for an increased release of dopamine, a slight increase of dopamine D2 receptors and an increase of dopamine D2High receptors in schizophrenia, all possibly explaining the clinical supersensitivity to dopamine. The elevation in apparent D2High receptors in vivo in schizophrenia matches the elevation in D2High receptors in many animal models of psychosis. The increased amounts of D2High receptors in psychotic-like behavior in animals may result from a loss of control of D2 by various factors. These factors include the rate of phosphorylation and desensitization of D2 receptors by kinases, the attachment of arrestin to D2 receptors, internalization of D2 receptors, the rate of receptor de-phosphorylation, formation of D2 receptor dimers, and GTP regulation by various GTPases. While at present there are no statistically significant associations of any of these controlling factors and their genes with schizophrenia, investigation of D2High receptors in schizophrenia will require a new radioligand in order to selectively label D2High receptors in vivo in patients. Finally, haloperidol reduces the number of D2High receptors that are elevated by amphetamine, indicating that this therapeutic effect may occur clinically.

Loss of dopamine neuron terminals in antipsychotic-treated schizophrenia; relation to tardive dyskinesia.

The in vivo labeling and brain imaging of dopamine transporters measure the density of dopamine neuron terminals in the human caudate/putamen. A review of such studies shows that the long-term use of antipsychotics had no major effect on the density of the dopamine terminals in individuals who had no tardive dyskinesia, but had reduced the density in those patients with tardive dyskinesia. In addition, the normal loss of dopamine terminals in healthy individuals was approximately 5% per decade. However, this rate of cell loss was apparently increased by approximately three-fold, to about 15% per decade, in schizophrenia patients using antipsychotics on a long-term basis, as measured by the in vivo imaging of the dopamine transporters in the dopamine neuron terminals. While an apparent reduction in dopamine transporters may result from reduced expression of the transporters secondary to antipsychotic treatment, the seemingly increased loss rate is consistent with the accumulation of antipsychotics in the neuromelanin of the substantia nigra, subsequent injury to the dopamine-containing neurons, and the development of extrapyramidal motor disturbances such as tardive dyskinesia or Parkinson's disease.