Structure-guided development of dual β2 adrenergic/dopamine D2 receptor agonists

Aiming to discover dual-acting β₂ adrenergic/dopamine D₂ receptor ligands, a structure-guided approach for the evolution of GPCR agonists that address multiple targets was elaborated. Starting from GPCR crystal structures, we describe the design, synthesis and biological investigation of a defined set of compounds leading to the identification of the benzoxazinone (R)-3, which shows agonist properties at the adrenergic β₂ receptor and substantial G protein-promoted activation at the D₂ receptor. This directed approach yielded molecular probes with tuned dual activity. The congener desOH-3 devoid of the benzylic hydroxyl function was shown to be a β₂ adrenergic antagonist/D₂ receptor agonist with K_i values in the low nanomolar range. The compounds may serve as a promising starting point for the investigation and treatment of neurological disorders.     

α-Synuclein and dopamine metabolism

α-Synuclein (α-Syn), a 140-amino-acid protein richly expressed in presynaptic terminals in the central nervous system, has been shown to play a central role in the pathogenesis of Parkinson’s disease. Although the normal functions of α-Syn remain elusive, accumulating evidence shows that the molecule is involved in multiple steps related to dopamine metabolism, including dopamine synthesis, storage, release, and uptake. The regulatory effect of α-Syn on dopamine metabolism is likely to tone down the amount of cytoplasmic dopamine at nerve terminals, thereby limiting its conversion to highly reactive oxidative molecules. Formation of α-Syn protofibrils triggered by factors such as gene mutations and environmental toxins can make the molecule lose its normal functions, leading to disrupted homeostasis of dopamine metabolism, increased cytoplasmic dopamine levels, and enhanced oxidative stress in dopaminergic neurons. The enhanced oxidative stress will, in turn, exacerbate the formation of α-Syn protofibrils and drive the neurons into a vicious cycle, which will finally result in the selective degeneration of the dopaminergic neurons associated with Parkinson’s disease.     

D-2 dopamine autoreceptor selective drugs: do they really exist?

The catecholamine dopamine plays an important role as a neurotransmitter or neurohormone in the brain and pituitary gland. Dopamine exerts its effects through activation of two types of receptors called D-1 and D-2. These receptors are distinguished by their different pharmacological characteristics and signal transduction mechanism(s). Release of dopamine inhibits the activity of dopaminergic neurons through activation of so-called dopamine autoreceptors which are of the D-2 type. In general, these receptors occur both in the soma-dendritic region of the dopaminergic neuron, where they are involved in the inhibition of the firing rate and on the dopaminergic terminals where they mediate the inhibition of dopamine synthesis and release. D-2 receptors occur also on the target cells of dopaminergic neurons both in the brain (postsynaptic D-2 receptors) and pituitary gland. On the basis of data gathered from in vivo (behavioral- as well as electrophysiological) studies it has been concluded that D-2 agonists are much more potent at dopamine autoreceptors as compared to postsynaptic D-2 receptors, indicating the possibility of a pharmacological distinction between these differentially located D-2 receptors. This concept led to the introduction of a whole group of drugs allegedly displaying a selective agonist profile at the dopamine autoreceptor. In contrast, biochemical (in vitro) studies with brain tissue as well as the pituitary gland, did not reveal any significant difference between the pharmacological profiles of autoreceptors and postsynaptic D-2 receptors. In the present minireview a balanced discussion is presented of these in vivo and in vitro findings and it is concluded that both autoreceptors as well as postsynaptic D-2 receptors are similar if not identical entities.     

Is dopamine important in nicotine dependence?

Nicotine, like other drugs when abused, can produce a wide array of behaviours, some of which collectively propel ‘drug-seeking behaviour’. This review focuses on three stimulus properties of nicotine and examines the role of dopamine in mediating each affect with respect to D1 and D2 receptor subtypes. Dopamine appears to be critical in mediating the reinforcing effects of nicotine, which is in line with other commonly abuse psychomotor stimulants. However, evidence derived from studies with local microinjections of nicotine suggests that the origin of nicotine action to produce its other stimulus properties may be via multiple neuroanatomical substrates. The aversive simulus effects are resistant to dopamine receptor antagonists. The discriminative stimulus effects of nicotine, despite showing some modification with dopaminergic compounds, appear not to be solely mediated via the mesolimbic dopamine system. Taken together, the neurobiology of nicotine dependence remains complex. Nonetheless, such association between stimulus properties may permit the development of more effective therapies in combating tobacco dependence.     

β-endorphin-induced increase in striatal dopamine turnover

Human β-endorphin administered intracisternally in a dose of 15 μg per rat increased striatal concentrations of the dopamine metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) as well as producing catalepsy. These effects were inhibited by naloxone. Pargyline-induced decreases in striatal DOPAC and HVA were greater in endorphin-treated than in saline-treated animals, supporting the concept that β-endorphin increases striatal dopamine turnover. β-endorphin increased the rate of decline in striatal dopamine concentration following synthesis inhibition with α-methyltyrosine, further suggesting that endorphin increases striatal dopamine turnover. β-endorphin and probenecid interacted competitively to decrease the effects of each other to increase striatal HVA. Naloxone prevented the effect of endorphin to decrease the HVA response to probenecid. Thus, probenecid cannot be used to assess the effects of endorphin on striatal dopamine turnover. If β-endorphin acts presynaptically to decrease dopamine release in striatum, the increases in striatal DOPAC and HVA probably represent a compensatory attempt to increase dopamine synthesis. Although turnover of dopamine to its metabolites is increased, dopamine release may be suppressed by β-endorphin.     

Alpha-ethyltryptamines as dual dopamine–serotonin releasers

The dopamine (DA), serotonin (5-HT), and norepinephrine (NE) transporter releasing activity and serotonin-2A (5-HT_2A) receptor agonist activity of a series of substituted tryptamines are reported. Three compounds, 7b, (+)-7d and 7f, were found to be potent dual DA/5-HT releasers and were >10-fold less potent as NE releasers. Additionally, these compounds had different activity profiles at the 5-HT_2A receptor. The unique combination of dual DA/5-HT releasing activity and 5-HT_2A receptor activity suggests that these compounds could represent a new class of neurotransmitter releasers with therapeutic potential.     

A QM protein–ligand investigation of antipsychotic drugs with the dopamine D2 Receptor (D2R)

The dopamine D2 Receptor (D2R) is a member of the G-Protein-Coupled Receptor family and plays a critical role in neurotransmission activities in the human brain. Dysfunction in dopamine receptor signaling may lead to mental health illnesses such as schizophrenia and Parkinson’s disease. D2R is the target protein of the commonly used antipsychotic drugs such as risperidone, clozapine, aripiprazole, olanzapine, ziprasidone, and quetiapine. Due to their significant side effects and non-selective profiles, the discovery of novel drugs has become a challenge for researchers working in this field. Recently, our group has focused on the interactions of these drug molecules in the active site of the D2R using different in silico approaches. We here compare the performances of different approaches in estimating the drug binding affinities using quantum chemical approaches. Conformations of drug molecules (ligands) at the binding site of the D2R taken from the preliminary docking studies and molecular dynamics simulations were used to generate protein–ligand interaction models. In a first approach, the BSSE-corrected interaction energies of the ligands with the most critical amino acid Asp114 and with the other amino acids closest to ligands in the binding cavity were calculated separately by density functional theory method in implicit water environment at the M06-2X/6-31 g(d,p) level of the theory. In a second approach, ligand binding affinities were calculated by taking into consideration not only the interaction energies but also deformation and desolvation energies of ligands with surrounding amino acid residues, in a radius of 5 Å of the protein-bound ligand. The quantum mechanically obtained results were compared with the experimentally obtained binding affinity values. We concluded that although H-bond interactions of ligands with Asp114 are the most dominant interaction in the binding site, if van der Waals and steric interactions of ligands which have cumulative effect on the ligand binding are not included in the calculations, the interaction energies are overestimated.     

Preparation of 123I-labeled 2′-iodospiperone and imaging of D2 dopamine receptors in the human brain using SPECT

[¹²³I]2′-ISP was readily prepared using a radioiodine exchange reaction with a radiochemical yield of approx. 50% after HPLC purification. The radiochemical purity of the product was more than 98% and the specific activity was 5.55–11.1 GBq/μmol. Biodistribution studies performed in mice indicated that injection of [¹²³I]2′-ISP with albumin produced a higher gastric uptake and a lower brain uptake than injection of the radioligand in a weakly acidic solution. In addition, toxicity tests performed in mice demonstrated that acute toxic effects would be very unlikely to be encountered if 2′-ISP was used for diagnostic purposes. A preliminary imaging study with [¹²³I]2′-ISP in a healthy human volunteer showed its specific uptake by the basal ganglia, a region of the brain known to have a high density of D₂ dopamine receptors.     

Nonidentity of chromogranin a and dopamine β-monooxygenase

Dopamine β-monooxygenase (EC 1.14.17.1) and chromogranin A from bovine adrenal chromaffin granules were purified by established procedures and examined for evidence of structural identity. The minimum molecular weights were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and found to be 74,000 and 35,000, respectively. Amino acid analyses of the two proteins are distinct. Dopamine β-monooxygenase does not possess a free amino terminus, whereas chromogranin A has a leucine amino terminus. Analysis in the protein sequencer showed that chromogranin A contains only a single degradable polypeptide chain. Radioactive S-carboxymethyl derivatives of the two proteins were prepared to compare the soluble peptides after thermolysin digestion. These thermolytic peptides were isolated from columns of Dowex 50-X8 resin and both the peptide and radioactive traces revealed no evidence for similarity of the two proteins, either in toto or in part. Thus, although dopamine β-monooxygenase and chromogranin A may sometimes be copurified, they are distinct entities.     

The structure of dopamine induced α-synuclein oligomers

Inclusions of aggregated α-synuclein (α-syn) in dopaminergic neurons are a characteristic histological marker of Parkinson’s disease (PD). In vitro, α-syn in the presence of dopamine (DA) at physiological pH forms SDS-resistant non-amyloidogenic oligomers. We used a combination of biophysical techniques, including sedimentation velocity analysis, small angle X-ray scattering (SAXS) and circular dichroism spectroscopy to study the characteristics of α-syn oligomers formed in the presence of DA. Our SAXS data show that the trimers formed by the action of DA on α-syn consist of overlapping worm-like monomers, with no end-to-end associations. This lack of structure contrasts with the well-established, extensive β-sheet structure of the amyloid fibril form of the protein and its pre-fibrillar oligomers. We propose on the basis of these and earlier data that oxidation of the four methionine residues at the C- and N-terminal ends of α-syn molecules prevents their end-to-end association and stabilises oligomers formed by cross linking with DA-quinone/DA-melanin, which are formed as a result of the redox process, thus inhibiting formation of the β-sheet structure found in other pre-fibrillar forms of α-syn.     

The Caenorhabditis elegans D2-like dopamine receptor DOP-2 physically interacts with GPA-14, a Gαi subunit

Dopaminergic inputs are sensed on the cell surface by the seven-transmembrane dopamine receptors that belong to a superfamily of G-protein-coupled receptors (GPCRs). Dopamine receptors are classified as D1-like or D2-like receptors based on their homology and pharmacological profiles. In addition to well established G-protein coupled mechanism of dopamine receptors in mammalian system they can also interact with other signaling pathways. In C. elegans four dopamine receptors (dop-1, dop-2, dop-3 and dop-4) have been reported and they have been implicated in a wide array of behavioral and physiological processes. We performed this study to assign the signaling pathway for DOP-2, a D2-like dopamine receptor using a split-ubiquitin based yeast two-hybrid screening of a C. elegans cDNA library with a novel dop-2 variant (DOP-2XL) as bait. Our yeast two-hybrid screening resulted in identification of gpa-14, as one of the positively interacting partners. gpa-14 is a G?? coding sequence and shows expression overlap with dop-2 in C. elegans ADE deirid neurons. In-vitro pull down assays demonstrated physical coupling between dopamine receptor DOP-2XL and GPA-14. Further, we sought to determine the DOP-2 region necessary for GPA-14 coupling. We generated truncated DOP-2XL constructs and performed pair-wise yeast two-hybrid assay with GPA-14 followed by in-vitro interaction studies and here we report that the third intracellular loop is the key domain responsible for DOP-2 and GPA-14 coupling. Our results show that the extra-long C. elegans D2-like receptor is coupled to gpa-14 that has no mammalian homolog but shows close similarity to inhibitory G-proteins. Supplementing earlier investigations, our results demonstrate the importance of an invertebrate D2-like receptor's third intracellular loop in its G-protein interaction.     

The Caenorhabditis elegans D2-like dopamine receptor DOP-2 physically interacts with GPA-14, a Gαi subunit

ABSTRACT: Dopaminergic inputs are sensed on the cell surface by the seven-transmembrane dopamine receptors that belong to a superfamily of G-protein-coupled receptors (GPCRs). Dopamine receptors are classified as D1-like or D2-like receptors based on their homology and pharmacological profiles. In addition to well established G-protein coupled mechanism of dopamine receptors in mammalian system they can also interact with other signaling pathways. In C. elegans four dopamine receptors (dop-1, dop-2, dop-3 and dop-4) have been reported and they have been implicated in a wide array of behavioral and physiological processes. We performed this study to assign the signaling pathway for DOP-2, a D2-like dopamine receptor using a split-ubiquitin based yeast two-hybrid screening of a C. elegans cDNA library with a novel dop-2 variant (DOP-2XL) as bait. Our yeast two-hybrid screening resulted in identification of gpa-14, as one of the positively interacting partners. gpa-14 is a Gα coding sequence and shows expression overlap with dop-2 in C. elegans ADE deirid neurons. In-vitro pull down assays demonstrated physical coupling between dopamine receptor DOP-2XL and GPA-14. Further, we sought to determine the DOP-2 region necessary for GPA-14 coupling. We generated truncated DOP-2XL constructs and performed pair-wise yeast two-hybrid assay with GPA-14 followed by in-vitro interaction studies and here we report that the third intracellular loop is the key domain responsible for DOP-2 and GPA-14 coupling. Our results show that the extra-long C. elegans D2-like receptor is coupled to gpa-14 that has no mammalian homolog but shows close similarity to inhibitory G-proteins. Supplementing earlier investigations, our results demonstrate the importance of an invertebrate D2-like receptor's third intracellular loop in its G-protein interaction.     

P25α / TPPP expression increases plasma membrane presentation of the dopamine transporter and enhances cellular sensitivity to dopamine toxicity

Parkinson's disease is characterized by preferential degeneration of the dopamine-producing neurons of the brain stem substantia nigra. Imbalances between mechanisms governing dopamine transport across the plasma membrane and cellular storage vesicles increase the level of toxic pro-oxidative cytosolic dopamine. The microtubule-stabilizing protein p25α accumulates in dopaminergic neurons in Parkinson's disease. We hypothesized that p25α modulates the subcellular localization of the dopamine transporter via effects on sorting vesicles, and thereby indirectly affects its cellular activity. Here we show that co-expression of the dopamine transporter with p25α in HEK-293-MSR cells increases dopamine uptake via increased plasma membrane presentation of the transporter. No direct interaction between p25α and the dopamine transporter was demonstrated, but they co-fractionated during subcellular fractionation of brain tissue from striatum, and direct binding of p25α peptides to brain vesicles was demonstrated. Truncations of the p25α peptide revealed that the requirement for stimulating dopamine uptake is located in the central core and were similar to those required for vesicle binding. Co-expression of p25α and the dopamine transporter in HEK-293-MSR cells sensitized them to the toxicity of extracellular dopamine. Neuronal expression of p25α thus holds the potential to sensitize the cells toward dopamine and toxins carried by the dopamine transporter.     

Inhibition of dopamine β-hydroxylase by bidentate chelating agents

1-2H-Phthalazine hydrazone (hydralazine; HYD), 2-1H-pyridinone hydrazone (2-hydrazinopyridine; HP), 2-quinoline-car☐ylic acid (QCA), 1-isoquinolinecar☐ylic acid (IQCA), 2,2′-bi-1H-imidazole (2,2′-biimidazole; BI), and 1H-imidazole-4-acetic acid (imidazole-4-acetic acid; IAA) directly and reversibly inhibit homogeneous soluble bovine dopamine β-hydroxylase (3,4-dihydroxyphenethylamine, ascorbate:oxygen oxidoreductase (β-hydroxylating), EC 1.14.17.1). HYD, QCA and IAA show competitive allosteric inhibition of dopamine β-hydroxylase with respect to ascorbate (K_is = 5.7(±0.9) μM, 0.14(±0.03) mM, 0.80(±0.20) mM; n_H= 1.4(±0.1), 1.8(±0.4), 2.8(±0.6), respectively). HYD and IAA show slope and intercept mixed-type allosteric inhibition of dopamine β-hydroxylase with respect to tyramine. QCA shows allosteric uncompetitive inhibition of dopamine β-hydroxylase with respect to tyramine. HP, BI and IQCA all show linear competitive inhibition (K_is = 1.9(±0.3) μM, 21(±6) μM, and 0.9(±0.3) μM, respectively) with respect to ascorbate. HP and BI show linear mixed-type while IQCA shows linear uncompetitive inhibition of dopamine β-hydroxylase with respect to tyramine. In the presence of HP, HYD or IAA intersecting double-reciprocal plots of the initial velocity as a function of tyramine concentration at differing fixed levels of ascorbate are observed. These findings are consistent with a uni-uni-ping-pong-ter-bi kinetic mechanism for dopamine β-hydroxylase that involves a ternary enzyme-ascorbate-tyramine-oxygen complex. The results for HYD, QCA and IAA are the first examples of allosteric inhibitor interactions with dopamine β-hydroxylase.     

Determination of key receptor–ligand interactions of dopaminergic arylpiperazines and the dopamine D2 receptor homology model

Interest in structure-based G-protein-coupled receptor (GPCR) ligand discovery is huge, given that almost 30 % of all approved drugs belong to this category of active compounds. The GPCR family includes the dopamine receptor subtype D2 (D2DR), but unfortunately—as is true of most GPCRs—no experimental structures are available for these receptors. In this publication, we present the molecular model of D2DR based on the previously published crystal structure of the dopamine D3 receptor (D3DR). A molecular modeling study using homology modeling and docking simulation provided a rational explanation for the behavior of the arylpiperazine ligand. The observed binding modes and receptor–ligand interactions provided us with fresh clues about how to optimize selectivity for D2DR receptors.

Repeated exposure to cocaine alters medial prefrontal cortex dopamine D₂-like receptor modulation of glutamate and dopamine neurotransmission within the mesocorticolimbic system

Repeated exposure to cocaine progressively increases drug-induced locomotor activity, which is termed behavioral sensitization. Previous studies have demonstrated that sensitization to cocaine is associated with a decrease in dopamine D? receptor function in the medial prefrontal cortex. The present report tested the hypothesis that reduced medial prefrontal cortex D? receptor function as a result of repeated cocaine exposure results in augmented excitatory transmission to the nucleus accumbens and ventral tegmental area, possibly as a partial result of enhanced inhibition of local dopamine release. Dual probe microdialysis experiments were conducted in male Sprague-Dawley rats 1, 7 or 30 days following the last of four daily injections of saline (1.0 mL/kg) or cocaine (15 mg/kg). Infusion of quinpirole (0.01, 1.0 and 100 μM), a D?-like receptor agonist, into the medial prefrontal cortex produced a dose-dependent decrease in cortical, nucleus accumbens and ventral tegmental area extracellular glutamate levels in control but not sensitized animals. Quinpirole also reduced basal dopamine levels in the medial prefrontal cortex in sensitized animals following 1 day of withdrawal from cocaine. Following 30 days of withdrawal, quinpirole also reduced dopamine levels in sensitized animals relative to saline controls, but not relative to baseline levels. These findings indicate that the expression of sensitization to cocaine is associated with altered modulation of mesocorticolimbic glutamatergic transmission at the level of the medial prefrontal cortex.     

Is clozapine selective for the dopamine D4 receptor?

Binding of ³H-spiperone and ³H-raclopride to membranes of cells stably-transfected with a human dopamine D₂ receptor clone was investigated, as was that of ³H-spiperone to those stably-transfected with a human D₄ receptor clone. ³H-spiperone and ³H-raclopride labeled the same number of sites in the D₂ receptor preparation. The inhibition of binding by clozapine, spiperone, (−) eticlopride, haloperidol and the novel substituted benzamide 1192U90 was also investigated. Clozapine and 1192U90 showed greater inhibition of ³H-raclopride binding than ³H-spiperone binding to the D₂ receptor. Comparison with inhibition of ³H-spiperone binding to the D₄ receptor revealed that clozapine and 1192U90 displayed apparent selectivity (as assessed by K_i ratios) for the D₄ receptor when compared with binding of ³H-spiperone, but not ³H-raclopride, to the D₂ receptor.     

Endogenous RGS proteins facilitate dopamine D2S receptor coupling to Gαo proteins and Ca2+ responses in CHO-K1 cells

The role of RGS proteins on dopaminergic D_2S receptor (D_2SR) signalling was investigated in Chinese hamster ovary (CHO)-K1 cells, using recombinant RGS protein- and PTX-insensitive G_αo proteins. Dopamine-mediated [³⁵S]GTPγS binding was attenuated by more than 60% in CHO-K1 D_2SR cells coexpressing a RGS protein- and PTX-insensitive G_αoGly¹⁸⁴Ser:Cys³⁵¹Ile protein versus cells coexpressing a similar amount of PTX-insensitive G_αoCys³⁵¹Ile protein. Dopamine-agonist-mediated Ca²⁺ responses were dependent on the coexpression with a G_αoCys³⁵¹Ile protein and were fully abolished upon coexpression with a G_αoGly¹⁸⁴Ser:Cys³⁵¹Ile protein. These results suggest that interactions between the G_αo protein and RGS proteins are involved in efficient D_2SR signalling.     

Contrast enhancement: a physiological effect of striatal dopamine?

Dopamine functions as an important neuromodulator in the dorsal striatum and ventral striatum/nucleus accumbens. Evidence is accumulating for the idea that striatal neurons compete with each other for control over the animals motor resources, and that dopamine plays an important modulatory role that allows a particular subset of neurons, encoding a specific behavior, to predominate in this competition. One means by which dopamine could facilitate selection among competing neurons is to enhance the contrast between stronger and weaker excitations (or to increase the signal to noise ratio among neurons, where the firing of the most excited neurons is assumed to transmit signal and the firing of the least excited to transmit noise). Here, we review the electrophysiological evidence for this hypothesis and discuss potential cellular mechanisms by which dopamine-mediated contrast enhancement could occur.This work was supported by funds provided by the State of California for medical research on alcohol and substance abuse through the University of California, San Francisco, and by NIH grant DA15676 to GOH.