Binding of [3H]SCH23390 in Rat Brain: Regional Distribution and Effects of Assay Conditions and GTP Suggest Interactions at a D1-Like Dopamine Receptor

The compound [9-3H]SCH23390 [R-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepine-7- ol] was synthesized, and the binding of this purportedly selective antagonist of D1 3,4-dihydroxyphenylethylamine (dopamine) receptors was characterized. The regional distribution of high-affinity, specific [3H]SCH23390 binding sites in the rat brain correlated well with levels of endogenous dopamine. Receptor densities were greatest in corpus striatum, nucleus accumbens, and olfactory tubercle; intermediate levels were found in several limbic and cortical areas, whereas few sites were detectable in cerebellum, brainstem, and ol-factory bulb. Specific binding in caudate-putamen was found to be both temperature- and pH-dependent, with optima at 25-30 degrees C and pH 7.8-8.0. Scatchard or Woolf analyses of binding in caudate-putamen suggest that most of the sites are either of a single class or of classes with similar characteristics (KD = 0.7 +/- 0.1 nM; Bmax = 347 +/- 35 fmol/mg of protein). Both dopamine and cis-flupenthixol altered the slope but not the intercept of lines generated by Scatchard analysis, suggesting a competitive mode of inhibition of [3H]SCH23390 binding. Competition for binding by dopamine or the D1 agonist SKF38393 was inhibited by guanine nucleotides, whereas GTP had little effect on the competition for binding by the antagonist cis-flupenthixol. The competition for [3H]SCH23390 binding sites by dopamine was much more sensitive to GTP than was competition for [3H]spiperone binding. These data support the hypotheses that [3H]SCH23390 binds to recognition sites that differ from those previously described using other radiolabeled dopamine antagonists and that these sites have the characteristics expected of dopamine receptors.     

Dopamine induces hyperpolarization of locust salivary gland acinar cells via D(1)-like receptors

The effect of dopamine on the salivary gland acinar cells of the locust was examined using conventional intracellular recording techniques. Application of dopamine induced a reversible, dose-dependent hyperpolarization of the acinar cells, with an EC(50) of 0.1 &mgr;M dopamine. We investigated the pharmacology of the dopamine receptor mediating hyperpolarization of the acinar cells using a range of dopaminergic agonists and antagonists. The effect of dopamine could be mimicked by the selective D(1) receptor agonist SKF82958, whilst the D(2) receptor agonists PPHT-HCl and TNPA-HBr were far less potent at inducing hyperpolarization. The receptor also showed selectivity to certain synthetic D(1)-like agonists. SKF82958 was much more effective at inducing a hyperpolarization than SKF81297. The dopamine-induced hyperpolarization of locust acinar cells could be blocked using the selective D(1) receptor antagonist SCH23390 whilst the D(2) receptor antagonists sulpiride and spiperone were inactive. The rank order of potency of several dopaminergic agonists and antagonists was obtained and suggests that the dopamine receptor mediating the hyperpolarization in locust salivary gland acinar cells is similar to a mammalian D(1) receptor. Stimulation of the salivary nerve mimicked the effect of dopamine on the acinar cells, inducing a rapid reversible hyperpolarization. This neurally-evoked hyperpolarization of the locust acinar cells was suppressed using 1.0 &mgr;M SCH23390, whilst 10 &mgr;M sulpiride was inactive. This demonstrated that both exogenously applied dopamine and endogenously released dopamine are probably acting on the same receptor.     

Endogenous Dopamine Increases Extracellular Concentrations of Glutamate and GABA in Striatum of the Freely Moving Rat: Involvement of D1 and D2 Dopamine Receptors

Interactions between endogenous dopamine, glutamate, GABA, and taurine were investigated in striatum of the freely moving rat by using microdialysis. Intrastriatal infusions of the selective dopamine uptake inhibitor nomifensine (NMF) were used to increase the endogenous extracellular dopamine. NMF produced a dose-related increase in extracellular dopamine and also increased extracellular concentrations of glutamate, GABA, and taurine. Extracellular increases of dopamine were significantly correlated with extracellular increases of glutamate and GABA, but not taurine. To investigate whether the increased extracelular dopamine produced by NMF was responsible for the concomitant increase of glutamate and GABA, D1, and D2 receptor antagonists were used. Dopamine receptor antagonists D1 (SCH23390) and D2 (sulpiride) significantly attenuated the increases of glutamate and GABA produced by NMF. These data suggest that endogenous dopamine, through both D1 and D2 dopamine receptors, plays a role in releasing glutamate and GABA in striatum of the freely moving rat.     

D1 and D2 Dopamine Receptors in Caudate-Putamen of Nonhuman Primates (Macaca fascicularis)

D1 and D2 dopamine receptors were characterized in the caudate-putamen region of nonhuman primate brains (Macaca fascicularis). D1 dopamine receptors were identified with [3H]SCH 23390 and D2 receptors with [3H]-spiperone. Scatchard analysis of [3H]SCH 23390 saturation data using washed membranes revealed a single high-affinity binding site (KD, 0.352 +/- 0.027 nM) with a density (Bmax) of 35.7 +/- 2.68 pmol/g original wet tissue weight (n = 10). The affinity of [3H]spiperone for the D2 site was 0.039 +/- 0.007 nM and the density was 25.7 +/- 1.97 pmol/g original wet tissue weight (n = 10). D1 and D2 receptors in nonhuman primates may be differentiated on the basis of drug affinities and stereoselectivity. In competition experiments, RS-SKF 38393 was the most selective D1 agonist, whereas (+)-4-propyl-9-hydroxynaphthoxazine [(+)-PHNO] was the most selective D2 agonist. Apomorphine was essentially nonselective for D1 or D2 binding sites. Of the antagonists, R-SKF 83566 and SCH 23390 were the most selective for the D1 site, whereas YM-09151-2 was the most selective for the D2 site. cis-Flupentixol and (S)-butaclamol were the least selective dopamine antagonists. D1 receptors bound benzazepine antagonists (SCH 23390/SCH 23388, R-SKF 83692/RS-SKF 83692) stereoselectively whereas D2 receptors did not. Conversely D2 receptors bound (S)-sulpiride and (+)-PHNO more potently than their enantiomers whereas D1 receptors showed little stereoselectively for each of these isomeric pairs. These binding characteristics may be utilized for evaluation of individual receptor function in vivo.     

Three-dimensional Pharmacophore Hypotheses for the Locust Neuronal Octopamine Receptor (OAR3): 1. Antagonists

Three-dimensional pharmacophore hypotheses were built from a set of 17 mianserin-like antagonists against octopamine receptor class 3 (OAR3) in locust nervous tissue. Among the ten chemical-featured models generated by program Catalyst/Hypo, three hypotheses were considered to be important and predictive in evaluating OAR3 antagonists. Predictions were fairly precise for all molecules but the three outliners including eresepine, metoclopramide and yohimbine. While the ideal and null hypotheses had a cost of 66.50 and 124.97, respectively, the ten resulting hypotheses possessed costs from 78.96 to 92.04. The best hypothesis that was confirmed to have a 95% chance of true correlation yielded a low RMS of 1.05 and high regression r of 0.934. Active antagonists mapped well onto all the features of the hypothesis such as hydrophobic, aromatic ring or positive ionizable features. On the other hand, inactive compounds lack of binding affinity were shown to be poorly capable of achieving an energetically favorable conformation shared by the active molecules in order to fit the 3D chemical feature pharmacophore models. In addition, from the comparison and conformation analysis it was proposed that positive ionizable feature contained a lower weight than hydrophobic or an aromatic ring one. Further research on the comparison of models from agonists and antagonists may help elucidate the mechanisms of OAR3 and other types of octopamine receptor-ligand interactions.     

A two-step target binding and selectivity support vector machines approach for virtual screening of dopamine receptor subtype-selective ligands

Target selective drugs, such as dopamine receptor (DR) subtype selective ligands, are developed for enhanced therapeutics and reduced side effects. In silico methods have been explored for searching DR selective ligands, but encountered difficulties associated with high subtype similarity and ligand structural diversity. Machine learning methods have shown promising potential in searching target selective compounds. Their target selective capability can be further enhanced. In this work, we introduced a new two-step support vector machines target-binding and selectivity screening method for searching DR subtype-selective ligands, which was tested together with three previously-used machine learning methods for searching D1, D2, D3 and D4 selective ligands. It correctly identified 50.6%-88.0% of the 21-408 subtype selective and 71.7%-81.0% of the 39-147 multi-subtype ligands. Its subtype selective ligand identification rates are significantly better than, and its multi-subtype ligand identification rates are comparable to the best rates of the previously used methods. Our method produced low false-hit rates in screening 13.56 M PubChem, 168,016 MDDR and 657,736 ChEMBLdb compounds. Molecular features important for subtype selectivity were extracted by using the recursive feature elimination feature selection method. These features are consistent with literature-reported features. Our method showed similar performance in searching estrogen receptor subtype selective ligands. Our study demonstrated the usefulness of the two-step target binding and selectivity screening method in searching subtype selective ligands from large compound libraries.     

A novel structural framework for α(1A/D)-adrenoceptor selective antagonists identified using subtype selective pharmacophores

In this study four and five-feature pharmacophores for selective antagonists at each of the three α(1)-adrenoceptor (AR) subtypes were used to identify novel α(1)-AR subtype selective compounds in the National Cancer Institute and Tripos LeadQuest databases. 12 compounds were selected, based on diversity of structure, predicted high affinity and selectivity at the α(1D)- subtype compared to α(1A)- and α(1B)-ARs. 9 out of 12 of the tested compounds displayed affinity at the α(1A) and α(1D) -AR subtypes and 6 displayed affinity at all three α(1)-AR subtypes, no α(1B)-AR selective compounds were identified. 8 of the 9 compounds with α(1)-AR affinity were antagonists and one compound displayed partial agonist characteristics. This virtual screening has successfully identified an α(1A/D)-AR selective antagonist, with low μM affinity with a novel structural scaffold of a an isoquinoline fused three-ring system and good lead-like qualities ideal for further drug development.     

Synthesis and SAR of highly potent and selective dopamine D3-receptor antagonists: variations on the 1H-pyrimidin-2-one theme

In our efforts to further pursue one of the most selective dopamine D(3)-receptor antagonists reported to date, we now describe the synthesis and SAR of novel and highly selective dopamine D(3) antagonists based on a 1H-pyridin-2-one or on a urea scaffold. The most potent compounds exhibited K(i) values toward the D(3) receptor in the nano- to subnanomolar range and high selectivity versus the related D(2) dopamine receptor. Thus, 1H-pyridin-2-one 7b displays oral bioavailability (F=37%) as well as brain penetration (brain plasma ratio 3.7) in rat. Within the urea series, an excellent D(3) versus D(2) selectivity (>100-fold) could be achieved by removal of one NH group (compound 6), although bioavailability (rat) was suboptimal (F<10%). These data significantly enhance our understanding of the D(3) pharmacophore and are expected to lead to novel approaches for the treatment of schizophrenia.     

Synthesis and characterization of selective dopamine D2 receptor antagonists

A series of indole compounds have been prepared and evaluated for affinity at D2-like dopamine receptors using stably transfected HEK cells expressing human D2, D3, or D4 dopamine receptors. These compounds share structural elements with the classical D2-like dopamine receptor antagonists, haloperidol, N-methylspiperone, and benperidol. The compounds that share structural elements with N-methylspiperone and benperidol bind non-selectively to the D2 and D3 dopamine receptor subtypes. However, several of the compounds structurally similar to haloperidol were found to (a) bind to the human D2 receptor subtype with nanomolar affinity, (b) be 10- to 100-fold selective for the human D2 receptor compared to the human D3 receptor, and (c) bind with low affinity to the human D4 dopamine receptor subtype. Binding at sigma (sigma) receptor subtypes, sigma1 and sigma2, were also examined and it was found that the position of the methoxy group on the indole was pivotal in both (a) D2 versus D3 receptor selectivity and (b) affinity at sigma1 receptors. Adenylyl cyclase studies indicate that our indole compounds with the greatest D2 receptor selectivity are neutral antagonists at human D2 dopamine receptor subtypes. With stably transfected HEK cells expressing human D2 (hD2-HEK), these compounds (a) have no intrinsic activity and (b) attenuated quinpirole inhibition of adenylyl cyclase. The D2 receptor selective compounds that have been identified represent unique pharmacological tools that have potential for use in studies on the relative contribution of the D2 dopamine receptor subtypes in physiological and behavioral situations where D2-like dopaminergic receptor involvement is indicated.