Development of an aequorin luminescence calcium assay for high-throughput screening using a plate reader, the LumiLux.

A luminescence assay using a new plate reader, the LumiLux (PerkinElmer, Waltham, MA), has been validated for high-throughput screening (HTS). In this study, we compared the aequorin luminescence-based calcium mobilization assay to the fluorescence-based calcium assay. A cell line stably co-expressing apo-aequorin, a chimeric G-protein, and a G-protein-coupled dopamine receptor was used to screen a collection of 8,106 compounds using the Hamamatsu Photonics (Bridgewater, NJ) FDSS6000 and LumiLux as the plate readers. The assay parameters evaluated included hit rate correlation, signal-to-noise ratio, and overall assay performance calculated by Z and standard deviation. The average Z values and hit rates were comparable between assay platforms;however, the standard deviation for the agonist aequorin assay was significantly smaller. There was also a significant decrease in the number of false-positives with the aequorin assay. These results suggest that the aequorin assay in combination with the new plate reader, LumiLux, provides a simple, cost-effective, robust, and sensitive assay for HTS

Hydrazides of clozapine: a new class of D1 dopamine receptor subtype selective antagonists.

Acylated and aroylated hydrazinoclozapines are highly potent dopamine D(1) antagonists that show remarkable selectivity over other dopamine receptors. The most potent compound in this series is the 2,6-dimethoxybenzhydrazide 33 with a D(1)K(i) of 1.6 nM and 212-fold selectivity over D(2) receptor.

Assessing the scaffold diversity of screening libraries.

Medicinal chemists have traditionally realized assessments of chemical diversity and subsequent compound acquisition, although a recent study suggests that experts are usually inconsistent in reviewing large data sets. To analyze the scaffold diversity of commercially available screening collections, we have developed a general workflow aimed at (1) identifying druglike compounds, (2) clustering them by maximum common substructures (scaffolds), (3) measuring the scaffold diversity encoded by each screening collection independently of its size, and finally (4) merging all common substructures in a nonredundant scaffold library that can easily be browsed by structural and topological queries. Starting from 2.4 million compounds out of 12 commercial sources, four categories of libraries could be identified: large- and medium-sized combinatorial libraries (low scaffold diversity), diverse libraries (medium diversity, medium size), and highly diverse libraries (high diversity, low size). The chemical space covered by the scaffold library can be searched to prioritize scaffold-focused libraries.

Classification of dopamine, serotonin, and dual antagonists by decision trees.

Dopamine antagonists (DA), serotonin antagonists (SA), and serotonin-dopamine dual antagonists (Dual) are being used as antipsychotics. A lot of dopamine and serotonin antagonists reveal non-selective binding affinity against these two receptors because the antagonists share structurally common features originated from conserved residues of binding site of the aminergic receptor family. Therefore, classification of dopamine and serotonin antagonists into their own receptors can be useful in the designing of selective antagonist for individual therapy of antipsychotic disorders. Data set containing 1135 dopamine antagonists (D2, D3, and D4), 1251 serotonin antagonists (5-HT1A, 5-HT2A, and 5-HT2C), and 386 serotonin-dopamine dual antagonists was collected from the MDDR database. Cerius2 descriptors were employed to develop a classification model for the 2772 compounds with antipsychotic activity. LDA (linear discriminant analysis), SIMCA (soft independent modeling of class analogy), RP (recursive partitioning), and ANN (artificial neural network) algorithms successfully classified the active class of each compound at the average 73.6% and predicted at the average 69.8%. The decision trees from RP, the best model, were generated to identify and interpret those descriptors that discriminate the active classes more easily. These classification models could be used as a virtual screening tool to predict the active class of new candidates.

Classification of dopamine antagonists using functional feature hypothesis and topological descriptors.

The designing of selective dopamine antagonists for their own subreceptors can be useful in individual therapy of various neuropsychiatric disorders. Three-dimensional pharmacophore hypothesis and two-dimensional topological descriptors were used to investigate and compare different classes of dopamine antagonists. The structurally diverse D(3) and D(4) antagonists above preclinical trials were selected to map common structural features of highly selective and efficacious antagonists. The generated pharmacophore hypotheses were successfully employed as discriminative probe for database screening. To filter out the false positive from screening hits, the classification models by two-dimensional topological descriptors were built. Molconn-Z and BCUT topological descriptors were employed to develop a classification model for 1328 dopamine antagonists from MDDR database. The soft independent modeling of class analogy and artificial neural network, two supervised classification techniques, successfully classified D(1), D(3), and D(4) antagonists at the average of 80% rates into their own active classes. The mean classification rates for D(2) antagonists were obtained to 60% due to insufficient selective D(2) antagonists. In this paper, we report the validity of our models generated using functional feature hypotheses and topological descriptors. The combining both of classification using functional feature hypotheses and topological descriptors would be a useful tool to predict selective antagonists.

Selective dopamine D3 receptor antagonists: a review 2001-2005.

A growing body of evidence indicates that dopamine (DA) D(3) receptors are significantly involved in the control of drug-seeking behavior, and may play an important role in the pathophysiology of impulse control disorders and schizophrenia. This hypothesis has been difficult to test due to the lack of compounds with high selectivity for central DA D(3) receptors. Recently, however, the synthesis and characterization of new highly potent and selective DA D(3) receptor antagonists has permitted to characterize the role of the DA D(3) receptor in a wide range of preclinical animal models. Although the proof of efficacy of pharmacotherapeutic agents is to be derived ultimately from clinical trials, the preclinical findings that selective antagonism at DA D(3) receptors reduces the reinforcing efficacy of drugs of abuse, reverses cognitive deficits, and shows efficacy in animal models of schizophrenia add to an accumulating body of evidence that selective DA D(3) receptor antagonists may hold highest promise in the treatment of several neuropsychiatric diseases. The present review is aiming at describing current areas of interest and the possible future development of selective DA D(3) receptor antagonists by outlining about 40 patents and 100 publications in this research field between 2001 and 2005.

G-protein coupled receptors: SAR analyses of neurotransmitters and antagonists.

BACKGROUND: From the deductive point of view, neurotransmitter receptors can be divided into categories such as cholinergic (muscarinic, nicotinic), adrenergic (alpha- and beta-), dopaminergic, serotoninergic (5-HT1 approximately 5-HT5), and histaminergic (H1 and H2). Selective agonists and antagonists of each receptor subtype can have specific useful therapeutic applications. For understanding the molecular mechanisms of action, an inductive method of analysis is useful. OBJECTIVE: The aim of the present study is to examine the structure-activity relationships of agents acting on G-protein coupled receptors. METHOD: Representative sets of G-PCR agonists and antagonists were identified from the literature and Medline [P.M. Walsh (2003) Physicians' Desk Reference; M.J. O'Neil (2001) The Merck Index]. The molecular weight (MW), calculated logarithm of octanol/water partition coefficient (C log P) and molar refraction (CMR), dipole moment (DM), E(lumo) (the energy of the lowest unoccupied molecular orbital, a measure of the electron affinity of a molecule and its reactivity as an electrophile), E(homo) (the energy of the highest occupied molecular orbital, related to the ionization potential of a molecule, and its reactivity as a nucleophile), and the total number of hydrogen bonds (H(b)) (donors and receptors), were chosen as molecular descriptors for SAR analyses. RESULTS: The data suggest that not only do neurotransmitters share common structural features but their receptors belong to the same ensemble of G-protein coupled receptor with seven to eight transmembrane domains with their resultant dipoles in an antiparallel configuration. Moreover, the analysis indicates that the receptor exists in a dynamic equilibrium between the closed state and the open state. The energy needed to open the closed state is provided by the hydrolysis of GTP. A composite 3-D parameter frame setting of all the neurotransmitter agonists and antagonists are presented using MW, Hb and mu as independent variables. CONCLUSION: It appears that all neurotransmitters examined in this study operate by a similar mechanism with the G-protein coupled receptors.

Classification of dopamine antagonists using TFS-based artificial neural network.

In the former work, the authors proposed the Topological Fragment Spectral (TFS) method as a tool for the description of the topological structure profile of a molecule. This paper describes the TFS-based artificial neural network (TFS/ANN) approach for the classification and the prediction of pharmacological active classes of chemicals. Dopamine antagonists of 1227 that interact with different types of receptors (D1, D2, D3, and D4) were used for the training. The TFS/ANN successfully classified 89% of the drugs into their own active classes. Then, the trained model was used for predicting the class of unknown compounds. For the prediction set of 137 drugs that were not included in the training set, the TFS/ANN model predicted 111 (81%) drugs of them into their own active classes correctly.

Differential sensitivity to acute administration of Ritalin, apomorphine, SCH 23390, but not raclopride in mice selectively bred for hyperactive wheel-running behavior.

RATIONALE: Previous studies of mice ( Mus domesticus) selectively bred for high voluntary wheel running have suggested that the hyperactivity is associated with dysfunction in the dopaminergic neuromodulatory system and that high-running mice may represent a useful genetic model for attention deficit hyperactivity disorder (ADHD). OBJECTIVES: We tested the hypothesis that mice from the four replicate hyperactive lines would respond differently to methylphenidate (Ritalin), apomorphine (non-selective dopamine agonist), SCH 23390 (selective D1-like dopamine antagonist), and raclopride (selective D2-like dopamine antagonist) than individuals from the four replicate, randomly bred, control lines. METHODS: After animals were habituated (3 weeks) to their cages with attached wheels, drugs were administered via intraperitoneal injections, at night, during peak wheel-running activity. Revolutions on wheels 10-70 min post-injection were used to quantify drug responses. RESULTS: Ritalin (15 mg/kg and 30 mg/kg) increased wheel running in control lines but decreased running in selected lines. A low-dose (0.125 mg/kg) of apomorphine reduced wheel running by a similar amount in control and selected lines; however, higher doses of apomorphine (0.25 mg/kg and 0.5 mg/kg) produced greater reductions in wheel running in the control lines. SCH 23390 (0.025, 0.05, and 0.1 mg/kg) caused greater reductions in wheel running in control than in selected lines. Raclopride (0.5, 1, and 2 mg/kg) reduced wheel running by a similar amount in control and selected lines. CONCLUSIONS: These results support the interpretation that genetically determined hyperactive wheel-running behavior is associated with altered dopaminergic function in this mouse model. More specifically, results suggest that D1-like (D1 or D5), but not D2-like (D2, D3, or D4), dopamine receptors have reduced function in the high-running mice. The fact that Ritalin decreased wheel running in selected lines further supports their use as an animal model of ADHD.

Nonlinear analysis of partial dopamine agonist effects on cAMP in C6 glioma cells.

Most drugs have some efficacy so that improved methods to determine the relative intrinsic efficacy of partial agonists should be of benefit to preclinical and clinical investigators. We examined the effects of partial D(1) or partial D(2) dopamine agonists using a partial agonist interaction model. The dependent variable was the modulation of the dopamine-receptor-mediated cAMP response in C6 glioma cells selectively and stably expressing either D(1) or D(2) recombinant dopamine receptors. The dissociation constant (K(B)) and relative intrinsic efficacy (E(r)) for each partial agonist were calculated using a partial agonist interaction null model in which the effects of fixed concentrations of each partial agonist on the dopamine dose-response curve were evaluated. This model is an extension of the competitive antagonist null model to drugs with efficacy and assumes only that the log-dose--response curve is monotonic. Generally, the partial agonist interaction model fit the data, as well as fits of the independent logistic curves. Furthermore, the partial agonist K(B) values could be shared across partial agonist concentrations without worsening the model fit (by increasing the residual variance). K(B) values were also similar to drug affinities reported in the literature. The model was validated in three ways. First, we assumed a common tissue stimulus parameter (beta) and calculated the E(r) values. This provided a qualitative check on the interaction model results. Second, we calculated new relative efficacy values, E(r)(beta), using the beta estimate. Third, we calculated relative efficacy using relative maxima times midpoint shift ratios (J. Theor. Biol. 198 (1999) 347.). All three methods indicated that the present model yielded reasonable estimates of affinity and relative efficacy for the set of compounds studied. Our results provide a quick and convenient method of quantification of partial agonist efficacy. Special applications and limitations of the model are discussed. In addition, the present results are the first report of the relative intrinsic efficacy values for this set of D(2) ligands.

Differential response of cortical-limbic neuropotentiated compulsive mice to dopamine D1 and D2 receptor antagonists.

We previously created transgenic mice in which dopamine D1 receptor-expressing (D1+) neurons in regional subsets of the cortex and amygdala express a neuropotentiating cholera toxin (CT) transgene. These 'D1CT' mice engage in complex biting, locomotor and behavioral perseverance-repetition abnormalities that resemble symptoms of human compulsive disorders associated with cortical-limbic hyperactivity. Because excessive cortical-limbic stimulation of striatal motor pathways may play a critical role in causing compulsive disorders, we examined the responsiveness of D1CT mice to dopamine D1 and D2 receptor antagonists. D1CT mice were found to be largely resistant to the cataleptic action of the D1 receptor antagonist SCH23390. The abnormal repetitive leaping of D1CT mice was similarly unaffected by SCH23390. In contrast, the D1CT mice displayed supersensitivity to cataleptic induction by the D2 receptor antagonist sulpiride. These data are consistent with the hypothesis that complex compulsions are mediated by chronic excessive corticostriatal (and/or amygdalostriatal) glutamatergic stimulation of the striatal direct and indirect motor pathways.

Antipsychotic drugs which elicit little or no parkinsonism bind more loosely than dopamine to brain D2 receptors, yet occupy high levels of these receptors.

This review addresses two questions. First, why does clozapine apparently occupy low levels of dopamine D2 receptors in patients, in contrast to all other antipsychotic drugs which occupy 70-80% of brain dopamine D2 receptors? Second, what is the receptor basis of action of antipsychotic drugs which elicit low levels of Parkinsonism? Antipsychotic doses of clozapine occupy between 0% and 50% of D2 receptors, as measured in patients by a variety of radioligands. It has recently been found, however, that the percent occupancy of a receptor by a drug depends on the radioligand used to measure that receptor. Based on this new finding, this review concludes that clozapine clinically occupies high levels of D2 receptors in the absence of any radioligand. This occupancy is estimated to be of the order of 70-80% in the dopamine-rich region of the human striatum, and even higher in the limbic D2-containing regions which are low in endogenous synaptic dopamine. This conclusion arises from two different approaches. One approach is to relate the reported clozapine occupancies in the human striatum with the dissociation constants of the various radioligands at the D2 receptor. This relation extrapolates to approximately 70-80% occupancy by clozapine when clozapine competes with endogenous dopamine at the D2 receptor. The second approach is to calculate the D2 occupancy of each antipsychotic drug, using the average spinal fluid concentration and the correct dissociation constant of the antipsychotic, thereby revealing that all antipsychotic drugs, including clozapine, occupy approximately 70-80% of dopamine D2 receptors in the human striatum, and possibly higher in the limbic regions. As determined by the new dissociation constants, antipsychotic drugs which elicit Parkinsonism (trifluperazine, chlorpromazine, raclopride, haloperidol, fluphenazine, risperidone) bind more tightly than dopamine to D2, while those antipsychotic drugs which elicit little or no Parkinsonism (melperone, seroquel, perlapine, clozapine, remoxipride, molindone, sulpiride, olanzapine, sertindole) bind more loosely than dopamine to D2 receptors. Compared to the tightly bound antipsychotic drugs, the more loosely bound antipsychotics generally require higher clinical doses, require fewer days for clinical adjustment, but may dissociate from the D2 receptor more rapidly and could lead to clinical relapse somewhat earlier than that found with the traditional tightly bound antipsychotic drugs.

New classes of selective D-1 dopamine receptor antagonist provide further evidence for two directions of D-1:D-2 interaction.

Initial notions of cooperative/synergistic interactions between D-1 and D-2 dopamine receptors have been followed by recent evidence suggesting more complex forms of D-1:D-2 interaction. Each of the new, putative selective D-1 antagonists SCH 39166, NO 756 and A-69024 antagonised typical behavioural responses to the selective D-2 agonist RU 24213 and concurrently released atypical behaviour. These data extend new notions of both cooperative/synergistic and oppositional D-1:D-2 interactions in the regulation of typical and atypical behaviours, which may involve further subtypes of D-1 and of D-2 receptor.