Human D2 Research Articles

Behavioral Effects of a Novel Allosteric Potentiator of the Dopamine D1 Receptor in Human D1 Knock‐in Mice and Rhesus Monkeys

Allosteric potentiators increase the affinity of endogenousagonist, in effect amplifying physiological control circuits. Because a potentiator should depend onendogenous tone, its effects are predicted to be self‐limiting and less proneto rapid tolerance development compared to direct‐acting agonists. Our objective was to test this hypothesis using DETQ, a novel allosteric potentiator of the dopamine D1 receptor.Although DETQ has high affinity for the human D1 receptor, it is 70‐fold less potent at the mouse and rat D1 receptors, limiting its use as a pharmacological tool in rodents. To overcome this limitation, we created a transgenic knock‐in mouse expressing the human D1 receptor (hD1). Homozygotes showed normal behavior and breeding.After oral dosing, DETQ caused a dose‐dependent 10‐fold increase in locomotor activity in habituated hD1 mice but not in wild‐type mice, implying a requirement for the human D1 receptor. The increase in locomotor activity was blocked by the D1 antagonist SCH39166 and by pretreatment with a high dose of reserpine, indicating that the behavioral response is dependent on endogenous dopamine release. At higher doses, the response to DETQ reached a plateau even though brain concentrations of unbound drug continued to rise. In contrast, the D1 agonists SKF82958 and A‐77636 showed bell‐shaped dose‐response curves, with a profound decrease in locomotor activity at the highest doses. The suppression of locomotor activity at high doses was due to engagement of competing stereotyped behaviors such as intense grooming. The stereotyped behaviors were not seen with DETQ, providing evidence that the response to DETQ is less liable to cause over stimulation. In repeated dosing over four days, the locomotor response to DETQ was maintained, whereas the response to A‐77636 showed rapid tolerance.In hD1 mice treated with a low dose of reserpine, DETQ restored locomotor activity to untreated control levels; this model is relevant to stand‐alone therapy in mild‐to‐moderate Parkinson's disease. Versus a higher dose of reserpine, DETQ acted synergistically with L‐DOPA in restoring locomotor activity. DETQ also increased wakefulness and decreased sleep and was found to be effective in a behavioral despair model. We also compared DETQ with SKF82958 for efficacy in the Y‐maze. While both compounds enhanced the number of arm entries in a dose–dependent fashion, mice treated with DETQ maintained spontaneous alternation even at high doses, whereas mice treated with higher doses of SKF82958 showed an increased prevalence to return to the same arm (asign of cognitive dysfunction). Finally, DETQ increased spontaneous eye blink rate in the rhesus monkey, a response related to central D1 activation.These results confirm that D1 potentiators may possess advantages over D1 agonists for the treatment of Parkinson's disease and other CNS disorders.

A Novel Class of Dopamine D4 Receptor Ligands Bearing an Imidazoline Nucleus.

Over the years, the 2-substituted imidazoline nucleus has been demonstrated to be a bioversatile structural motif. In this study, novel imidazoline derivatives bearing a 3- and/or 4-hydroxy- or methoxy-substituted phenyl ring, linked by an ethylene bridge to position 2 of an N-benzyl- or N-phenethyl-substituted imidazoline nucleus, were prepared and studied against D2 -like receptor subtypes. Binding studies highlighted that a set of N-phenethylimidazoline compounds are selective for D4 over D2 and D3 receptors. In functional assays, the 3-methoxy-substituted derivative, endowed with the highest D4 affinity value, and its 3-hydroxy analogue behaved as partial agonists with low intrinsic efficacy and as competitive D4 antagonists when tested in the presence of the D2 -like receptor agonist quinpirole. Molecular docking analysis, performed using a homology model of the human D4 receptor developed using the X-ray crystal structure of the antagonist-bound human D3 receptor as a template, was in accordance with the binding results and provided useful information for the design of novel imidazoline D4 receptor ligands based on this new scaffold.

Erratum to: Translational Modeling in Schizophrenia: Predicting Human Dopamine D2 Receptor Occupancy

Erratum to: Pharm Res DOI 10.1007/s11095-015-1846-4 There occurred two errors in the above manuscript (Pharm Res. 2015 Dec 30 [Epub ahead of print]). The correct information is as follows: The differential equations in Appendix 2 should read: dAbv/dt=CLbv/Vplasma∗Aplasma−CLbv/Vbv∗Abv−CLbev/Vbv∗fuplasma∗Abv+CLbev/Vbev∗fubrain∗Abev+CLeff/Vbev∗fubrain∗AbevdAbev/dt=CLbev/Vbv∗fuplasma∗Abv−CLbev/Vbev∗fubrain∗Abev−CLeff/Vbev∗fubrain∗Abev−CLst/Vbev∗fubrain∗Abev+CLst/Vstf∗fubrain∗AstfdAstf/dt=CLst/Vbev∗fubrain∗Abev−CLst/Vstf∗fubrain∗Astf−kon∗fubrain∗Astf∗Bmax−CB+koff∗AstbdAstb/dt=kon∗fubrain∗Astf∗Bmax−CB−koff∗Astb Table III should read: Table III In vitro, in vivo and ex vivo values estimates used in the human D2RO predictive model.

Exploring quantitative structure–activity relationship (QSAR) models for some biologically active catechol structures using PC-LS-SVM and PC-ANFIS

Exploring predictive QSAR models for dopamine catechol structures could be used in designing more potent ligands. In this study, efforts were taken to find out the most important molecular features responsible for the biological activity of catechol structures. All 2D descriptors of Dragon including constitutional, topological, molecular walk counts, BCUT descriptors, Galvez topological, 2D autocorrelations, functional groups, atom-centred fragments, empirical descriptors and properties were calculated for the structures. Two non-linear modelling methods (PC-LS-SVM and PC-ANFIS) were used and compared in this QSAR study. The results revealed the more predictive ability of PC-LS-SVM in the QSAR analysis of the compounds with catechol substructure. The roles of topological properties and number of hydrogen bond donors group as molecular features responsible for the activity of the compounds were discussed. The obtained QSAR models can be used in future studies of drug development for human dopamine D2 receptor.

Dysbindin-1 modifies signaling and cellular localization of recombinant, human D₃ and D₂ receptors.

Dystrobrevin binding protein-1 (dysbindin-1), a candidate gene for schizophrenia, modulates cognition, synaptic plasticity and frontocortical circuitry and interacts with glutamatergic and dopaminergic transmission. Loss of dysbindin-1 modifies cellular trafficking of dopamine (DA) D2 receptors to increase cell surface expression, but its influence upon signaling has never been characterized. Further, the effects of dysbindin-1 upon closely related D3 receptors remain unexplored. Hence, we examined the impact of dysbindin-1 (isoform A) co-expression on the localization and coupling of human D2L and D3 receptors stably expressed in Chinese hamster ovary or SH-SY5Y cells lacking endogenous dysbindin-1. Dysbindin-1 co-transfection decreased cell surface expression of both D3 and D2L receptors. Further, while their affinity for DA was unchanged, dysbindin-1 reduced the magnitude and potency of DA-induced adenylate cylase recruitment/cAMP production. Dysbindin-1 also blunted the amplitude of DA-induced phosphorylation of ERK1/2 and Akt at both D2L and D3 receptors without, in contrast to cAMP, affecting the potency of DA. Interference with calveolin/clathrin-mediated processes of internalization prevented the modification by dysbindin-1 of ERK1/2 and adenylyl cyclase stimulation at D2L and D3 receptors. Finally, underpinning the specificity of the influence of dysbindin-1 on D2L and D3 receptors, dysbindin-1 did not modify recruitment of adenylyl cyclase by D1 receptors. These observations demonstrate that dysbindin-1 influences cell surface expression of D3 in addition to D2L receptors, and that it modulates activation of their signaling pathways. Accordingly, both a deficiency and an excess of dysbindin-1 may be disruptive for dopaminergic transmission, supporting its link to schizophrenia and other CNS disorders. Dysbindin-1, a candidate gene for schizophrenia, alters D2 receptors cell surface expression. We demonstrate that dysbindin-1 expression also influences cell surface levels of D3 receptors. Further, Dysbindin-1 reduces DA-induced adenylate cylase recruitment/cAMP production and modifies major signaling pathways (Akt and extracellular signal-regulated kinases1/2 (ERK1/2)) of both D2 and D3 receptors. Dysbindin-1 modulates thus D2 and D3 receptor signaling, supporting a link to schizophrenia.

The effect of low-speed drilling without irrigation on heat generation: an experimental study.

ObjectivesIn this study we evaluated heat generation during the low-speed drilling procedure without irrigation.Materials and MethodsTen artificial bone blocks that were similar to human D1 bone were used in this study. The baseline temperature was 37.0℃. We drilled into 5 artificial bone blocks 60 times at the speed of 50 rpm without irrigation. As a control group, we drilled into an additional 5 artificial bone blocks 60 times at the speed of 1,500 rpm with irrigation. The temperature changes during diameter 2 mm drilling were measured using thermocouples.ResultsThe mean maximum temperatures during drilling were 40.9℃ in the test group and 39.7℃ in the control group. Even though a statistically significant difference existed between the two groups, the low-speed drilling did not produce overheating.ConclusionThese findings suggest that low-speed drilling without irrigation may not lead to overheating during drilling.

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.

Improving selectivity of dopamine D3 receptor ligands

The seminal human dopamine D3 receptor (hD3R) ligand BP 897 has shown interesting properties during clinical trials. However, its lack of selectivity towards human adrenergic receptor impedes further development. Two approaches were followed to increase hD3R selectivity. The lead optimisation succeeded, we disclose here ligands with subnanomolar potency for D3R, combined with a good selectivity for the closely related human dopamine D2 and human adrenergic alpha-1 receptors.

Aberrant regulation of synchronous network activity by the attention-deficit/hyperactivity disorder-associated human dopamine D4 receptor variant D4.7 in the prefrontal cortex.

The hD4.7 variant has been linked to attention-deficit/hyperactivity disorder (ADHD); however, the underlying mechanism is unknown. We found that activation of hD4.7 induced over-suppression of glutamatergic excitatory network bursts and under-suppression of GABAergic inhibitory network bursts in the prefrontal cortex (PFC) circuitry. Methylphenidate, a psychostimulant drug used to treat ADHD, normalized the effects of hD4.7 on synchronous network bursts in PFC pyramidal neurons. The findings of the present study suggest that the aberrant regulation of PFC synchronous network activity by hD4.7 may underlie its involvement in ADHD. A unique feature of the human D4 receptor (hD4 R) gene is the existence of a large number of polymorphisms in exon 3 coding for the third intracellular loop, which consists of a variable number of tandem repeats. The hD4 R variants with long repeats have been linked to attention-deficit/hyperactivity disorder (ADHD); however, the underlying mechanism is unknown. Emerging evidence suggests that selective attention is controlled by the rhythmic synchronization in the prefrontal cortex (PFC) and its connected networks. In the present study, we examined the role of hD4 R variants in regulating PFC synchronous network activity. D4 R knockout mice with viral infection of hD4.4 or hD4.7 in the medial PFC were used. Whole-cell patch-clamp recordings were performed to examine the effects of activating hD4.x on the spontaneous large scale correlated activity in PFC pyramidal neurons. We found that, compared to the normal four-repeat variant hD4.4, the ADHD-linked variant hD4.7 induces more suppression of glutamatergic excitatory network bursts and less suppression of GABAergic inhibitory network bursts in the PFC circuitry. Methylphenidate, a psychostimulant drug used to treat ADHD, normalized the effects of hD4.7 on synchronous network bursts in PFC pyramidal neurons. These results reveal the differential effects of hD4 R variants on the integrated excitability of PFC circuits. It is suggested that the aberrant regulation of PFC network activity by hD4.7 may underlie its involvement in ADHD. The methylphenidate-induced normalization of synaptic circuitry regulation may contribute to its effectiveness in ADHD treatment.

In Vitro and In Vivo Identification of Novel Positive Allosteric Modulators of the Human Dopamine D2 and D3 Receptor.

Agonists at dopamine D2 and D3 receptors are important therapeutic agents in the treatment of Parkinson's disease. Compared with the use of agonists, allosteric potentiators offer potential advantages such as temporal, regional, and phasic potentiation of natural signaling, and that of receptor subtype selectivity. We report the identification of a stereoselective interaction of a benzothiazol racemic compound that acts as a positive allosteric modulator (PAM) of the rat and human dopamine D2 and D3 receptors. The R isomer did not directly stimulate the dopamine D2 receptor but potentiated the effects of dopamine. In contrast the S isomer attenuated the effects of the PAM and the effects of dopamine. In radioligand binding studies, these compounds do not compete for binding of orthosteric ligands, but indeed the R isomer increased the number of high-affinity sites for [(3)H]-dopamine without affecting K(d). We went on to identify a more potent PAM for use in native receptor systems. This compound potentiated the effects of D2/D3 signaling in vitro in electrophysiologic studies on dissociated striatal neurons and in vivo on the effects of L-dopa in the 6OHDA (6-hydroxydopamine) contralateral turning model. These PAMs lacked activity at a wide variety of receptors, lacked PAM activity at related Gi-coupled G protein-coupled receptors, and lacked activity at D1 receptors. However, the PAMs did potentiate [(3)H]-dopamine binding at both D2 and D3 receptors. Together, these studies show that we have identified PAMs of the D2 and D3 receptors both in vitro and in vivo. Such compounds may have utility in the treatment of hypodopaminergic function.

Essential Role of GATA2 in the Negative Regulation of Type 2 Deiodinase Gene by Liganded Thyroid Hormone Receptor β2 in Thyrotroph.

The inhibition of thyrotropin (thyroid stimulating hormone; TSH) by thyroid hormone (T3) and its receptor (TR) is the central mechanism of the hypothalamus-pituitary-thyroid axis. Two transcription factors, GATA2 and Pit-1, determine thyrotroph differentiation and maintain the expression of the β subunit of TSH (TSHβ). We previously reported that T3-dependent repression of the TSHβ gene is mediated by GATA2 but not by the reported negative T3-responsive element (nTRE). In thyrotrophs, T3 also represses mRNA of the type-2 deiodinase (D2) gene, where no nTRE has been identified. Here, the human D2 promoter fused to the CAT or modified Renilla luciferase gene was co-transfected with Pit-1 and/or GATA2 expression plasmids into cell lines including CV1 and thyrotroph-derived TαT1. GATA2 but not Pit-1 activated the D2 promoter. Two GATA responsive elements (GATA-REs) were identified close to cAMP responsive element. The protein kinase A activator, forskolin, synergistically enhanced GATA2-dependent activity. Gel-shift and chromatin immunoprecipitation assays with TαT1 cells indicated that GATA2 binds to these GATA-REs. T3 repressed the GATA2-induced activity of the D2 promoter in the presence of the pituitary-specific TR, TRβ2. The inhibition by T3-bound TRβ2 was dominant over the synergism between GATA2 and forskolin. The D2 promoter is also stimulated by GATA4, the major GATA in cardiomyocytes, and this activity was repressed by T3 in the presence of TRα1. These data indicate that the GATA-induced activity of the D2 promoter is suppressed by T3-bound TRs via a tethering mechanism, as in the case of the TSHβ gene.

The protein arginine methyltransferase PRMT5 promotes D2-like dopamine receptor signaling.

Protein arginine methylation regulates diverse functions of eukaryotic cells, including gene expression, the DNA damage response, and circadian rhythms. We showed that arginine residues within the third intracellular loop of the human D2 dopamine receptor, which are conserved in the DOP-3 receptor in the nematode Caenorhabditis elegans, were methylated by protein arginine methyltransferase 5 (PRMT5). By mutating these arginine residues, we further showed that their methylation enhanced the D2 receptor-mediated inhibition of cyclic adenosine monophosphate (cAMP) signaling in cultured human embryonic kidney (HEK) 293T cells. Analysis of prmt-5-deficient worms indicated that methylation promoted the dopamine-mediated modulation of chemosensory and locomotory behaviors in C. elegans through the DOP-3 receptor. In addition to delineating a previously uncharacterized means of regulating GPCR (heterotrimeric guanine nucleotide-binding protein-coupled receptor) signaling, these findings may lead to the development of a new class of pharmacological therapies that modulate GPCR signaling by changing the methylation status of these key proteins.

Association of dopamine receptor D4 (DRD4) gene polymorphism with cardiovascular disease risk factors

Dopamine receptor genes are known to be candidate genes, mutations of which may be involved in the development of cardiovascular diseases. The human D4 receptor gene (DRD4) contains a variable number (two to ten copies) of tandem 48-bp repeats in exon 3. The goal of the study was to analyze the association of DRD4 gene polymorphism with cardiovascular disease risk factors in the Russian population: blood pressure (BP), high-density (HDL) and low-density (LDL) lipoprotein cholesterol levels, and the triglyceride level. We genotyped 257 women and 425 men for variable number tandem repeat polymorphism in DRD4. The subjects were Novosibirsk residents of Slavic ethnicity. They were divided either into 7+ (those having the allele of seven or eight repeats) and 7–(two to six repeats) groups or into 2+5+ (two or five repeats) and 2–5–(three, four, six, seven, or eight repeats) groups. We found associations between the 7+ allele and elevated systolic and diastolic BP in women but not in men. Neither sex showed associations between the long variants of the DRD4 gene and pulse blood pressure. The presence of the 2–or 5–allele of the DRD4 polymorphism was associated with low HDL cholesterol levels in women and low total cholesterol levels in men. The polymorphism exerted no significant effects on LDL, HDL, or triglyceride levels in men.

Molecular modelling studies on 2-substituted octahydropyrazinopyridoindoles for histamine H2 receptor antagonism

The human histamine H2 receptor (hH2HR) is a G-protein coupled receptor protein with seven transmembrane (TM)-spanning helices primarily involved in regulation of gastric acid secretion. Antagonists targeting hH2HR are useful in the treatment of hyperacidic conditions such as peptic ulcers, gastresophageal reflux disease and gastrointestinal bleeding. We have previously reported the antagonism of 2-substituted pyrazinopyridoindoles at the human histamine H1 receptor and mode of binding of these compounds at the hH1HR using in silico methods. Interestingly, some of the compounds in the series also showed promising activity towards hH2HR that prompted us to investigate the mode of binding of these compounds at hH2HR. In the absence of the crystal structure of hH2HR a homology model has been constructed using multiple sequence alignment, using the X-ray crystal structures of Turkey β1-adrenergic receptor (tβ1AR), Human histamine H1 receptor (hH1HR), Human β2-adrenergic receptor (hβ2AR) and Human D3 dopamine receptor (hD3R). The important residues for binding were depicted in TMIII, TMV, TMVI and TMVII by the homology modelled hH2HR for 2-substituted pyrazinopyridoindoles. A comparative study for deducing the selectivity regarding the binding towards hH1HR and hH2HR has been carried out, which may be useful in designing of selective hH1HR/hH2HR antagonists in these classes of compounds.

MS Binding Assays for D1 and D5 Dopamine Receptors.

MS Binding Assays are a label-free alternative to radioligand binding assays. They provide basically the same capabilities as the latter, but an unlabeled reporter ligand is used instead of a radioligand. The study presented herein describes the development of MS Binding Assays that address D1 and D5 dopamine receptors. A highly sensitive, rapid and robust LC-ESI-MS/MS quantification method for the selective D1 dopamine receptor antagonist SCH23390 ((5R)-8-chloro-3-methyl-5-phenyl-1,2,4,5-tetrahydro-3-benzazepin-7-ol) was established and validated, using its 8-bromo analogue SKF83566 as an internal standard. This quantification method proved to be suitable for the characterization of SCH23390 binding to human D1 and D5 receptors. Following the concept of MS Binding Assays, saturation experiments for D1 and D5 receptors were performed, as well as competition experiments for D1 receptors. The results obtained are in good agreement with results from radioligand binding assays and therefore indicate that the established MS Binding Assays addressing D1 and D5 receptors are well-suited substitutes for radioligand binding assays, the technique that has so far dominated affinity determinations toward these targets.

Abstract 3524: BAY 1238097, a novel BET inhibitor with strong efficacy in hematological tumor models

Abstract Several BET inhibitors with strong anti-tumor efficacy have been described in the last few years. Most of them are derived from diazepine and azepine scaffolds, but more recently quinazolinones and isoxazoles chemotypes have also been described. We have identified a novel scaffold with strong BET inhibitory activity. Here we describe the biochemical characteristics of BAY 1238097 and its anti-proliferative activity in acute myeloid leukemia (AML) and multiple myeloma (MM) models. In vitro, BAY 1238097 showed strong inhibitory activity (IC50 < 100 nM) in a TR-FRET assay using BET BRD4 bromodomain 1 and an acetylated peptide derived from histone H4. In the NanoBRET assay, the interaction between BRD4, BRD3 or BRD2 and H4 was inhibited with IC50 values of 63 nM, 609 nM and 2430 nM, showing selectivity of the compound for BRD4. A strong reduction of c-Myc transcript and protein levels was observed in treated MOLM-13 (AML) and MOLP-8 (MM) cell lines. ChIP experiments performed in these models additionally revealed that BAY 1238097 prevented binding of BRD4 to c-Myc regulatory regions. In vivo, BAY 1238097 showed strong efficacy in the AML models THP-1, MOLM-13 and KG-1, with T/C between 13 and 20%. Overall, the compound was well tolerated at MTD, with body weight losses of 5-9% at nadir. BAY 1238097 was also active in MM models. Efficacy was observed against a human IGH-cyclin D1 translocated MOLP-8 model with a T/C of 3%, whereas the standard-of-care agents bortezomib and lenalidomide were inactive or poorly active. In this model, BAY 1238097 was well tolerated at 10 mg/kg applied over 14 days, with no body weight loss measured. BAY 1238097 was also active against the FGFR/MMSET translocated model NCIH929, with 19% T/C versus 49% T/C for the standard-of-care lenalidomide. The compound was well tolerated applied at 12 mg/kg for 9 days (maximum body weight loss 6%). Gene expression profiling performed in liver, blood and duodenum of rats treated daily with 2 mg/kg BAY 1238097 for 14 days demonstrated substantial effects on genes involved in cell proliferation and in the immune response in vivo. Altogether, BAY 1238097 is a potent inhibitor of BET binding to histones and has strong anti-proliferative activity in different AML and MM models through down-regulation of c-Myc levels and its downstream transcriptome. Initiation of clinical studies with BAY 1238097 is planned for early 2015. Citation Format: Pascale Lejeune, Tatsuo Sugawara, Kathy A. Gelato, Heidrun Ellinger-Ziegelbauer, Amaury E. Fernandez-Montalvan, Norbert Schmees, Stephan Siegel, Hilmar Weinmann, Volker Gekeler, Annette O. Walter, Matthias Ocker, Stuart Ince, Bernard Haendler. BAY 1238097, a novel BET inhibitor with strong efficacy in hematological tumor models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3524. doi:10.1158/1538-7445.AM2015-3524

Discovery of D1 Dopamine Receptor Positive Allosteric Modulators: Characterization of Pharmacology and Identification of Residues that Regulate Species Selectivity.

The present studies represent the first published report of a dopamine D1 positive allosteric modulator (PAM). D1 receptors have been proposed as a therapeutic target for the treatment of cognitive deficits associated with schizophrenia. However, the clinical utility of orthosteric agonist compounds is limited by cardiovascular side effects, poor pharmacokinetics, lack of D1 selectivity, and an inverted dose response. A number of these challenges may be overcome by utilization of a selective D1 PAM. The current studies describe two chemically distinct D1 PAMs: Compound A [1-((rel-1S,3R,6R)-6-(benzo[d][1,3]dioxol-5-yl)bicyclo[4.1.0]heptan-3-yl)-4-(2-bromo-5-chlorobenzyl)piperazine] and Compound B [rel-(9R,10R,12S)-N-(2,6-dichloro-3-methylphenyl)-12-methyl-9,10-dihydro-9,10-ethanoanthracene-12-carboxamide]. Compound A shows pure PAM activity, with an EC50 of 230 nM and agonist activity at the D2 receptor in D2-expressing human embryonic kidney cells. Compound B shows superior potency (EC50 of 43 nM) and selectivity for D1 versus D2 dopamine receptors. Unlike Compound A, Compound B is selective for human and nonhuman primate D1 receptors, but lacks activity at the rodent (rat and mouse) D1 receptors. Using molecular biology techniques, a single amino acid was identified at position 130, which mediates the species selectivity of Compound B. These data represent the first described D1-selective PAMs and define critical amino acids that regulate species selectivity.

Identification of indole inhibitors of human hematopoietic prostaglandin D2 synthase (hH-PGDS).

Human H-PGDS has shown promise as a potential target for anti-allergic and anti-inflammatory drugs. Here we describe the discovery of a novel class of indole inhibitors, identified through focused screening of 42,000 compounds and evaluated using a series of hit validation assays that included fluorescence polarization binding, 1D NMR, ITC and chromogenic enzymatic assays. Compounds with low nanomolar potency, favorable physico-chemical properties and inhibitory activity in human mast cells have been identified. In addition, our studies suggest that the active site of hH-PGDS can accommodate larger structural diversity than previously thought, such as the introduction of polar groups in the inner part of the binding pocket.

Blonanserin extensively occupies rat dopamine D3 receptors at antipsychotic dose range

Antagonism of the dopamine D3 receptor has been hypothesized to be beneficial for schizophrenia cognitive deficits, negative symptoms and extrapyramidal symptoms. However, recent animal and human studies have shown that most antipsychotics do not occupy D3 receptors in vivo, despite their considerable binding affinity for this receptor in vitro. In the present study, we investigated the D3 receptor binding of blonanserin, a dopamine D2/D3 and serotonin 5-HT2A receptors antagonist, in vitro and in vivo. Blonanserin showed the most potent binding affinity for human D3 receptors among the tested atypical antipsychotics (risperidone, olanzapine and aripiprazole). Our GTPγS-binding assay demonstrated that blonanserin acts as a potent full antagonist for human D3 receptors. All test-drugs exhibited antipsychotic-like efficacy in methamphetamine-induced hyperactivity in rats. Treatment with blonanserin at its effective dose blocked the binding of [3H]-(+)-PHNO, a D2/D3 receptor radiotracer, both in the D2 receptor-rich region (striatum) and the D3 receptor-rich region (cerebellum lobes 9 and 10). On the other hand, the occupancies of other test-drugs for D3 receptors were relatively low. In conclusion, we have shown that blonanserin, but not other tested antipsychotics, extensively occupies D3 receptors in vivo in rats.

Modeling and protein engineering studies of active and inactive states of human dopamine D2 receptor (D2R) and investigation of drug/receptor interactions.

Homology model structures of the dopamine D2 receptor (D2R) were generated starting from the active and inactive states of β2-adrenergic crystal structure templates. To the best of our knowledge, the active conformation of D2R was modeled for the first time in this study. The homology models are built and refined using MODELLER and ROSETTA programs. Top-ranked models have been validated with ligand docking simulations and in silico Alanine-scanning mutagenesis studies. The derived extra-cellular loop region of the protein models is directed toward the binding site cavity which is often involved in ligand binding. The binding sites of protein models were refined using induced fit docking to enable the side-chain refinement during ligand docking simulations. The derived models were then tested using molecular modeling techniques on several marketed drugs for schizophrenia. Alanine-scanning mutagenesis and molecular docking studies gave similar results for marketed drugs tested. We believe that these new D2 receptor models will be very useful for a better understanding of the mechanisms of action of drugs to be targeted to the binding sites of D2Rs and they will contribute significantly to drug design studies involving G-protein-coupled receptors in the future.