Innovative Bioluminescence Resonance Energy Transfer Assay Reveals Differential Agonist-Induced D2 Receptor Intracellular Trafficking and Arrestin-3 Recruitment.

The dopamine D2 receptor (D2R) mediates ligand-biased signaling with potential therapeutic implications. However, internalization, choice of endocytic routes, and degradation of the D2R in lysosomes may also participate in agonist-directed trafficking. We developed bioluminescence resonance energy transfer (BRET) assays that measure relative distances between Renilla luciferase8-tagged D2R and green fluorescent protein 2 (GFP2)-tagged K-Ras (plasma membrane marker), and between luciferase8-tagged D2R and GFP2-Rab5 (early), GFP2-Rab4 (recycling), or GFP2-Rab7 (late) endosomal markers. The BRET signal between D2R-Luc and GFP2-K-Ras was robustly diminished after receptor internalization induced by dopamine, with subsequent BRET signals increasing when luciferase8-tagged D2R approached GFP2-Rab proteins in endosomal compartments. All BRET signals were blocked by the selective D2R antagonist haloperidol and were decreased by low temperature and high sucrose blocks, two parameters interfering with internalization. Some antipsychotic drugs, such as aripiprazole, are less efficacious in internalizing D2R than most of the antiparkinsonian agents. However, antipsychotics were nearly as efficacious as antiparkinsonians in directing the D2R toward early and recycling endosomes. The Rab7 marker for the late endosome/lysosome route was also capable of discriminating between D2R compounds. We could show that some drugs engaged the D2R either to interact preferentially with arrestin-3 or to internalize. Our study revealed that D2R trafficking in cells was differentially regulated by antipsychotic and antiparkinsonian drugs. Taken together, the BRET assays reported here could further help decipher D2R ligand-induced arrestin-3 recruitment and trafficking, with potentially more selective therapeutic profiles and fewer undesired side effects.

Distinctive in vitro signal transduction profile of NLX-112, a potent and efficacious serotonin 5-HT1A receptor agonist.

OBJECTIVES: NLX-112 (befiradol, F13640) is a selective serotonin 5-HT1A receptor agonist. Although it has been tested in vivo, little has been reported on its in vitro signal transduction profile. METHODS: NLX-112 was tested on G-protein activation, inhibition of adenylyl cyclase, ERK1/2 phosphorylation (pERK) and receptor internalization in recombinant cell lines. NLX-112 was also tested on G-protein activation in rat hippocampal membranes. Gα subunit mRNA expression in cell lines and rat brain tissue was quantified by quantitative PCR. KEY FINDINGS: For all signalling measures, NLX-112 exhibited agonist efficacy greater than for reference compounds ((±)8-OH-DPAT or buspirone), but similar to the endogenous agonist, serotonin, and was more potent for pERK than other responses. In rat hippocampal membranes, NLX-112 stimulated 'total G-proteins' but, unlike (±)8-OH-DPAT and buspirone, was more potent for Gαo activation. Cell lines predominantly expressed Gαi1 and Gαi2 mRNA, with low levels of Gαo, whereas in rat brain Gαo subunits showed highest mRNA expression. CONCLUSIONS: Unlike reference compounds, NLX-112 was a highly efficacious agonist in vitro, preferentially activating pERK in cell lines and Gαo proteins in rat hippocampal membranes. However, Gα subunit mRNA levels differ markedly between rat brain and cell lines, warranting caution when extrapolating from recombinant systems to native tissues.

Contribution of muscarinic receptors to in vitro and in vivo effects of Ruscus extract.

The objectives of this study were to evaluate, in vitro and in vivo, the contribution of muscarinic receptors to the effects of Ruscus extract. Ruscus extract was tested in competition binding experiments at recombinant human muscarinic receptors, heterologous expressed in Chinese Hamster Ovary (CHO) cells and in cellular assays measuring Ca2+ liberation and activator protein-1 (AP-1) reporter gene activation. The impact of muscarinic blockade on prolonged treatment outcome was evaluated using the hamster cheek pouch (HCP) microcirculation examining macromolecular permeability increase induced by histamine or ischemia/reperfusion (I/R), mean arteriolar and venular diameters, functional capillary density and I/R-induced leukocyte rolling and sticking. Ruscus extract exhibited affinities for muscarinic receptor subtypes at a range of 50-100µg/ml and behaved as partial agonist at human recombinant M1 and M3 receptors for Ca2+ liberation, confirmed in an AP-1 reporter gene assay. In the HCP model, topical application of atropine completely or partially blocked Ruscus extract-induced reductions of histamine- and I/R-induced increases of macromolecular permeability and leukocyte-endothelium interaction. Our results showed that Ruscus extract in vitro binds and activates different subtypes of muscarinic receptors and in vivo its anti-inflammatory effects are, at least partially, mediated via muscarinic receptors.

Pharmacological evaluation of a series of smoothened antagonists in signaling pathways and after topical application in a depilated mouse model.

The Hedgehog (HH) pathway has been linked to the formation of basal cell carcinoma (BCC), medulloblastoma, and other cancers. The recently approved orally active drugs vismodegib (GDC-0449) and sonidegib (LDE-225) were not only efficacious for the treatment of advanced or metastatic BCC by antagonizing the smoothened (SMO) receptor, but also produced important side effects, limiting their use for less invasive BCC. Herein, we compared a large series of SMO antagonists, including GDC-0449 and LDE-225, the clinically tested BMS-833923, CUR-61414, cyclopamine, IPI-926 (saridegib), itraconazole, LEQ-506, LY-2940680 (taladegib), PF-04449913 (glasdegib), and TAK-441 as well as preclinical candidates (PF-5274857, MRT-83) in two SMO-dependent cellular assays and for G-protein activation. We report marked differences in inhibitor potencies between compounds as well as a notable disparity between the G-protein assay and the cellular tests, suggesting that classification of drugs is assay dependent. Furthermore, we explored topical efficacies of SMO antagonists on depilated mice using Gli1 and Ptch1 mRNA quantification in skin as biomarkers of the HH signaling inhibition. This topical model rapidly discriminated drugs in terms of efficacies and potencies for inhibition of both biomarkers. SMO antagonists showed also a large variation in their blood and skin partition, suggesting that some drugs are more favorable for topical application. Overall, our data suggested that in vitro and in vivo efficacious drugs such as LEQ-506 and TAK-441 may be of interest for topical treatment of less invasive BCC with minimal side effects.

Agonist stimulation at human µ opioid receptors in a [(35)S]GTPγS incorporation assay: observation of "bell-shaped" concentration-response relationships under conditions of strong receptor G protein coupling.

CONTEXT: The appearance of "bell"- (or "inverted U"-) shaped agonist concentration-response curves (CRCs) in in vitro pharmacological experiments is a frequently observed but poorly communicated phenomenon. In the context of G protein coupled receptor research, it is commonly attributed to the recruitment of secondary targets or to desensitization or feedback processes, but the concrete background of these observations often remains intriguing. OBJECTIVE: Here, we addressed the subject of bell-shaped agonist CRCs at the µ opioid receptor (µOR) by testing the impact of experimental conditions favoring G protein coupling. METHODS: G protein activation by recombinant human µORs heterologously expressed in CHO cells was assessed in [(35)S]GTPγS binding assays using the opioid ligands DAMGO, morphine, fentanyl and naloxone. Experimental conditions were varied by changing the NaCl (10-300 mM) and the GDP concentration (0.3-30 µM). RESULTS: Both the sodium and the GDP concentration were inversely related to G protein coupling, as evident by an increase in basal [(35)S]GTPγS incorporation at low sodium and low GDP levels and by the concomitant appearance of the partial agonist activity of the µOR antagonist, naloxone. Bell-shaped CRCs were observed for the efficacious agonists DAMGO, fentanyl and morphine, and this phenomenon was promoted by low sodium as well as by low GDP concentrations. CONCLUSION: µOR agonist CRCs show a non-monotonic behavior with a decline of maximal stimulation under conditions of strong receptor-G protein coupling, and this behavior is visible at the level of G protein activation itself.

Characterization of V0162, a new long-acting antagonist at human M3 muscarinic acetylcholine receptors.

The anticholinergic properties of the mequitazine enantiomer V0162 make it a drug candidate for the treatment of chronic obstructive airway diseases. Here, we compared V0162's in vitro pharmacological activity at recombinant human M3 muscarinic acetylcholine receptors (hM3Rs) with that of other anticholinergics, using (i) a radioligand binding assay, (ii) a functional reporter gene assay and (iii) a bronchoconstriction inhibition assay on human bronchial preparations. V0162 had high affinity for hM3Rs, with a pKi varying from 9.01 after a 2 h incubation to 9.21 after 23 h. The other mequitazine enantiomer (V0114) was less potent. V0162 displayed rapid off-kinetics and a biphasic time course of binding. V0162 was found to be an antagonist behaving as an inverse agonist for hM3R-mediated reporter gene activation, with much the same efficacy as atropine, ipratropium and tiotropium. However, in contrast to ipratropium and atropine, V0162's inhibitory potency was only slightly affected by compound washout. V0162 antagonized acetylcholine-mediated contractions in a human bronchial preparation; the pA2 values increased with the incubation time (up to 2 h). Moreover, there was a progressive increase in V0162's ability to inhibit electrically-induced contractions, which persisted after compound washout. In conclusion, V0162 is the most active mequitazine enantiomer at hM3Rs and shows a complex pattern of binding to the membrane compartment. These particular features may be of therapeutic value when persistent antagonism at hM3Rs is required.

Consequences of combining siRNA-mediated DNA methyltransferase 1 depletion with 5-aza-2'-deoxycytidine in human leukemic KG1 cells.

5-azacytidine and 5-aza-2'-deoxycytidine are clinically used to treat patients with blood neoplasia. Their antileukemic property is mediated by the trapping and the subsequent degradation of a family of proteins, the DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) leading to DNA demethylation, tumor suppressor gene re-expression and DNA damage. Here we studied the respective role of each DNMT in the human leukemia KG1 cell line using a RNA interference approach. In addition we addressed the role of DNA damage formation in DNA demethylation by 5-aza-2'-deoxycytidine. Our data show that DNMT1 is the main DNMT involved in DNA methylation maintenance in KG1 cells and in mediating DNA damage formation upon exposure to 5-aza-2'-deoxycytidine. Moreover, KG1 cells express the DNMT1 protein at a level above the one required to ensure DNA methylation maintenance, and we identified a threshold for DNMT1 depletion that needs to be exceeded to achieve DNA demethylation. Most interestingly, by combining DNMT1 siRNA and treatment with low dose of 5-aza-2'-deoxycytidine, it is possible to uncouple DNA damage formation from DNA demethylation. This work strongly suggests that a direct pharmacological inhibition of DNMT1, unlike the use of 5-aza-2'-deoxycytidine, should lead to tumor suppressor gene hypomethylation and re-expression without inducing major DNA damage in leukemia.

Potencies and unblocking kinetic properties of antagonists at recombinant human NMDA receptors in a Xenopus oocytes model.

N-methyl-D-aspartate (NMDA) receptor channels are implicated in a wide range of physiological and pathophysiological processes, and a large number of pharmacological agents have been introduced that target the receptor via diverse mechanisms of action. Amongst others, subunit selectivity (in particular for the NR2B receptor subunit) and rapid unblocking kinetics have been put forward as favourable pharmacological properties of NMDA receptor-targeting drugs. Here, we describe a pharmacological characterization of human recombinant NMDA receptors expressed in Xenopus oocytes in an electrophysiological set-up. Using this approach, we compare inhibitor potencies of several known NMDA receptor ligands as well as unblocking kinetic properties of selected compounds. All compounds tested had similar potencies at receptors containing NR2A or NR2B receptors with the exception of traxoprodil, which was selective for NR2B. The rank order of potency was (+)MK-801 > phencyclidine (PCP) ≈ traxoprodil > memantine ≈ ketamine > duloxetine. In line with its proposed rapid dissociation properties, the relatively well-tolerated drug memantine exhibits markedly faster unblocking than ketamine and PCP, similar to the low-affinity compound, duloxetine. Electrophysiological recording in Xenopus oocytes thus allows a relatively convenient comparison of key pharmacological parameters at recombinant human NMDA receptors.

µ-Opioid and 5-HT1A receptors heterodimerize and show signalling crosstalk via G protein and MAP-kinase pathways.

µ-opioid receptors have been shown to form heterodimers with several G protein coupled receptors involved in pain regulation such as α(2A)-adrenergic and neurokinin 1 receptors. Because the 5-HT(1A) receptor is also involved in pain control, we investigated whether it can interact with the µ-opioid receptor in cell lines. Using epitope-tagged µ-opioid and 5-HT(1A) receptors, we show that both receptors can co-immunoprecipate when expressed in the same cells. This physical interaction was corroborated by a Bioluminescence Resonance Energy Transfer signal between the µ-opioid receptor fused to Renilla luciferase and the 5-HT(1A) receptor fused to the Green Fluorescent Protein. Consistent with the presence of functional heterodimers, the µ-opioid receptor activated a Gα(o) protein covalently fused to the 5-HT(1A) receptor in membrane preparations as well as a Gα(15) protein fused to the 5-HT(1A) receptor in living cells. We demonstrate that both receptors can coexerce control of the ERK1/2 pathway: for example, µ-opioid receptor-induced ERK1/2 phosphorylation was selectively desensitized by 5-HT(1A) receptor activation. Although 5-HT(1A) and µ-opioid receptors were capable to internalize in response to their own activation, they were ineffective to induce the co-internalization of their partners. Thus, we show a functional heterodimerization of µ-opioid and 5-HT(1A) receptors in cell lines, a complex that might play a role in the control of pain in vivo. These results also support the potential therapeutic action of 5-HT(1A) agonists against nociceptive processes.

Does endoplasmic reticulum stress participate in APD-induced hepatic metabolic dysregulation?

Metabolic side effects caused by certain antipsychotic drugs (APDs), in particular clozapine and olanzapine, are now clinically well-documented. However, the potential mechanisms implicated in the metabolic disturbances of these drugs on peripheral tissues remain obscure. Here, we investigated the effects of five frequently prescribed APDs on the Sterol Regulatory Element Binding Protein (SREBP) transcription factor pathways which control lipogenesis and cholesterogenesis, using the Immortalized Human Hepatocyte cell model (IHH). First, clozapine, haloperidol, olanzapine and risperidone activated, at different levels, SREBP-1 activity reflected by an increased expression of SREBP-1 target genes involved in fatty acid biosynthesis (SREBP-1, FAS and/or SCD1) resulting in an accumulation of intracellular lipids. Second, clozapine and haloperidol also stimulated the SREBP-2 pathway associated with an increase in HMGCoAR expression. In contrast, quetiapine did not affect either the SREBP-1 or -2 pathways, but induced a slight accumulation of intracellular lipids. Interestingly, clozapine, haloperidol and olanzapine induced Endoplasmic Reticulum (ER) stress and, more precisely, initiation of the ER stress-activated eIF2α kinase (PERK) branch of the Unfolded Protein Response (UPR). Furthermore, treatment with thapsigargin, which increases intracellular calcium release, induced both ER stress and SREBP-1 and -2 pathway activation, whereas Ca(2+) chelation by BAPTA completely reversed the lipogenic effects and ER stress induction produced by clozapine. Based on these results, we propose that certain APDs induce ER stress via changes in Ca(2+) homeostasis in hepatocytes. This phenomenon potentially underlies a part of their known undesirable hepatic metabolic side effects. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.

S32212, a novel serotonin type 2C receptor inverse agonist/α2-adrenoceptor antagonist and potential antidepressant: I. A mechanistic characterization.

Although most antidepressants suppress serotonin (5-HT) and/or noradrenaline reuptake, blockade of 5-HT(2C) receptors and α(2)-adrenoceptors likewise enhances monoaminergic transmission. These sites are targeted by the urea derivative N- [4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]-1,2-dihydro-3-H-benzo[e]indole-3-carboxamide (S32212). S32212 was devoid of affinity for monoamine reuptake sites, yet displayed pronounced affinity (pK(i), 8.2) for constitutively active human 5-HT(2CINI) (h5-HT(2CINI)) receptors, behaving as an inverse agonist in reducing basal Gα(q) activation, [(3)H]inositol-phosphate production, and the spontaneous association of h5-HT(2CINI)-Renilla luciferase receptors with ß-arrestin2-yellow fluorescent protein. Furthermore, upon 18-h pretreatment, S32212 enhanced the plasma membrane expression of h5-HT(2CINI) receptors as visualized by confocal microscopy and quantified by enzyme-linked immunosorbent assay. Its actions were prevented by the neutral antagonist 6-chloro-5-methyl-N-[6-(2-methylpyridin-3-yloxy)pyridin-3-yl]indoline-1-carboxamide (SB242,084), which also impeded the induction by long-term exposure to S32212 of otherwise absent Ca(2+) mobilization in mouse cortical neurones. In vivo, S32212 blunted the inhibitory influence of the 5-HT(2C) agonist 2-(3-chlorobenzyloxy)-6-(1-piperazinyl)pyrazine (CP809,101) on ventrotegmental dopaminergic neurones. S32212 also blocked 5-HT-induced Gα(q) and phospholipase C activation at the h5-HT(2A) and, less potently, h5-HT(2B) receptors and suppressed the discriminative stimulus properties of the 5-HT(2A) agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane in rats. S32212 manifested marked affinity for human α(2A)- (pK(i) 7.2), α(2B)- (pK(i) 8.2), and α(2C)- (pK(i) 7.4) adrenoceptors, at which it abolished noradrenaline-induced recruitment of Gα(i3), Gα(o), adenylyl cyclase, and extracellular-regulated kinase1/2. Moreover, S32212 dose-dependently abolished the discriminative stimulus effects of the α(2)-adrenoceptor agonist (S)-spiro[(1-oxa-2-amino-3-azacyclopent-2-ene)-4,2'-(1',2',3',4'-tetrahydronaphthalene)] (S18616). Finally, S32212 displayed negligible affinity for α(1A)-adrenoceptors, histamine H(1) receptors, and muscarinic M(1) receptors. In conclusion, S32212 behaves as an inverse agonist at h5-HT(2C) receptors and as an antagonist at human α(2)-adrenoceptors (and h5-HT(2A) receptors). Its promising profile in preclinical models potentially relevant to the treatment of depression is described in J Pharmacol Exp Ther 340:765-780, 2012.

The clozapine metabolite N-desmethylclozapine displays variable activity in diverse functional assays at human dopamine D2 and serotonin 5-HT1A receptors.

N-desmethylclozapine (NDMC or norclozapine) is the major active metabolite of the antipsychotic clozapine in humans. The activity of NDMC differs from clozapine at a number of neurotransmitter receptors, probably influencing the pharmacological effects of clozapine treatment. Here, we tested the properties of NDMC in comparison with clozapine at recombinant human dopamine D(2) and serotonin 5-HT(1A) receptors, using a panel of functional assays implicating diverse signalling pathways. At dopamine D(2) receptors, NDMC as well as clozapine did not display agonist activity in measures of G protein activation by [(35)S]GTPγS binding and in the sensitive Extracellular Signal-Regulated Kinase 1/2 (ERK1/2) phosphorylation assay. In contrast, there were weak partial agonist actions of NDMC (but not of clozapine) for dopamine D(2)-dependent activation of Ca(2+) liberation via coexpressed chimeric Gα(q/o) proteins and for G protein-coupled inward rectifier potassium channel (GIRK) current induction in Xenopus oocytes. Intriguingly, GIRK currents induced by NDMC via dopamine D(2) receptors showed a rapid and transient time course, strikingly different from currents recorded with other receptor agonists. At serotonin 5-HT(1A) receptors, NDMC was a more efficacious partial agonist than clozapine for [(35)S]GTPγS binding, ERK1/2 phosphorylation and GIRK activation. Respective low and moderate partial agonist properties of NDMC at dopamine D(2) and serotonin 5-HT(1A) receptors thus differentiate the metabolite from its parent drug and may contribute to the overall effects of clozapine pharmacotherapy.

Overweight induced by chronic risperidone exposure is correlated with overexpression of the SREBP-1c and FAS genes in mouse liver.

Weight gain and metabolic disturbances, such as dyslipidemia and hyperglycaemia, are common side effects of most antipsychotic drugs, including risperidone. The aim of this study was to investigate the effects of chronic treatment with risperidone on body weight, fat accumulation, liver weight, and hepatic expression of key genes involved in lipid metabolism in female mice. We also addressed the mechanism of risperidone induction of metabolic side effects by exploring its effect on lipid and cholesterol metabolism in primary cultures of rat hepatocytes. Eleven weeks of treatment with long-acting risperidone (12.5 mpk/week) resulted in a significant weight gain associated with an increase of liver and adipose tissue weight. These effects were positively correlated with hepatic mRNA induction of two key genes involved in lipogenesis: sterol regulatory element binding protein-1c (SREBP-1c) and fatty acid synthase (FAS). Furthermore, in line with these in vivo results, risperidone elicited significant inductions of SREBP-1 maturation and FAS mRNA expression in primary cultures of rat hepatocytes associated with an increase of free fatty acid, triacylglycerol, and phospholipid synthesis as assessed by acetate incorporation. The current investigations underscore the usefulness of a mouse model to study the weight gain observed with risperidone treatment in humans. This study shows that risperidone induces similar effects in the liver (in vivo) and in hepatocyte cell cultures (in vitro) on the expression of key genes and/or proteins that control lipid metabolism. This suggests that risperidone could alter lipid metabolism in the liver and induce weight gain in a way that is partly independent of its action on the central nervous system.

Competitive interaction of 5-HT(1A) receptors with G-protein subtypes in CHO cells demonstrated by RNA interference.

Following agonist action, G-protein-coupled receptors may exhibit differential coupling to G-proteins or second messenger pathways, supporting the notion of agonist-directed trafficking. To explore these mechanisms, we have designed and transfected synthetic siRNA duplexes to knockdown different G(α) subunits in Chinese hamster ovary (CHO) cells expressing human (h)5-hydroxytryptamine 1A receptors (CHO-h5-HT(1A)). siRNAs against G(αi2) and G(αi3) transfected alone or in combination caused a large decrease in the corresponding mRNA level (64-80%) and also at the protein level for G(αi3) (60-70%), whereas a non-specific siRNA showed no effect. In membranes of CHO-h5-HT(1A), 5-HT stimulated guanosine-5'-O-(3-[(35)S]thio)-triphosphate ([(35)S]GTPγS) binding was differentially affected by transfection of siRNAs against G(αi) protein, siRNAs against G(αi2) inducing a more important decrease in the efficacy of 5-HT than transfection of siRNAs against G(αi3). The high potency component was abolished after transfection of siRNAs against G(αi3) and the lower potency component was suppressed after transfection of siRNAs against G(αi2). To directly investigate G(αi3) activation we used an antibody-capture/scintillation proximity assay. (+)8-OH-DPAT yielded bell-shaped curves for G(αi3) activation, a response that was abolished after transfection of siRNAs against G(αi3) protein. Interestingly, (+)8-OH-DPAT yielded a sigmoidal response when only G(αi3) protein was expressed. These data suggest that when efficacious agonists attain a high level of occupation of h5-HT(1A) receptors, a change occurs that induces coupling to G(αi2) protein and suppresses signalling through G(αi3) subunits.

Rigid analogues of the α2-adrenergic blocker atipamezole: small changes, big consequences.

We report the discovery of a new family of α(2) adrenergic receptor antagonists derived from atipamezole. Affinities of the compounds at human α(2) and α(1b) receptors as well as their functional activities at hα(2A) receptors were determined in competition binding and G-protein activation assays, respectively. Central α(2) antagonist activities were confirmed in mice after oral administration. Further studies on a selected example: (+)-4-(1a,6-dihydro-1H-cyclopropa[a]inden-6a-yl)-1H-imidazole, (+)-1 (F 14805), were undertaken to probe the potential of the series. On the one hand, (+)-1 increased the release of noradrenaline in mouse frontal cortex following acute systemic administration, the magnitude of this effect being much larger than that obtained with reference agents. On the other, (+)-1 produced minimal cardiovascular effects in intact, anesthetized rat, a surprising outcome that might be explained by its differential action at peripheral and central α(2) receptors. A strategy for improving the therapeutic window of α(2) antagonists is put forward.

[(3)H]-F13640, a novel, selective and high-efficacy serotonin 5-HT(1A) receptor agonist radioligand.

F13640 is a selective and high-efficacy serotonin 5-HT(1A) receptor agonist that demonstrates outstanding analgesic potential in different animal models. Here, we use the radiolabelled compound to further characterise its binding properties at 5-HT(1A) receptors. F13640 was tritium-labelled to 47 and 64 Ci/mmol specific activity and used as radioligand at membrane preparations of CHO cells expressing human (h) 5-HT(1A) receptors. The K (d) of [(3)H]-F13640 was 1.8 nM at h5-HT(1A) receptors as determined from saturation binding experiments. In association time-course experiments, k (obs) of [(3)H]-F13640 was 0.06 min(-1). Dissociation experiments performed in the presence of unlabelled F13640 as competing ligand yielded a k (off) value of 0.05 min(-1), resulting in a calculated K (d) of 1.4 nM. In comparison, [(3)H]-8-OH-DPAT had a k (obs) of 0.50 min(-1), a k (off) of 0.25 min(-1) and a calculated K (d) of 0.37 nM. Surprisingly, [(3)H]-F13640 dissociation kinetics were distinctly slower in the presence of WAY-100635 and spiperone as competing ligands when compared with the agonist competitors, F13640 and (+)8-OH-DPAT. The competitive binding profile of [(3)H]-F13640 with eight chemically diverse 5-HT(1A) receptor agonists and antagonists correlated highly (r = 0.996) with that of [(3)H]-8-OH-DPAT. In conclusion, [(3)H]-F13640 is a potent agonist radioligand at 5-HT(1A) receptors and may be a useful tool in pharmacological studies at native and recombinant 5-HT(1A) receptors. In addition, [(3)H]-F13640 dissociates more slowly from h5-HT(1A) receptors than [(3)H]-8-OH-DPAT, a kinetic property that might be related to its powerful analgesic effects as observed in vivo.

Actions of the prototypical 5-HT1A receptor agonist 8-OH-DPAT at human alpha2-adrenoceptors: (+)8-OH-DPAT, but not (-)8-OH-DPAT is an alpha2B subtype preferential agonist.

8-OH-DPAT [8-hydroxy-2-(di-n-propylamino)tetralin] is the prototypical agonist at serotonin 5-HT1A receptors; however, activity at other targets contributes to the functional effects of the compound as well. We examined the properties of 8-OH-DPAT and its enantiomers at recombinant human (h)alpha2-adrenoceptor subtypes, using a panel of radioligand binding and functional tests. In competition binding experiments using [3H]-RX821002, about 10-fold selectivity of (+)8-OH-DPAT for the halpha2B subtype (pKi about 7) over halpha2A- and halpha2C-adrenoceptors was observed. In contrast, the S(-) enantiomer of 8-OH-DPAT showed similar weak affinities for the three receptor subtypes (pKis<6). The binding affinity of (+)8-OH-DPAT at the halpha2B- and the halpha2A-adrenoceptor was found sensitive to GTPgammaS, a receptor/G protein-uncoupling agent, indicating agonist properties of the drug. Furthermore, using [35S]GTPgammaS binding determination at CHO-halpha2B or CHO-halpha2A cell membranes and G protein coupled inwardly rectifying potassium (GIRK) current recordings in Xenopus oocytes expressing halpha2B, partial agonist activity of (+)8-OH-DPAT at the respective receptors was confirmed in these two different functional assays. Potency of (+)8-OH-DPAT for stimulation of [35S]GTPgammaS incorporation was lower at the halpha2A- than at the halpha2B-adrenoceptor, consistent with binding affinities. Thus, (+)8-OH-DPAT and, as a consequence, racemic (+/-)8-OH-DPAT are partial agonists at halpha2-adrenoceptors with selectivity for the halpha2B subtype, a property that might contribute to the effects of the compound described in native systems.

Antipsychotic drug action on SREBPs-related lipogenesis and cholesterogenesis in primary rat hepatocytes.

The use of some of antipsychotic drugs (APDs) in humans has been hampered by the induction of metabolic disorders such as weight gain, dyslipidemia, and diabetes. In primary rat hepatocytes, we investigated the actions of several APDs on lipid and cholesterol metabolism using [(14)C]acetate incorporation, quantitative reverse transcription-polymerase chain reaction, and western blotting. Clozapine and olanzapine, known to have significant metabolic side effects in man, strongly increased de novo lipid and cholesterol synthesis in rat hepatocytes. Haloperidol, which has less impact in metabolic disorders, enhanced lipogenesis without altering cholesterol production. By contrast, quetiapine, which exhibits few metabolic side effects in man, did not affect lipid and cholesterol synthesis. Interestingly, aripiprazole, which has not yet been reported to induce metabolic disorders in humans, strongly decreases cholesterol synthesis. Furthermore, these inductions of lipid and cholesterol synthesis observed with clozapine and olanzapine were also associated with up-regulation of the transcription factors sterol regulatory element-binding protein (SREBP)-1 and/or SREBP-2 and their associated target genes. Part of the APD-induced metabolic disorders in humans may be due to direct effects on liver metabolism. Our model may also be of interest to assess the action of future drugs on metabolic parameters.

Cellular BRET assay suggests a conformational rearrangement of preformed TrkB/Shc complexes following BDNF-dependent activation.

We developed a cellular Bioluminescent Resonance Energy Transfer (BRET) assay based on the interaction of TrkB fused to Renilla luciferase with the intracellular adaptor protein Shc fused to Enhanced Yellow Fluorescent Protein (EYFP). The TrkB agonist Brain Derived Neurotrophic Factor (BDNF) induced a maximum BRET signal as of 10 min with an EC(50) value of 1.4 nM, similar to the other endogenous agonists NT-3 and NT-4/5, 1.5 nM and 0.34 nM, respectively. Interestingly, measure of the BRET signal with increasing expression of Shc-EYFP, in the presence or absence of BDNF, suggested a conformational change of preformed TrkB/Shc complexes rather than Shc recruitment. Furthermore, the Y516F TrkB mutant deficient to bind Shc as well as the kinase-dead K572R TrkB mutant was unable to respond to BDNF and exhibited a lower basal BRET signal than that of the wild-type TrkB receptor, again suggesting a preformed complex with constitutive activity. The double YY706/707FF TrkB mutant in the kinase activation loop also showed reduced basal activity but surprisingly kept its capacity to enhance BDNF-induced interaction with Shc, though with less efficacy. The Trk selective kinase inhibitors K252a and BMS-9 blocked BDNF-induced BRET signal with similar potency (100-150 nM), the preferential c-Met inhibitor PF-2341006 being one order of magnitude less potent. Remarkably, in the absence of BDNF, K252a and BMS-9 also reduced basal activity to the level of the Y516F TrkB mutant, suggesting that these compounds were able to reduce the TrkB constitutive activity. BRET responses of mutants and to kinase inhibitors thus reveal a complex level of interaction between TrkB and Shc and suggest that this BRET assay could be of great utility to test blockers of TrkB signalling in a physiologically relevant context.

Discovery of novel protease activated receptors 1 antagonists with potent antithrombotic activity in vivo.

Protease activated receptors (PARs) or thrombin receptors constitute a class of G-protein-coupled receptors (GPCRs) implicated in the activation of many physiological mechanisms. Thus, thrombin activates many cell types such as vascular smooth muscle cells, leukocytes, endothelial cells, and platelets via activation of these receptors. In humans, thrombin-induced platelet aggregation is mediated by one subtype of these receptors, termed PAR1. This article describes the discovery of new antagonists of these receptors and more specifically two compounds: 2-[5-oxo-5-(4-pyridin-2-ylpiperazin-1-yl)penta-1,3-dienyl]benzonitrile 36 (F 16618) and 3-(2-chlorophenyl)-1-[4-(4-fluorobenzyl)piperazin-1-yl]propenone 39 (F 16357), obtained after optimization. Both compounds are able to inhibit SFLLR-induced human platelet aggregation and display antithrombotic activity in an arteriovenous shunt model in the rat after iv or oral administration. Furthermore, these compounds are devoid of bleeding side effects often observed with other types of antiplatelet drugs, which constitutes a promising advantage for this new class of antithrombotic agents.