Delphine S. Dupuis

Activation of KCNQ5 channels stably expressed in HEK293 cells by BMS-204352.

The novel anti-ischemic compound, BMS-204352 ((3S)-(+)-(5-chloro-2-methoxyphenyl)-1,3-dihydro-3-fluoro-6-(trifluoromethyl)-2H-indol-2-one)), strongly activates the voltage-gated K+ channel KCNQ5 in a concentration-dependent manner with an EC50 of 2.4 microM. At 10 microM, BMS-204352 increased the steady state current at -30 mV by 12-fold, in contrast to the 2-fold increase observed for the other KCNQ channels [Schrøder et al., 2001]. Retigabine ((D-23129; N-(2-amino-4-(4-fluorobenzylamino)-phenyl) carbamic acid ethyl ester) induced a smaller, yet qualitatively similar effect on KCNQ5. Furthermore, BMS-204352 (10 microM) did not significantly shift the KCNQ5 activation curves (threshold and potential for half-activation, V1/2), as observed for the other KCNQ channels. In the presence of BMS-204352, the activation and deactivation kinetics of the KCNQ5 currents were slowed as the slow activation time constant increased up to 10-fold. The M-current blockers, linopirdine (DuP 996; 3,3-bis(4-pyridinylmethyl)-1-phenylindolin-2-one) and XE991 (10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone), inhibited the activation of the KCNQ5 channel induced by the BMS-204352. Thus, BMS-204352 appears to be an efficacious KCNQ channels activator, and the pharmacological properties of the compound on the KCNQ5 channel seems to be different from what has been obtained on the other KCNQ channels.

An overview on functional receptor autoradiography using [35S]GTPγS

Abstract [35S]GTPγS binding autoradiography is a novel method to study the distribution and function of neurotransmitter receptors in tissue sections. This technique unifies the advantages of receptor-autoradiography and [35S]GTPγS binding, providing anatomical and functional information at the same time. Due to these two main features, it can also be called ‘functional autoradiography’. [35S]GTPγS binding has long been used to study the first step of the intracellular signaling pathway, but until the mid 1990s it has only been performed on cell membrane extracts. Functional autoradiography evolved from this biochemical assay and ligand autoradiography, and is based on the increase in guanine nucleotide exchange at G-proteins upon agonist stimulation. With the technique, activation of G-protein-coupled receptors upon agonist binding can be detected, and, at the same time, the location of activated receptors can also be visualized. Thus only those presumably active G-protein-coupled receptors are visualized that can be involved in signal transduction. In the past 5 years the technique has become more and more frequently used in neuroscience, and it has been adapted to several receptors in different species, including also the human brain. [35S]GTPγS binding autoradiography can be used to describe the distribution of G-protein-coupled receptors. Some inferences on their coupling efficiency can also be drawn. Besides the localization of ligand binding sites, it provides information on the action of the ligand on the receptor: agonists, antagonists, and inverse agonists can clearly be distinguished. Moreover, [35S]GTPγS binding autoradiography can successfully be combined with other in vitro assays, like receptor autoradiography, in situ hybridization histochemistry, or even with biochemical and electrophysiological experiments. This review presents an overview on the history and the development of this technique. Its main advantages and limitations are summarized, together with a few basic technical questions. A number of experiments performed with [35S]GTPγS binding autoradiography so far, and some possible applications for the future, are also reviewed.

Basolateral localisation of KCNQ1 potassium channels in MDCK cells: molecular identification of an N-terminal targeting motif

KCNQ1 potassium channels are expressed in many epithelial tissues as well as in the heart. In epithelia KCNQ1 channels play an important role in salt and water transport and the channel has been reported to be located apically in some cell types and basolaterally in others. Here we show that KCNQ1 channels are located basolaterally when expressed in polarised MDCK cells. The basolateral localisation of KCNQ1 is not affected by co-expression of any of the five KCNE β-subunits. We characterise two independent basolateral sorting signals present in the N-terminal tail of KCNQ1. Mutation of the tyrosine residue at position 51 resulted in a non-polarized steady-state distribution of the channel. The importance of tyrosine 51 in basolateral localisation was emphasized by the fact that a short peptide comprising this tyrosine was able to redirect the p75 neurotrophin receptor, an otherwise apically located protein, to the basolateral plasma membrane. Furthermore, a di-leucine-like motif at residues 38-40 (LEL) was found to affect the basolateral localisation of KCNQ1. Mutation of these two leucines resulted in a primarily intracellular localisation of the channel.

Actions of novel agonists, antagonists and antipsychotic agents at recombinant rat 5-HT6 receptors: A comparative study of coupling to Gαs

Though 5-HT6 receptors are targets for the treatment of schizophrenia and other psychiatric disorders, the influence of drugs upon signal transduction has not been extensively characterized. Herein, we employed a Scintillation Proximity Assay (SPA)/antibody-immunocapture procedure of coupling to G alpha s to evaluate the interaction of a broad range of novel agonists, antagonists and antipsychotics at rat 5-HT(6) receptors stably expressed in HEK293 cells. Serotonin (pEC(50), 7.7) increased [35S]GTP gamma S binding to G alpha s by ca 2-fold without affecting binding to Gi/o or Gq. LSD (9.2), 5-MeODMT (7.9), 5-CT (7.0) and tryptamine (6.1) were likewise full agonists. In contrast, the novel sulfonyl derivatives, WAY181,187 (9.1) and WAY208,466 (7.8), behaved as partial agonists and attenuated the actions of 5-HT. SB271,046 and SB258,585 abolished activation of G alpha s by 5-HT with pKb values of 10.2 and 9.9, respectively, actions mimicked by the novel antagonist, SB399,885 (10.9). SB271,046 likewise blocked partial agonist properties of WAY181,187 and WAY208,466 with pKb values of 9.8 and 9.0, respectively. 5-HT-stimulated [35S]GTP gamma S binding to G alpha s was antagonised by various antipsychotics including olanzapine (7.8), asenapine (9.1) and SB737,050 (7.8), whereas aripiprazole and bifeprunox were inactive. Further, antagonist properties of clozapine (8.0) were mimicked by its major metabolite, N-desmethylclozapine (7.9). In conclusion, the novel ligands, WAY208,466 and WAY181,187, behaved as partial agonists at 5-HT6 receptors coupled to G alpha s, while SB399,885 was a potent antagonist. Though 5-HT6 receptor blockade is not indispensable for therapeutic efficacy, it may well play a role in the functional actions of certain antipsychotic agents.

Autoradiography of Serotonin 5-HT1A Receptor-Activated G Proteins in Guinea Pig Brain Sections by Agonist-Stimulated [35S]GTPγS Binding

Abstract: G protein activation mediated by serotonin 5‐HT1A and 5‐HT1B/D receptors in guinea pig brain was investigated by using quantitative autoradiography of agonist‐stimulated [35S]GTPγS binding to brain sections. [35S]GTPγS binding was stimulated by the mixed 5‐HT1A/5‐HT1B/D agonist L694247 in brain structures enriched in 5‐HT1A binding sites, i.e., hippocampus (+140 ± 14%), dorsal raphe (+70 ± 8%), lateral septum (+52 ± 12%), cingulate (+36 ± 8%), and entorhinal cortex (+34 ± 5%). L694247 caused little or no stimulation of [35S]GTPγS binding in brain regions with high densities of 5‐HT1B/D binding sites (e.g., substantia nigra, striatum, central gray, and dorsal subiculum). The [35S]GTPγS binding response was antagonized by WAY100635 (10 µM) and methiothepin (10 µM). In contrast, the 5‐HT1B inverse agonist SB224289 (10 µM) did not affect the L694247‐mediated [35S]GTPγS binding response, and the mixed 5‐HT1B/D antagonist GR127935 (10 µM) yielded a partial blockade. The distribution pattern of the [35S]GTPγS binding response and the antagonist profile suggest the L694247‐mediated response in guinea pig brain to be mediated by 5‐HT1A receptors. In addition to L694247, 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin, and flesinoxan also stimulated [35S]GTPγS binding; their maximal responses varied between 46 and 52% compared with L694247, irrespective of the brain structure being considered. Sumatriptan, rizatriptan, and zolmitriptan (10 µM) stimulated [35S]GTPγS binding in the hippocampus by 20–50%. Naratriptan, CP122638, and dihydroergotamine stimulated [35S]GTPγS binding to a similar level as L694247 in hippocampus, lateral septum, and dorsal raphe. It appears that under the present experimental conditions, G protein activation through 5‐HT1A but not 5‐HT1B/D receptors can be measured in guinea pig brain sections.

Autoradiographic studies of 5-HT1A-receptor-stimulated [35S]GTPγS-binding responses in the human and monkey brain

G‐protein activation mediated by serotonin 5‐HT1A receptors in human and monkey brain was investigated by using quantitative autoradiography of agonist‐stimulated [35S]GTPγS binding to whole‐hemisphere brain sections. [35S]GTPγS binding was stimulated by the mixed 5‐HT1A/1B/1D agonist L 694247 (10 μm) in human brain regions enriched in 5‐HT1A binding sites [e.g. hippocampus (132–137%), superficial layers of the neocortex (37–61%), and cingulate and entorhinal cortex (34 and 32%, respectively)]. L 694247 caused virtually no stimulation in regions with 5‐HT1B/1D receptors, such as substantia nigra, caudate nucleus and putamen. Similar results were obtained with monkey brain sections. The L 694247‐mediated [35S]GTPγS‐binding responses in human and monkey brain sections were antagonized by the selective, silent 5‐HT1A antagonist WAY 100635 (10 μm). The 5‐HT1B inverse agonist SB 224289 (10 μm) did not affect the [35S]GTPγS‐binding response of L 694247. The distribution pattern of the [35S]GTPγS‐binding response and the antagonist profile suggest the L 694247‐induced response in human and monkey brain is mediated by 5‐HT1A receptors. A weak stimulation of [35S]GTPγS binding was also observed in human hippocampus with either 10 μm 8‐OH‐DPAT (25 ± 4%) or naratriptan (42 ± 2%) compared with that obtained with L 694247. In conclusion, G‐protein activation by 5‐HT1A receptors can be measured in human and monkey brain sections. L 694247 appears to possess higher efficacy at 5‐HT1A receptors compared with 8‐OH‐DPAT and naratriptan.

Modulation of 5-HT1A receptor activation by its interaction with wild-type and mutant Gαi3 proteins

Abstract Constitutive and agonist-dependent activation of the recombinant human 5-HT 1A receptor (RC: 2.1.5HT.01A) was investigated by co-expression with a rat G αi3 protein in Cos-7 cells. The interaction between the 5-HT 1A receptor and rat G αi3 protein was modulated by substitution of the G αi3 protein site for pertussis toxin-catalysed ADP-ribosylation (cysteine 351 ) by each of the natural amino acids. Enhanced basal [ 35 S]GTPγS binding responses (+24 to +189%) were observed with the mutant G αi3 proteins containing at position 351 either a histidine, glutamine, serine, tyrosine or a nonpolar amino acid with the exception of a proline. With each of these mutant G αi3 proteins, spiperone (10 μM), but not WAY 100635 (10 μM), reduced (−22 to −60%, p 35 S]GTPγS binding response. 5-HT (10 μM)-mediated [ 35 S]GTPγS binding responses attained for some of the mutant G αi3 Cys 351 proteins (Phe, Met, Val and Ala) more than 300% of that obtained with the wt G αi3 protein. Similar results were also obtained with the prototypical 5-HT 1A agonist 8-OH-DPAT and the partial agonist (−)-pindolol. Fusion proteins assembled from the 5-HT 1A receptor and either the wt G αi3 Cys 351 , mutant G αi3 Cys 351 Gly or G αi3 Cys 351 Ile protein displayed similar observations for these ligands as obtained by co-expression of the 5-HT 1A receptor with each of these G αi3 proteins. Both the degree of 5-HT 1A receptor activation by 8-OH-DPAT and (−)-pindolol, and its inhibition by spiperone, strongly correlate ( r 2 : 0.78–0.81) with the octanol/water partition coefficients of the mutated amino acid at position 351 of the G αi3 protein. The present data also suggest the wt G αi3 protein does not result in maximal activation of the 5-HT 1A receptor by the agonists being investigated.

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-HT2C receptors and α2-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 (pKi, 8.2) for constitutively active human 5-HT2CINI (h5-HT2CINI) receptors, behaving as an inverse agonist in reducing basal Gαq activation, [3H]inositol-phosphate production, and the spontaneous association of h5-HT2CINI-Renilla luciferase receptors with β-arrestin2-yellow fluorescent protein. Furthermore, upon 18-h pretreatment, S32212 enhanced the plasma membrane expression of h5-HT2CINI 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 Ca2+ mobilization in mouse cortical neurones. In vivo, S32212 blunted the inhibitory influence of the 5-HT2C agonist 2-(3-chlorobenzyloxy)-6-(1-piperazinyl)pyrazine (CP809,101) on ventrotegmental dopaminergic neurones. S32212 also blocked 5-HT-induced Gαq and phospholipase C activation at the h5-HT2A and, less potently, h5-HT2B receptors and suppressed the discriminative stimulus properties of the 5-HT2A agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane in rats. S32212 manifested marked affinity for human α2A- (pKi 7.2), α2B- (pKi 8.2), and α2C- (pKi 7.4) adrenoceptors, at which it abolished noradrenaline-induced recruitment of Gαi3, Gαo, adenylyl cyclase, and extracellular-regulated kinase1/2. Moreover, S32212 dose-dependently abolished the discriminative stimulus effects of the α2-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 α1A-adrenoceptors, histamine H1 receptors, and muscarinic M1 receptors. In conclusion, S32212 behaves as an inverse agonist at h5-HT2C receptors and as an antagonist at human α2-adrenoceptors (and h5-HT2A 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.

Modulations of the amide function of the preferential dopamine D3 agonist (R,R)-S32504: Improvements of affinity and selectivity for D3 versus D2 receptors

Starting with the preferential dopamine (DA) D(3) agonist S32504, we prepared two series of derivatives of the general formula I-A and I-B, in an effort to improve both potency and selectivity. For the first set of derivatives, where the primary amide function of S32504 was replaced by either secondary and tertiary amide or ester, acid, nitrile and ketone, no improvement was obtained. Conversely, when the primary amide function was integrated in a lactam ring, an enhancement of affinity and selectivity was attained for the five-membered ring lactam but also for its five-membered ring lactone analogue.

Design and synthesis of novel N-sulfonyl-2-indoles that behave as 5-HT6 receptor ligands with significant selectivity for D3 over D2 receptors

All clinically-used antipsychotics display similar affinity for both D2 (D2R) and D3 (D3R) receptors, and they likewise act as 5-HT2A receptor antagonists. They provide therapeutic benefit for positive symptoms, but no marked or consistent improvement in neurocognitive, social cognitive or negative symptoms. Since blockade of D3 and 5-HT6 (5-HT6R) receptors enhances neurocognition and social cognition, and potentially improves negative symptoms, a promising approach for improved treatment for schizophrenia would be to develop drugs that preferentially act at D3R versus D2R and likewise recognize 5-HT6R. Starting from the high affinity 5-HT6R ligands I and II, we identified compounds 11a and 14b that behave as 5-HT6R ligands with significant selectivity for D3R over D2R.