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Iolanda Nistor,
Pierre Lebrun,
Attilio Ceccato,
Frédéric Lecomte,
Ines Slama,
Radu Oprean,
Eduard Badarau,
Fabien Dufour,
Katina Sourou Sylvestre Dossou,
Marianne Fillet, Jean-François Liégeois,
Philippe Hubert,
Eric Rozet
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ABSTRACT: This paper focuses on implementing a design space approach and on the critical process parameters (CPPs) to consider when applying the Quality by Design (QbD) concepts outlined in ICH Q8(R2), Q9 and Q10 to analytical method development and optimization for three chiral compounds developed as modulators of small conductance calcium-activated potassium (SK) channels. In this sense, an HPLC method using a polysaccharide-based stationary phase containing a cellulose tris (4-chloro-3-methylphenylcarbamate) chiral selector in polar organic solvent chromatography mode was considered. The effects of trifluoroacetic acid (TFA) and n-hexane concentration in an acetonitrile (MeCN) mobile phase were investigated under a wide range of column temperatures. Good correlations were found between the observed data obtained after using a central composite design and the expected chromatographic behaviours predicted by applying the design of experiments-design space (DoE-DS) methodology. The critical quality attribute represented here by the separation criterion (S(crit)) allowed assessing the quality of the enantioseparation. Baseline separation for the compounds of interest in an analysis time of less than 20min was possible due to the original and powerful tools applied which facilitated an enhanced method comprehension. Finally, the advantage of the DoE-DS approach resides in granting the possibility to concurrently assess robustness and identify the optimal conditions which are compound dependent.
Journal of pharmaceutical and biomedical analysis 02/2013; 74:273-83. · 2.45 Impact Factor
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ABSTRACT: Valine residues in the pore region of SK2 (V366) and SK3 (V520) were replaced by either an alanine or a phenylalanine to evaluate the impact on the interactions with the allosteric blocker apamin. Unlike TEA which showed high sensitivity to phenylalanine mutated channels, the binding affinity of apamin to the phenylalanine mutants was strongly reduced. In addition, currents from phenylalanine mutants were largely resistant to block by apamin. On the other hand, when the valine residue was replaced by an alanine residue, an increase of the binding affinity and the amount of block by apamin was observed for alanine mutated SK2 channels, but not for mutated SK3 channels. Interestingly, the VA mutation reduced the sensitivity to TEA. In silico data confirmed these experimental results. Therefore, such mutations in the pore region of SK channels show that the three-dimensional structure of the SK tetramers can be disorganized in the outer pore region leading to reduced interaction of apamin with its target.
Biochemical pharmacology 12/2012; · 4.25 Impact Factor
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ABSTRACT: The selectivity for 5-HT(1A) versus D(4) receptors is significantly increased when the basic side chain of WAY-100635 is replaced by a 4-phenylpiperazine (3e) or a 4-phenyl-1,2,3,6-tetrahydropyridine moiety (3i). The 4-phenyl-1,2,3,6-tetrahydropyridine compounds (3i-l) have a higher affinity for 5-HT(1A) receptors than do the corresponding unsubstituted phenylpiperazine analogues (3e-h). Compounds 3e and 3i appear to be selective for 5-HT(1A) receptors over other relevant receptors and still behave as neutral antagonists.
Bioorganic & medicinal chemistry letters 06/2012; 22(14):4550-4. · 2.65 Impact Factor
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ABSTRACT: Various types of ion channels are involved in the control of neuronal activity. Among them, SK channels represent an interesting therapeutic target. Indeed, they underlie medium duration after hyperpolarizations in many types of neurons, thus inhibiting cell excitability. A thorough knowledge of the physiology of these channels and the discovery of non-peptidic selective modulators able to cross the blood-brain barrier are essential in view of developing future drugs for brain diseases such as those related to a dysfunction of dopaminergic and serotonergic systems.
Medecine sciences: M/S 04/2012; 28(4):395-402. · 0.64 Impact Factor
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ABSTRACT: A series of new pyridobenzoxazepine derivatives with various heterocyclic amine side chains were synthesized to explore two main parameters related to the distal basic nitrogen. These compounds were tested for their affinity for dopamine D(2L) and D(4), serotonin 5-HT(1A) and 5-HT(2A), and adrenergic α(2A) receptors in comparison with 5-(4-methylpiperazin-1-yl)-8-chloro-pyrido[2,3-b][1,5]benzoxazepine, JL13 (1), and other diarylazepine derivatives. In terms of multireceptor target strategy, 2 and 5 present the most promising in vitro binding profile. Bulky, polar, and more flexible side chains are not favorable in this context. Compounds 2 and 5 were tested in adult rats to evaluate their long-term effects on dopamine and serotonin receptors density in different brain areas. Similar to 1 and other second-generation antipsychotic drugs, repeated treatment with 2 significantly increased D(1) and D(4) receptors in nucleus accumbens and caudate putamen and D(2) receptors in medial prefrontal cortex and hippocampus, while 5 significantly increased D(2) and D(4) receptors in nucleus accumbens. In addition, 2 increased 5-HT(1A) and decreased 5-HT(2A) receptors in cerebral cortex. In contrast, 5 did not alter levels of any 5-HT receptor subtype in any brain region examined. These results encourage further development of 2 as a novel second-generation antipsychotic agent.
Journal of Medicinal Chemistry 02/2012; 55(4):1572-82. · 4.80 Impact Factor
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ABSTRACT: A structure-activity relationship study of N-methyl-laudanosine, a SK channel blocker, has indicated that the 6,7-dimethoxy group could be successfully replaced by a hydrophobic moiety such as an isopropyl substituent in position 8 of the isoquinoline ring. In the present study, bis-(8-isopropyl-isoquinolinium) derivatives (2a-e) were synthesized and tested for their affinity for cloned SK2 and SK3 channels in comparison with their 6,7-dimethoxy analogues (4a-f). Several ligands were investigated, both in flexible (propyl, butyl and pentyl) and rigid (m- or p-xylyl) series, the m-xylyl derivative (2d) having the best profile in terms of affinity and selectivity for SK3/SK2 channels. Molecular studies showed that the optimal conformation of compound 2d fits well with our SK pharmacophore model.
Bioorganic & medicinal chemistry letters 11/2011; 21(22):6756-9. · 2.65 Impact Factor
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ABSTRACT: Activation of small-conductance calcium (Ca(2+))-dependent potassium (K(Ca)2) channels (herein called "SK") produces membrane hyperpolarization to regulate membrane excitability. Three subtypes (SK1-3) have been cloned and are distributed throughout the nervous system, smooth muscle, and heart. It is difficult to discern the physiological role of individual channel subtypes as most blockers or enhancers do not discriminate between subtypes. The archetypical blocker apamin displays some selectivity between SK channel subtypes, with SK2 being the most sensitive, followed by SK3 and then SK1. Sensitivity of SK1 is species specific, with the human isoform being blocked by the toxin, whereas the rat is not. Mutation studies have identified residues within the outer pore that suggest apamin blocks by an allosteric mechanism. Apamin also uses a residue within the S3-S4 extracellular loop to produce a high-sensitivity block. We have identified that a 3-amino acid motif within this loop regulates the shape of the channel pore. This motif is required for binding and block by apamin, suggesting that a change in pore shape underlies allosteric block. This motif is absent in rat SK1, explaining why it is insensitive to block by apamin. The overlapping distribution of SK channel subtype expression suggests that native heteromeric channels may be common. We show that the S3-S4 loop of one subunit overlaps the outer pore of the adjacent subunit, with apamin interacting with both regions. This arrangement provides a unique binding site for each combination of SK subunits within a coassembled channel that may be targeted to produce blockers specific for heteromeric SK channels.
Proceedings of the National Academy of Sciences 11/2011; 108(45):18494-9. · 9.68 Impact Factor
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ABSTRACT: In a series of carboxamide and sulphonamide alkyl (ethyl to hexyl) piperazine analogues, although the size of the linker is very different, ethyl and hexyl derivatives possess a high affinity for 5-HT(1A) receptors. Docking studies clearly show that hexyl and ethyl compounds favorably interact with the binding site of the active conformation of 5-HT(1A) receptors, thus confirming a possible agonist profile. This activity is effectively detected in electrophysiological experiments in which all four compounds inhibit the activity of rat dorsal raphe serotonergic neurons.
Journal of Chemical Information and Modeling 09/2011; 51(11):2961-6. · 4.68 Impact Factor
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ABSTRACT: Ion-channel function can be modified in various ways. For example, numerous studies have shown that currents through voltage-gated ion channels are affected by pore block or modification of voltage dependence of activation/inactivation. Recent experiments performed on various ion channels show that allosteric modulation is an important mechanism for affecting channel function. For instance, in K(Ca)2 (formerly SK) channels, the prototypic "blocker" apamin prevents conduction by an allosteric mechanism, while TRPV1 channels are prevented from closing by a tarantula toxin, DkTx, through an interaction with residues located away from the selectivity filter. The recent evidence, therefore, suggests that in several ion channels, the region around the outer mouth of the pore is rich in binding sites and could be exploited therapeutically. These discoveries also suggest that the pharmacological vocabulary should be adapted to define these various actions.
ChemBioChem 07/2011; 12(12):1808-12. · 3.94 Impact Factor
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ABSTRACT: The interaction of diazepine analogues like clozapine or olanzapine with D2 receptor was greatly affected by a mixture of HRP/H(2)O(2) known to induce the formation of nitrenium ion. Unlike diazepine derivatives, the oxidative mixture had low impact on the affinity of oxa- and thiazepine derivatives such as loxapine, clothiapine or JL13 for the D2 receptor. Molecular docking simulations revealed a huge difference between the mode of interaction of clozapine nitrenium ion and the parent drug. Electronic and geometric changes of the tricyclic ring system caused by the oxidation appeared to prevent the compound finding the correct binding mode and could therefore explain the difference observed in binding affinities.
Journal of Computer-Aided Molecular Design 02/2011; 25(2):163-9. · 3.39 Impact Factor
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ABSTRACT: Small conductance Ca(2+)-activated K(+) (SK) channels are widely expressed in the brain and underlie medium-duration afterhyperpolarizations (mAHPs) in many types of neurons. It was recently reported that the activation of sigma-1 (sigma(1)) receptors inhibits SK currents in rat hippocampus. Because many interactions between sigma receptors and brain dopaminergic systems have been reported, we set out to examine putative effects of sigma receptor ligands on the SK mediated mAHP in midbrain dopaminergic neurons. We found that 1,3-di-o-tolyl-guanidine (DTG) inhibited the mAHP in a concentration-dependent manner (approximately 60% inhibition at 100 microM), while other sigma receptor agonists (carbetapentane, (+)-SKF10047 and PRE-084) had little effect. Moreover, the effect of DTG was not affected by high concentrations of the sigma(1) receptor antagonist BD 1047. A role for sigma(2) receptors could also be excluded by the lack of effect of the sigma(2) receptor ligand 5-bromo-tetrahydroisoquinolinylbenzamide. These results argue against a coupling of sigma receptors to SK channels in dopaminergic neurons. We next hypothesized that DTG could directly block the channel. This hypothesis was tested in HEK-293 cells which were transiently transfected with rSK2 or hSK3 subunits. DTG inhibited the current flowing through both subtypes with mean IC(50)s approximately 200 microM. This action was also unaffected by BD 1047. Other sigma receptor ligands had little or no effect. We conclude that DTG directly blocks SK channels. This pharmacological action may be important to consider in future experimental settings.
European journal of pharmacology 09/2010; 641(1):23-8. · 2.59 Impact Factor
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ABSTRACT: A series of carboxamide and sulphonamide alkyl(ethyl to hexyl)piperazine analogues were prepared and tested for their affinity to bind to a range of receptors potentially involved in psychiatric disorders. These chemical modifications led us to explore the impact of homology and bioisosteric replacement of the amide group. All of these compounds possessed a high affinity for 5-HT(1A) receptors, irrespective of the size of the linker, the carboxamide derivative with a pentyl linker had the highest affinity for alpha(2A) receptor sites and also a high affinity for 5-HT(1A) and D3 receptors. The sulphonamide analogue with a hexyl linker possessed a high affinity for 5-HT(1A), D4.2 and D3 receptors.
Bioorganic & medicinal chemistry letters 09/2010; 20(17):5199-202. · 2.65 Impact Factor
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ABSTRACT: Activation of small conductance calcium-activated potassium (K(Ca)2) channels can regulate neuronal firing and synaptic plasticity. They are characterized by their high sensitivity to the bee venom toxin apamin, but the mechanism of block is not understood. For example, apamin binds to both K(Ca)2.2 and K(Ca)2.3 with the same high affinity (K(D) approximately 5 pM for both subtypes) but requires significantly higher concentrations to block functional current (IC(50) values of approximately 100 pM and approximately 5 nM, respectively). This suggests that steps beyond binding are needed for channel block to occur. We have combined patch clamp and binding experiments on cell lines with molecular modeling and mutagenesis to gain more insight into the mechanism of action of the toxin. An outer pore histidine residue common to both subtypes was found to be critical for both binding and block by the toxin but not for block by tetraethylammonium (TEA) ions. These data indicated that apamin blocks K(Ca)2 channels by binding to a site distinct from that used by TEA, supported by a finding that the onset of block by apamin was not affected by the presence of TEA. Structural modeling of ligand-channel interaction indicated that TEA binds deep within the channel pore, which contrasted with apamin being modeled to interact with the channel outer pore by utilizing the outer pore histidine residue. This multidisciplinary approach suggested that apamin does not behave as a classical pore blocker but blocks using an allosteric mechanism that is consistent with observed differences between binding affinity and potency of block.
Journal of Biological Chemistry 08/2010; 285(35):27067-77. · 4.77 Impact Factor
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ABSTRACT: Crystal structure of the three stereoisomers of 1,1'-(propane-1,3-diyl)-bis-(6,7-dimethoxy-2-methyl-1,2,3,4-tetrahydroisoquinoline) hydrochloride after resolution by semi-preparative chiral HPLC establishes the absolute configuration and conformation.
European journal of medicinal chemistry 03/2010; 45(7):3240-4. · 3.27 Impact Factor
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ABSTRACT: Midbrain dopaminergic neurons in the substantia nigra, pars compacta and ventral tegmental area are critically important in many physiological functions. These neurons exhibit firing patterns that include tonic slow pacemaking, irregular firing and bursting, and the amount of dopamine that is present in the synaptic cleft is much increased during bursting. The mechanisms responsible for the switch between these spiking patterns remain unclear. Using both in-vivo recordings combined with microiontophoretic or intraperitoneal drug applications and in-vitro experiments, we have found that M-type channels, which are present in midbrain dopaminergic cells, modulate the firing during bursting without affecting the background low-frequency pacemaker firing. Thus, a selective blocker of these channels, 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride, specifically potentiated burst firing. Computer modeling of the dopamine neuron confirmed the possibility of a differential influence of M-type channels on excitability during various firing patterns. Therefore, these channels may provide a novel target for the treatment of dopamine-related diseases, including Parkinson's disease and drug addiction. Moreover, our results demonstrate that the influence of M-type channels on the excitability of these slow pacemaker neurons is conditional upon their firing pattern.
European Journal of Neuroscience 02/2010; 31(5):827-35. · 3.63 Impact Factor
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ABSTRACT: The main feature of many drugs having a 5-HT1A affinity is the presence of an arylpiperazine moiety. Indeed, the protonated nitrogen and the aromatic ring of the arylpiperazine compounds are considered crucial for the interaction with the receptor. However, the replacement of the piperazine moiety by a 1,2,3,6-tetrahydropyridine ring in 4-arylpiperazine-ethyl carboxamide derivatives was recently shown to be highly favourable for 5-HT1A affinity. In order to better understand the favourable effect of this chemical modification, we performed a conformational analysis of these compounds mainly based on the position of the phenyl ring relative to the piperazine and tetrahydropyridine moiety. In the piperazine compounds, the phenyl ring preferentially adopts a perpendicular orientation, whereas an almost planar orientation seems to be the most favourable conformation for the tetrahydropyridine compounds. Therefore, this conformational difference appears as a key for a better interaction with the receptor binding site. This result will serve for the designing high-affinity 5-HT1A ligands.
Bioorganic & Medicinal Chemistry Letters - BIOORG MEDICINAL CHEM LETTER. 01/2010; 20(3):1118-1123.
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ABSTRACT: A series of carboxamide and sulphonamide alkyl(ethyl to hexyl)piperazine analogues were prepared and tested for their affinity to bind to a range of receptors potentially involved in psychiatric disorders. These chemical modifications led us to explore the impact of homology and bioisosteric replacement of the amide group. All of these compounds possessed a high affinity for 5-HT1A receptors, irrespective of the size of the linker, the carboxamide derivative with a pentyl linker had the highest affinity for α2A receptor sites and also a high affinity for 5-HT1A and D3 receptors. The sulphonamide analogue with a hexyl linker possessed a high affinity for 5-HT1A, D4.2 and D3 receptors.
Bioorganic & Medicinal Chemistry Letters - BIOORG MEDICINAL CHEM LETTER. 01/2010; 20(17):5199-5202.
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ABSTRACT: The main feature of many drugs having a 5-HT(1A) affinity is the presence of an arylpiperazine moiety. Indeed, the protonated nitrogen and the aromatic ring of the arylpiperazine compounds are considered crucial for the interaction with the receptor. However, the replacement of the piperazine moiety by a 1,2,3,6-tetrahydropyridine ring in 4-arylpiperazine-ethyl carboxamide derivatives was recently shown to be highly favourable for 5-HT(1A) affinity. In order to better understand the favourable effect of this chemical modification, we performed a conformational analysis of these compounds mainly based on the position of the phenyl ring relative to the piperazine and tetrahydropyridine moiety. In the piperazine compounds, the phenyl ring preferentially adopts a perpendicular orientation, whereas an almost planar orientation seems to be the most favourable conformation for the tetrahydropyridine compounds. Therefore, this conformational difference appears as a key for a better interaction with the receptor binding site. This result will serve for the designing high-affinity 5-HT(1A) ligands.
Bioorganic & medicinal chemistry letters 12/2009; 20(3):1118-23. · 2.65 Impact Factor
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ABSTRACT: Previous in vivo studies have shown that blockade of small-conductance Ca(2+)-activated potassium (SK) channels enhances burst firing in dopaminergic neurons. As bursting has been found to be physiologically relevant for the synaptic release of serotonin (5-HT), we investigated the possible role of SK channels in the control of this firing pattern in 5-HT neurons of the dorsal raphe nucleus. In these cells, bursts are usually composed of doublets consisting of action potentials separated by a small interval (< 20 ms). Both in vivo and in vitro extracellular recordings were performed, using anesthetized rats and rat brain slices, respectively. In vivo, the specific SK blocker UCL 1684 (200 microm) iontophoresed onto presumed 5-HT neurons significantly increased the production of bursts in 13 out of 25 cells. Furthermore, the effect of UCL 1684 persisted in the presence of both the GABA(A) antagonist SR 95531 (10 mm) and the GABA(B) antagonist CGP 35348 (10 mm), whereas these agents by themselves did not significantly influence the neuronal firing pattern. In vitro, bath superfusion of the SK channel blocker apamin (300 nm) induced bursting in only three out of 18 neurons, although it increased the coefficient of variation of the interspike intervals in all the other cells. Our results suggest that SK channel blockade promotes bursting activity in 5-HT neurons via a direct action. An input which is present only in vivo seems to be important for the induction of this firing pattern in these cells.
European Journal of Neuroscience 10/2008; 28(6):1108-15. · 3.63 Impact Factor
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ABSTRACT: So far, small conductance Ca(2+)-activated K(+) channel (SK) blockers mostly consist of quaternary ammonium derivatives or peptides. Due to their physicochemical properties, these blockers are not suitable to study the physiological roles of SK channels in the central nervous system in vivo. Herein, we report the discovery of a chiral bis-tertiary amine with SK blocking properties from chemical modulation of laudanosine. AG525E1 has an affinity for SK channels (K(i)=293nM) approximately 100-fold higher than the tertiary compound laudanosine (K(i) approximately 30muM) and similar to the charged compound dequalinium (K(i)=221nM). AG525E1 equipotently blocks SK1, SK2 and SK3 currents in transfected cell lines. Because of its basic and lipophilic properties, it can reach central SK targets.
Bioorganic & medicinal chemistry letters 07/2008; 18(11):3440-5. · 2.65 Impact Factor
