Ballerup, Denmark
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Vinkers C.H.,University Utrecht | van Oorschot R.,University Utrecht | Nielsen E.O.,Neurosearch | Cook J.M.,University of Wisconsin - Milwaukee | And 7 more authors.
PLoS ONE | Year: 2012

Background: Within the GABAA-receptor field, two important questions are what molecular mechanisms underlie benzodiazepine tolerance, and whether tolerance can be ascribed to certain GABAA-receptor subtypes. Methods: We investigated tolerance to acute anxiolytic, hypothermic and sedative effects of diazepam in mice exposed for 28-days to non-selective/selective GABAA-receptor positive allosteric modulators: diazepam (non-selective), bretazenil (partial non-selective), zolpidem (α1 selective) and TPA023 (α2/3 selective). In-vivo binding studies with [3H]flumazenil confirmed compounds occupied CNS GABAA receptors. Results: Chronic diazepam treatment resulted in tolerance to diazepam's acute anxiolytic, hypothermic and sedative effects. In mice treated chronically with bretazenil, tolerance to diazepam's anxiolytic and hypothermic, but not sedative, effects was seen. Chronic zolpidem treatment resulted in tolerance to diazepam's hypothermic effect, but partial anxiolytic tolerance and no sedative tolerance. Chronic TPA023 treatment did not result in tolerance to diazepam's hypothermic, anxiolytic or sedative effects. Conclusions: Our data indicate that: (i) GABAA-α2/α3 subtype selective drugs might not induce tolerance; (ii) in rodents quantitative and temporal variations in tolerance development occur dependent on the endpoint assessed, consistent with clinical experience with benzodiazepines (e.g., differential tolerance to antiepileptic and anxiolytic actions); (iii) tolerance to diazepam's sedative actions needs concomitant activation of GABAA-α1/GABAA-α5 receptors. Regarding mechanism, in-situ hybridization studies indicated no gross changes in expression levels of GABAA α1, α2 or α5 subunit mRNA in hippocampus or cortex. Since selective chronic activation of either GABAA α2, or α3 receptors does not engender tolerance development, subtype-selective GABAA drugs might constitute a promising class of novel drugs. © 2012 Vinkers et al.


Ussing C.A.,Copenhagen University | Hansen C.P.,Copenhagen University | Hansen C.P.,Control Data | Petersen J.G.,Copenhagen University | And 10 more authors.
Journal of Medicinal Chemistry | Year: 2013

In our search for selective agonists for the α4β 2 subtype of the nicotinic acetylcholine receptors (nAChRs), we have synthesized and characterized a series of novel heterocyclic analogues of 3-(dimethylamino)butyl dimethylcarbamate (DMABC, 4). All new heterocyclic analogues, especially N,N-dimethyl-4-(1-methyl-1H-imidazol-2-yloxy)butan-2-amine (7), showed an improved binding selectivity profile in favor of α4β2 over other nAChR subtypes, primarily due to impaired binding at β4 containing receptors. This observation can be rationalized based on cocrystal structures of (R)-4 and (R)-7 bound to acetylcholine binding protein from Lymnaea stagnalis. Functional characterization at both (α4)2(β2) 3 and (α4)3(β2) 2 receptors using two-electrode voltage clamp techniques in Xenopus laevis oocytes indicates that the investigated compounds interact differently with the two receptor stoichiometries. Compound 7 is an efficacious agonist at both α4-β2 and α4- α4 binding sites, while the close analogue N,N-dimethyl-4-(1,4- dimethyl-1H-imidazol-2-yloxy)butan-2-amine (9) primarily activates via α4-β2 binding sites. The results suggest that it may be possible to rationally design compounds with specific stoichiometry preferences. © 2012 American Chemical Society.


Krintel C.,Copenhagen University | Harpsoe K.,Copenhagen University | Harpsoe K.,Novo Nordisk AS | Zachariassen L.G.,Copenhagen University | And 7 more authors.
Acta Crystallographica Section D: Biological Crystallography | Year: 2013

Positive allosteric modulators of the ionotropic glutamate receptor A2 (GluA2) can serve as lead compounds for the development of cognitive enhancers. Several benzamide-type (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor modulators such as aniracetam, CX516 and CX614 have been shown to inhibit the deactivation of AMPA receptors with a less pronounced effect on desensitization. Despite CX516 being an extensively investigated AMPA receptor modulator and one of the few clinically evaluated compounds, the binding mode of CX516 to AMPA receptors has not been reported. Here, the structures of a GluA2 ligand-binding domain mutant in complex with CX516 and the 3-methylpiperidine analogue of CX516 (Me-CX516) are reported. The structures show that the binding modes of CX516 and Me-CX516 are similar to those of aniracetam and CX614 and that there is limited space for substitution at the piperidine ring of CX516. The results therefore support that CX516, like aniracetam and CX614, modulates deactivation of AMPA receptors. © 2013 International Union of Crystallography Printed in Singapore-all rights reserved.


Olsen J.A.,Neurosearch | Olsen J.A.,Copenhagen University | Kastrup J.S.,Copenhagen University | Peters D.,Neurosearch | And 4 more authors.
Journal of Biological Chemistry | Year: 2013

Positive allosteric modulators (PAMs) of α4α2 nicotinic acetylcholine receptors have the potential to improve cognitive function and alleviate pain. However, only a few selective PAMs of α4β2 receptors have been described limiting both pharmacological understanding and drug-discovery efforts. Here, we describe a novel selective PAM of α4β2 receptors, NS206, and compare with a previously reported PAM, NS9283. Using two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes, NS206 was observed to positively modulate acetylcholine (ACh)-evoked currents at both known α4β2 stoichiometries (2α:3β and 3α:2β). In the presence of NS206, peak current amplitudes surpassed those of maximal efficacious ACh stimulations (Emax(ACh)) with no or limited effects at potencies and current waveforms (as inspected visually). This pharmacological action contrasted with that of NS9283, which only modulated the 3α:2β receptor and acted by left shifting the ACh concentration-response relationship. Interestingly, the two modulators can act simultaneously in an additive manner at 3α:2β receptors, which results in current levels exceeding Emax(ACh) and a left-shifted ACh concentration-response relationship. Through use of chimeric and point-mutated receptors, the binding site of NS206 was linked to the α4-subunit transmembrane domain, whereas binding of NS9283 was shown to be associated with the αα-interface in 3α:2β receptors. Collectively, these data demonstrate the existence of two distinct modulatory sites in α4β2 receptors with unique pharmacological attributes that can act additively. Several allosteric sites have been identified within the family of Cys-loop receptors and with the present data, a detailed picture of allosteric modulatory mechanisms of these important receptors is emerging. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.


Olsen J.A.,Copenhagen University | Olsen J.A.,Neurosearch | Balle T.,University of Sydney | Gajhede M.,Copenhagen University | And 3 more authors.
PLoS ONE | Year: 2014

Despite extensive studies on nicotinic acetylcholine receptors (nAChRs) and homologues, details of acetylcholine binding are not completely resolved. Here, we report the crystal structure of acetylcholine bound to the receptor homologue acetylcholine binding protein from Lymnaea stagnalis. This is the first structure of acetylcholine in a binding pocket containing all five aromatic residues conserved in all mammalian nAChRs. The ligand-protein interactions are characterized by contacts to the aromatic box formed primarily by residues on the principal side of the intersubunit binding interface (residues Tyr89, Trp143 and Tyr185). Besides these interactions on the principal side, we observe a cation-π interaction between acetylcholine and Trp53 on the complementary side and a water-mediated hydrogen bond from acetylcholine to backbone atoms of Leu102 and Met114, both of importance for anchoring acetylcholine to the complementary side. To further study the role of Trp53, we mutated the corresponding tryptophan in the two different acetylcholine-binding interfaces of the widespread α4β2 nAChR, i.e. the interfaces α4(+)β2(2) and α4(+)α4(2). Mutation to alanine (W82A on the b2 subunit or W88A on the a4 subunit) significantly altered the response to acetylcholine measured by oocyte voltageclamp electrophysiology in both interfaces. This shows that the conserved tryptophan residue is important for the effects of ACh at α4β2 nAChRs, as also indicated by the crystal structure. The results add important details to the understanding of how this neurotransmitter exerts its action and improves the foundation for rational drug design targeting these receptors. © 2014 Olsen et al.


Grant
Agency: Cordis | Branch: FP7 | Program: CP-IP | Phase: HEALTH-2011.2.2.1-2 | Award Amount: 24.91M | Year: 2012

The goal of this proposal (INMiND) is to carry out collaborative research on molecular mechanisms that link neuroinflammation with neurodegeneration in order to identify novel biological targets for activated microglia, which may serve for both diagnostic and therapeutic purposes, and to translate this knowledge into the clinic. The general objectives of INMiND are: (i) to identify novel mechanisms of regulation and function of microglia under various conditions (inflammatory stimuli; neurodegenerative and -regenerative model systems); (ii) to identify and implement new targets for activated microglia, which may serve for diagnostic (imaging) and therapeutic purposes; (iii) to design new molecular probes (tracers) for these novel targets and to implement and validate them in in vivo model systems and patients; (iv) to image and quantify modulated microglia activity in patients undergoing immune therapy for cognitive impairment and relate findings to clinical outcome. Within INMiND we bring together a group of excellent scientists with a proven background in efficiently accomplishing common scientific goals (FP6 project DiMI, www.dimi.eu), who belong to highly complementary fields of research (from genome-oriented to imaging scientists and clinicians), and who are dedicated to formulate novel image-guided therapeutic strategies for neuroinflammation related neurodegenerative diseases. The strength of this proposal is that, across Europe, it will coordinate research and training activities related to neuroinflammation, neurodegeneration/-regeneration and imaging with special emphasis on translating basic mechanisms into clinical applications that will provide health benefits for our aging population. With its intellectual excellence and its crucial mass the INMiND consortium will play a major role in the European Research Area and will gain European leadership in the creation of new image-guided therapy paradigms in patients with neurodegenerative diseases.


Rotering S.,Helmholtz Center Dresden | Scheunemann M.,Helmholtz Center Dresden | Fischer S.,Helmholtz Center Dresden | Hiller A.,Helmholtz Center Dresden | And 4 more authors.
Bioorganic and Medicinal Chemistry | Year: 2013

[18F]NS14490, a new potential radiotracer for neuroimaging of α7 nicotinic acetylcholine receptors (α7 nAChRs), was synthesized and evaluated in vitro and in vivo. Radioligand binding studies using [ 3H]methyllycaconitine and NS14490 as competitor showed a good target affinity (Ki,α7 = 2.5 nM) and a high selectivity towards other nAChRs. Radiosynthesis of [18F]NS14490 was performed by two different labelling procedures: a two-step synthesis using a prosthetic group, which led to 7% labelling yield, and the convenient direct nucleophilic substitution of the corresponding tosylate precursor, which resulted in 70% labelling yield. After optimisation of the isolation, purification and formulation process, biodistribution studies were performed in CD-1 mice. The brain uptake of [ 18F]NS14490 was comparably low (0.16% ID g-1 wet weight at 5 min p.i.). The radiotracer showed a high metabolic stability in plasma and brain. Also, the target specificity was proven by pre-administration of a highly affine α7 ligand providing a rationale basis for further in vivo evaluation. © 2013 Elsevier Ltd. All rights reserved.


Rasmussen A.H.,Aniona ApS | Strobaek D.,Aniona ApS | Dyhring T.,Aniona ApS | Jensen M.L.,Aniona ApS | And 5 more authors.
Brain Research | Year: 2014

Nicotinic acetylcholine receptors (nAChR's) containing the α6 subunit (α6â are putative drug targets of relevance to Parkinson's disease and nicotine addiction. However, heterologous expression of α6â receptors has proven challenging which has stifled drug discovery efforts. Here, we investigate potential new avenues for achieving functional α6 receptor expression. Combinations of chimeric and mutated α6, β2 and β3 subunits were co-expressed in the human HEK293 cell line and receptor expression was assessed using Ca2+-imaging (FLIPR™) and whole-cell patch-clamp electrophysiology. Transient transfections of a chimeric α6/α3 subunit construct in combination with β2 and β3 V9'S gave rise to significant acetylcholine-evoked whole-cell currents. Increasing the β3V9'S:β2:α6/α3 cDNA ratio, resulted in a significantly higher fraction of cells with robust current levels. Using an excess of wild-type β3, significant functional expression of α6/α3β2β3 was also demonstrated. Comparing the acetylcholine concentration-response relationship of α6/ α3β2β3V9'S to that of α6/α3β2β3 revealed the β3 point mutation to result in decreased current decay rate and increased ACh agonist potency. Ca2+-imaging experiments showed preservation of basic α6 receptor pharmacology. Our results establish that α6/α3β2β3V9'S replicate several basic features of native α6 receptors but also highlight several caveats associated with using this construct and may therefore provide guidance for future drug hunting efforts. © 2013 Elsevier B.V.


Grunnet M.,Lundbeck | Strobaek D.,Aniona Aps | Hougaard C.,Aniona Aps | Christophersen P.,Aniona Aps
European Journal of Pharmacology | Year: 2014

The Kv7 channels, a family of voltage-dependent K+ channels (Kv7.1-Kv7.5), have gained much attention in drug discovery especially because four members are genetically linked to diseases. For disorders of the CNS focus was originally on epilepsy and pain, but it is becoming increasingly evident that Kv7 channels can also be valid targets for psychiatric disorders, such as anxiety and mania. The common denominator is probably neuronal hyperexcitability in different brain areas, which can be successfully attenuated by pharmacological increment of K v7 channel activity. This perspective attempts to review the current status and challenges for CNS drug discovery based on Kv7 channels as targets for neurological and psychiatric indications with special focus on selectivity and mode-of-actions. © 2014 Elsevier B.V.

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