Jensen M.L.,Neurosearch |
Wafford K.A.,Eli Lilly and Company |
Brown A.R.,University of Dundee |
Belelli D.,University of Dundee |
And 3 more authors.
British Journal of Pharmacology | Year: 2013
Background and Purpose Most GABAA receptor subtypes comprise 2α, 2β and 1γ subunit, although for some isoforms, a δ replaces a γ-subunit. Extrasynaptic δ-GABAA receptors are important therapeutic targets, but there are few suitable pharmacological tools. We profiled DS2, the purported positive allosteric modulator (PAM) of δ-GABAA receptors to better understand subtype selectivity, mechanism/site of action and activity at native δ-GABAA receptors. Experimental Approach Subunit specificity of DS2 was determined using electrophysiological recordings of Xenopus laevis oocytes expressing human recombinant GABAA receptor isoforms. Effects of DS2 on GABA concentration-response curves were assessed to define mechanisms of action. Radioligand binding and electrophysiology utilising mutant receptors and pharmacology were used to define site of action. Using brain-slice electrophysiology, we assessed the influence of DS2 on thalamic inhibition in wild-type and δ0/0 mice. Key Results Actions of DS2 were primarily determined by the δ-subunit but were additionally influenced by the α, but not the β, subunit (α4/6βxδ > α1βxδ >> γ2-GABAA receptors > α4β3). For δ-GABAA receptors, DS2 enhanced maximum responses to GABA, with minimal influence on GABA potency. (iii) DS2 did not act via the orthosteric, or known modulatory sites on GABAA receptors. (iv) DS2 enhanced tonic currents of thalamocortical neurones from wild-type but not δ0/0 mice. Conclusions and Implications DS2 is the first PAM selective for α4/6βxδ receptors, providing a novel tool to investigate extrasynaptic δ-GABAA receptors. The effects of DS2 are mediated by an unknown site leading to GABAA receptor isoform selectivity. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.
Andreasen J.T.,Copenhagen University |
Redrobe J.P.,Lundbeck |
Nielsen E.O.,Neurosearch |
Christensen J.K.,Neurosearch |
And 3 more authors.
Neuropharmacology | Year: 2013
As affective and cognitive disturbances frequently co-occur in psychiatric disorders, research into opportunities to simultaneously target both entities is warranted. These disorders are typically treated with monoamine reuptake inhibitors (MRIs), whereas ongoing research suggests that symptoms also improve by nicotinic acetylcholine receptor (nAChR) activation. Preclinical studies have corroborated this and also demonstrated a synergistic antidepressant-like action when nAChR agonists and MRIs are combined. Here, we present the in vitro and in vivo profile of NS9775, a combined full α7 nAChR agonist and triple MRI. NS9775 potently inhibited [3H]α-bungarotoxin binding in vitro (Ki: 1.8 nM), and ex vivo (ED50: 3.6 mg/kg), showing negligible activity at α4β2-(Ki: 1720 nM) or a1-containing nAChRs (Ki: 12,200 nM). In α7-expressing oocytes, NS9775 displayed an EC50 value of 280 nM, with a maximal response of 77% relative to a saturating acetylcholine concentration. Furthermore, NS9775 inhibited cortical [3H]5-HT, [3H]NA and [3H]DA uptake equipotently (14-43 nM), and inhibited striatal [3H]WIN35,428 binding (ED50: 9.1 mg/kg). Behaviourally in mice, NS9775 (0.3-3.0 mg/kg) reversed scopolamine-induced deficits in a modified Y-maze and MK-801-induced learning deficits in 5-trial inhibitory avoidance. Swim distance in the forced swim test was increased by 30 mg/kg NS9775, and 10 and 30 mg/kg NS9775 reduced digging behaviour in the marble burying paradigm and increased the number of punished crossings in the four plate test. This pro-cognitive, antidepressant-like and anxiolytic-like effect of NS9775 suggests that combining α7 nAChR agonism and triple monoamine reuptake inhibition could be a step in the evolution of pharmacological treatments of affective and/or cognitive disturbances. © 2013 Elsevier Ltd. All rights reserved.
Hansen R.R.,Neurosearch |
Hansen R.R.,King's College London |
Erichsen H.K.,Neurosearch |
Brown D.T.,Neurosearch |
And 3 more authors.
Neuropharmacology | Year: 2012
GABA-A receptor positive allosteric modulators (PAMs) mediate robust analgesia in animal models of pathological pain, in part via enhancing injury-induced loss of GABA-A-α2 and -α3 receptor function within the spinal cord. As yet, a lack of clinically suitable tool compounds has prevented this concept being tested in humans. Prior to assessing the efficacy of GABA-A receptor PAMs in a human volunteer pain model we have compared compounds capable of variously modulating GABA-A receptor function in comparable rat models of capsaicin-induced acute nocifensive flinching behaviour and secondary mechanical hypersensitivity. The subtype-selective PAM NS11394 (0.3-10 mg/kg), and the non-selective PAM diazepam (1-5 mg/kg) variously reduced capsaicin-induced secondary mechanical hypersensitivity (180 min post-injection). However, the low efficacy subtype-selective PAM TPA023 (3-30 mg/kg) was completely ineffective. This was surprising as both NS11394 and TPA023 robustly attenuated late phase (6-30 min post-injection) capsaicin-induced flinching, a pain-like behaviour that is putatively driven by peripheral and central sensitizing mechanisms. Diazepam also attenuated capsaicin-induced nocifensive behaviours, albeit at doses previously shown to impair locomotor function. Our data indicate that GABA-A receptor PAMs with optimal selectivity and efficacy profiles reduce centrally-mediated mechanical hypersensitivity in capsaicin-injected rats, an observation that we expect can translate directly to human volunteer studies. © 2012 Elsevier Ltd. All rights reserved.
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.,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.
Agency: European Commission | 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.
Aniona Aps | Date: 2014-04-28
The present application discloses novel 8-aza-bicyclo[3.2.1]oct-3-yloxy)-chromen-2-one derivatives useful as monoamine neurotransmitter re-uptake inhibitors. In other aspects the application discloses the use of these compounds, a method for therapy and to pharmaceutical compositions comprising these compounds.