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A Photoswitchable GABA Receptor Channel Blocker

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Received: 24 December 2018 Revised: 3 March 2019 Accepted: 3 April 2019 DOI: 10.1111/bph.14689 RESEARCH PAPER BJP A photoswitchable GABA receptor channel blocker Galyna Maleeva1 | Daniel Wutz2 | Karin Rustler2 | Alba Nin‐Hill3 | Carme Rovira3,10 | Elena Petukhova6 | Antoni Bautista‐Barrufet7 | Alexandre Gomila‐Juaneda7 | Petra Scholze8 | Franck Peiretti9 | Mercedes Alfonso‐Prieto4,5 | Burkhard König2 | Pau Gorostiza7,10,11 | Piotr Bregestovski1,6,12 1 INSERM, INS, Institut de Neurosciences des Systèmes, Aix‐Marseille University, Marseille, France 2 Institute of Organic Chemistry, University of Regensburg, Regensburg, Germany 3 Departament de Química Inorgànica i Orgànica (Secció de Química Orgànica) and Institut de Química Teòrica i Computacional (IQTCUB), Universitat de Barcelona, Barcelona, Spain 4 Department of Computational Biomedicine, Institute for Advanced Simulations IAS‐5 and Institute of Neuroscience and Medicine INM‐9, Forschungszentrum Jülich GmbH, Jülich, Germany 5 Cécile and Oskar Vogt Institute for Brain Research, Medical Faculty, Heinrich Heine University Düsseldorf, Düsseldorf, Germany 6 Department of Normal Physiology, Kazan State Medical University, Kazan, Russia 7 Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST), Barcelona, Spain 8 Department of Pathobiology of the Nervous System, Center for Brain Research, Medical University Vienna, Vienna, Austria 9 INSERM 1263, INRA 1260, C2VN, Aix‐Marseille Université, Marseille, France 10 Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain 11 Network Biomedical Research Center in Biomaterials, Bioengineering and Nanomedicine (CIBER‐BBN), Spain 12 Institute of Neurosciences, Kazan State Medical University, Kazan, Russia Correspondence Background and Purpose: Anion‐selective Cys‐loop receptors (GABA and glycine Piotr Bregestovski, INSERM, INS, Institut de Neurosciences des Systèmes, Aix‐Marseille receptors) provide the main inhibitory drive in the CNS. Both types of receptor oper- University, 13005 Marseille, France. ate via chloride‐selective ion channels, though with different kinetics, pharmacologi- Email: piotr.bregestovski@univ‐amu.fr; [email protected] cal profiles, and localization. Disequilibrium in their function leads to a variety of Mercedes Alfonso‐Prieto, Department of disorders, which are often treated with allosteric modulators. The few available GABA Computational Biomedicine, Institute for Advanced Simulations IAS‐5 and Institute of and glycine receptor channel blockers effectively suppress inhibitory currents in Neuroscience and Medicine INM‐9, neurons, but their systemic administration is highly toxic. With the aim of developing Forschungszentrum Jülich GmbH, D‐52425 Jülich, Germany. an efficient light‐controllable modulator of GABA receptors, we constructed Email: [email protected] azobenzene‐nitrazepam (Azo‐NZ1), which is composed of a nitrazepam moiety Pau Gorostiza, Institute for Bioengineering of merged to an azobenzene photoisomerizable group. Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST), Barcelona, Experimental Approach: The experiments were carried out on cultured cells Spain. ‐ Email: [email protected] expressing Cys loop receptors of known subunit composition and in brain slices using patch‐clamp. Site‐directed mutagenesis and molecular modelling approaches were Funding information ‐ Agència de Gestió d'Ajuts Universitaris i de applied to evaluate the mechanism of action of Azo NZ1. Recerca, Grant/Award Number: 2017‐SGR‐ Abbreviations: Azo‐NZ, azobenzene‐nitrazepam; TM, transmembrane domain; Vh, holding membrane potential -------------------------------------------------------------------------------------------------------------------------------- This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. © 2019 Institut de Neurosciences des Systémes. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society. Br J Pharmacol. 2019;176:2661–2677. wileyonlinelibrary.com/journal/bph 2661 2662 BJP MALEEVA ET AL. 1442; Ministerio de Economía y Key Results: At visible light, being in trans‐configuration, Azo‐NZ1 blocked Competitividad, Grant/Award Number: ‐ CTQ2016 80066R; Barcelona Supercomputing heteromeric α1/β2/γ2 GABAA receptors, ρ2 GABAA (GABAC), and α2 glycine recep- Center, Grant/Award Numbers: BCV‐2016‐3‐ tors, whereas switching the compound into cis‐state by UV illumination restored the 0005 and BCV‐2016‐2‐0002; MINECO; Ramón Areces Foundation; Fundaluce Foundation; activity. Azo‐NZ1 successfully photomodulated GABAergic currents recorded from Human Brain Project WAVESCALES; FEDER; dentate gyrus neurons. We demonstrated that in trans‐configuration, Azo‐NZ1 blocks AGAUR/Generalitat de Catalunya, Grant/ Award Number: 2017‐SGR‐1442; Algorish; the Cl‐selective ion pore of GABA receptors interacting mainly with the 2′ level of the Russian Science Foundation, Grant/Award TM2 region. Number: 18‐15‐00313; ERA SynBIO, Grant/ Award Number: PCIN‐2015‐163‐C02‐01 Conclusions and Implications: Azo‐NZ1 is a soluble light‐driven Cl‐channel blocker, which allows photo‐modulation of the activity induced by anion‐selective Cys‐loop receptors. Azo‐NZ1 is able to control GABAergic postsynaptic currents and provides new opportunities to study inhibitory neurotransmission using patterned illumination. 1 | INTRODUCTION What is already known • Pharmacological activity of ligands can be regulated with The major inhibitory neurotransmission in the mammalian CNS is azobenzene photochromic group, which changes its provided by GABAA, GABAA ρ (also known as GABAC ρ receptor; configuration upon illumination with light of different see Alexander et al., 2015), and glycine receptors (Langosch, Becker, wavelength. & Betz, 1990). In different combinations, these receptors are widely expressed throughout the nervous system and are involved in physio- What this study adds logical functions such as cognition, learning, memory, and motor • Azobenzene‐nitrazepam based photochromic compound control. Disequilibrium in the functioning of GABA and glycine (Gly) Azo‐NZ1 is a light controllable channel blocker of receptors leads to important disorders including epilepsy, anxiety, heteromeric GABA , homomeric GABA ρ2 receptors, depression, misbalance of blood pressure, sleep disorders, schizophre- A C and α2 GlyRs. nia, and hyperekplexia (Johnston, Chebib, Hanrahan, & Mewett, 2003; Tan, Rudolph, & Lüscher, 2011; Sieghart, 2015; Lynch, 2004). • Azo‐NZ1 modulates in a light-dependent manner The activity of GABAA receptors can be successfully regulated synaptic GABAergic currents in dentate gyrus of with allosteric modulators like benzodiazepines, including flumazenil hippocampal brain slices. and diazepam. They are among the most popular pharmacological drugs and are commonly prescribed to treat insomnia, anxiety, and What is the clinical significance convulsions. Benzodiazepines can potentiate GABA receptors through • Azo‐NZ1 modulates in a light‐dependent manner interaction with different sites in the receptor displaying nano‐ and synaptic GABAergic currents in dentate gyrus of micromolar affinities (Sieghart, 2015). Therefore, benzodiazepine hippocampal brain slices. derivatives are convenient scaffolds to develop new modulators of GABA receptor function. Light‐switchable ligands have become a useful tool to specifically control ionic channels, cellular functions, the activity of neuronal characterized their properties. Herein, we describe the interaction of circuits, and behaviour (Gorostiza & Isacoff, 2008; Kramer, Mourot, & one of these compounds, azobenzene‐nitrazepam‐1 (Azo‐NZ1), with Adesnik, 2013). The pharmacological activity of ligands can be regulated several subtypes of anion‐selective Cys‐loop receptors (GABAA, with photochromic groups—molecules that change their configuration GABAC and glycine receptors). Azo‐NZ1 successfully photocontrolled upon illumination with different wavelengths. Azobenzene undergoes a GABAergic currents in cultured cells and in brain slices, but in rapid trans–cis photoisomerization and has been widely used for this pur- contrast to the intended design to obtain an allosteric ligand of the pose. In the dark, or under visible light irradiation, azobenzene is in the classical diazepam site, we found that the azobenzene sulfonyl group elongated trans‐configuration, while upon illumination with UV light introduced alters the binding properties of the compound, enabling (360–380 nm), it isomerizes to its cis‐state and becomes shorter by sev- light‐controlled blocking of the chloride‐selective ion pore. Thus, eral angstroms (Gorostiza & Isacoff, 2008; Yu & Ikeda, 2004). Azo‐NZ1 reveals a class of photochromic ligand with a structure capa- With the aim of regulating the activity of GABA receptors with light, ble of blocking the anion‐selective channel pore of several Cys‐loop we constructed a series of azobenzene derivatives of nitrazepam and receptors. 2663 MALEEVA ET AL. BJP 2 | METHODS used for recording mass spectra. UV/Vis absorption spectroscopy was accomplished using a Varian Cary BIO 50 UV/Vis spectrometer in 10‐ 2.1 | Synthesis of compounds mm quartz cuvettes. IR spectra were recorded on an Agilent Cary 630 FT‐IR spectrometer, and the peak positions are reported in wavenumbers
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