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Synthetic Neurochemistry - Introduction of Biophysical Tools into Ion Channels Using Chemical Approaches

English title Synthetic Neurochemistry - Introduction of Biophysical Tools into Ion Channels Using Chemical Approaches
Applicant Lochner Martin
Number 146321
Funding scheme SNSF Professorships
Research institution Departement für Chemie und Biochemie Universität Bern
Institution of higher education University of Berne - BE
Main discipline Organic Chemistry
Start/End 01.01.2014 - 31.12.2015
Approved amount 794'247.00
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All Disciplines (5)

Discipline
Organic Chemistry
Neurophysiology and Brain Research
Biophysics
Biochemistry
Pharmacology, Pharmacy

Keywords (8)

Biophysical Tools; Binding Assay; Organic Chemistry; Post-photoaffinity Modifications; Ligand-gated Ion Channels; Neuroscience; Serotonin 5-HT3 Receptor; Fluorescence Spectroscopy

Lay Summary (German)

Lead
Ionenkanäle sind Proteine, die essentiell sind für die Reizübertragung im Nervensystem. Obwohl Ionenkanäle vor einiger Zeit entdeckt wurden, versteht man ihre molekularen Mechanismen immer noch viel zu wenig, was z.B. bei der Entwicklung von neuen Medikamenten hinderlich ist. Das Ziel unserer Arbeit ist die Synthese von molekularen Werkzeugen (kleinen Molekülen) mit massgeschneiderten Eigenschaften, die für die zelluläre Untersuchung von Ionenkanälen verwendet werden können.
Lay summary

Inhalt und Ziel des Forschungsprojekts

Unsere Forschung fokussiert vor allem auf den Serotonin-Ionenkanal 5-HT3R, der in vielen Bereichen des Gehirns vorkommt. Wir entwickeln zum einen fluoreszierende Moleküle, die selektiv an den 5-HT3R binden. Damit kann der 5-HT3R in Zellen sichtbar gemacht werden und wir versuchen solche fluoreszierende 5-HT3R-Liganden soweit zu optimieren, dass sie für diagnostische Zwecke verwendet werden könnten. Ausserdem nutzen wir diese fluoreszierenden Liganden um Bindungs-Assays für den 5-HT3R zu entwickeln, d.h. wir können damit bestimmen, wie stark und wie selektive andere Moleküle (z.B. Medikament-Kandidaten) an den 5-HT3R binden. Wir entwickeln auch andere Werkzeuge, mit denen wir die Ionenkanäle gezielt chemisch modifizieren und mit neuen Eigenschaften (z.B. Fluoreszenz) ausstatten können.

Wissenschaftlicher und gesellschaftlicher Kontext des Forschungsprojekts

Ionenkanäle haben eine essentielle medizinische Funktion und sind deshalb wichtige Therapieziele. Wir sind zuversichtlich, dass unsere Forschunganstrengungen neue, nützliche molekulare Werkzeuge produzieren werden, welche mithelfen werden Ionenkanäle besser zu verstehen. Die verbesserte Kenntnis wird dazu beitragen, neue Neuropharmaka zu entwickeln, die effizienter sind und weniger Nebenwirkungen haben.

Direct link to Lay Summary Last update: 11.04.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
The Binding Orientation of Epibatidine at α7 nACh Receptors
Thompson Andrew J., Metzger Simon, Lochner Martin, Ruepp Marc-David (2017), The Binding Orientation of Epibatidine at α7 nACh Receptors, in Neuropharmacology, 116, 421-428.
The Binding Orientations of Structurally-related Ligands Can Differ; A Cautionary Note
Marc-David Ruepp, Hao Wei, Michele Leuenberger, Martin Lochner, Andrew J. Thompson (2017), The Binding Orientations of Structurally-related Ligands Can Differ; A Cautionary Note, in Neuropharmacology, 119, 48-61.
Concise Asymmetric Synthesis and Pharmacological Characterization of All Stereoisomers of Glutamate Transporter Inhibitor TFB-TBOA and Synthesis of EAAT Photoaffinity Probes
Leuenberger Michele, Ritler Andreas, Simonin Alexandre, Hediger Matthias A., Lochner Martin (2016), Concise Asymmetric Synthesis and Pharmacological Characterization of All Stereoisomers of Glutamate Transporter Inhibitor TFB-TBOA and Synthesis of EAAT Photoaffinity Probes, in ACS Chemical Neuroscience, 7, 534-539.
Synthesis and Pharmacological Evaluation of [11C]Granisetron and [18F]Fluoropalonosetron as PET Probes for 5-HT3 Receptor Imaging
Mu Linjing, Müller Herde Adrienne, Rüefli Pascal M., Sladojevich Filippo, Milicevic Sephton Selena, Krämer Stefanie D., Thompson Andrew J., Schibli Roger, Ametamey Simon M., Lochner Martin (2016), Synthesis and Pharmacological Evaluation of [11C]Granisetron and [18F]Fluoropalonosetron as PET Probes for 5-HT3 Receptor Imaging, in ACS Chemical Neuroscience, 7(11), 1552-1564.
The Muscarinic Antagonists Scopolamine and Atropine are Competitive Antagonists at 5-HT3 Receptors
Lochner Martin, Thompson Andrew J. (2016), The Muscarinic Antagonists Scopolamine and Atropine are Competitive Antagonists at 5-HT3 Receptors, in Neuropharmacology, 108, 220-228.
A Review of Fluorescent Ligands for Studying 5-HT3 Receptors
Lochner Martin, Thompson Andrew J. (2015), A Review of Fluorescent Ligands for Studying 5-HT3 Receptors, in Neuropharmacology, 98, 31-40.
Characterizing New Fluorescent Tools for Studying 5-HT3 Receptor Pharmacology
Jack Thomas, Simonin Jonathan, Ruepp Marc-David, Thompson Andrew J., Gertsch Jürg, Lochner Martin (corresponding author) (2015), Characterizing New Fluorescent Tools for Studying 5-HT3 Receptor Pharmacology, in Neuropharmacology, 90, 63-73.
Lighting up Neuroscience
Lochner Martin, Thompson Andrew J. (2015), Lighting up Neuroscience, Elsevier, London.
Tracking Individual Membrane Proteins and Their Biochemistry: The Power of Direct Observation
Barden A. O., Goler A. S., Humphreys S. C., Tabatabaei S., Lochner Martin, Ruepp Marc-David, Jack Thomas, Simonin Jonathan, Thompson Andrew J., Jones J. P., Brozik James A. (2015), Tracking Individual Membrane Proteins and Their Biochemistry: The Power of Direct Observation, in Neuropharmacology, 98, 22-30.
Synthesis and Characterization of Photoaffinity Probes that Target the 5-HT3 Receptor
Jack Thomas, Ruepp Marc-David, Thompson Andrew J., Mühlemann Oliver, Lochner Martin (corresponding author) (2014), Synthesis and Characterization of Photoaffinity Probes that Target the 5-HT3 Receptor, in Chimia, 68(4), 239-242.
The Antimalarial Drug Proguanil Is an Antagonist at 5-HT3 Receptors
Lochner Martin, Thompson Andrew J. (corresponding author) (2014), The Antimalarial Drug Proguanil Is an Antagonist at 5-HT3 Receptors, in Journal of Pharmacology and Experimental Therapeutics, 351(3), 674-684.
Discovery of Novel Adenosine Receptor Agonists that Exhibit Subtype Selectivity
Knight Anthony, Hemmings Jennifer L., Winfield Ian, Leuenberger Michele, Frattini Eugenia, Frenguelli Bruno G., Dowell Simon J., Lochner Martin, Ladds Graham, Discovery of Novel Adenosine Receptor Agonists that Exhibit Subtype Selectivity, in Journal of Medicinal Chemistry.

Collaboration

Group / person Country
Types of collaboration
Dr. Marc-David Ruepp, Department of Chemistry and Biochemistry, University of Bern Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Dr. Andrew J. Thompson, Department of Pharmacology, University of Cambridge Great Britain and Northern Ireland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Prof. Oliver Mühlemann, Dept. Chemistry and Biochemistry, University of Bern Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Dr. Chris N. Connolly, Medical School, Centre for Neuroscience, University of Dundee Great Britain and Northern Ireland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Dr. Graham Ladds, Department of Pharmacology, University of Cambridge Great Britain and Northern Ireland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Prof. James A. Brozik, Department of Chemistry, Washington State Univ. United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Prof. Matthias A. Hediger, Institute of Biochemistry and Molecular Medicine, University of Bern Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Prof. Hugues Abriel, Dept. Clinical Research, University of Bern Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Prof. Roger Schibli, Institute of Pharmacological Sciences, ETHZ Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
PD Dr. Manfred Heller, Inselspital, Bern Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Prof. Simon M. Ametamey, Institute of Pharmacological Sciences, ETHZ Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Prof. Sarah C. R. Lummis, Department of Biochemistry, University of Cambridge Great Britain and Northern Ireland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Prof. Erwin Sigel, Institute of Biochemistry and Molecular Medicine, University of Bern Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Prof. Bruno Frenguelli, School of Life Sciences, University of Warwick Great Britain and Northern Ireland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Prof. Magdalini Polymenidou, Institute of Molecular Life Sciences, University of Zürich Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Prof. Jürg Gertsch, Institute of Biochemistry and Molecular Medicine, Universtiy of Bern Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
GDCh/SCS conference on Frontiers in Medicinal Chemistry Poster Molecular Tools for the Cellular Study of Adenosine A1 Receptors 12.02.2017 Bern, Switzerland Perozzo Alyssa; Lochner Martin;
Swiss Chemical Society Fall Meeting Poster Fluorescent probes for the cellular study of the 5-HT3A receptor-synthesis and evaluation of near-infrared probes 04.09.2015 Lausanne, Switzerland Lochner Martin; Jonathan Simonin;
Swiss Chemical Society Fall Meeting Poster Synthesis of photo-crosslinking probes and their application for the site-selective chemical modification of the 5-HT3 receptor 04.09.2015 Lausanne, Switzerland Thompson Andrew J.; Lochner Martin;
Swiss Chemical Society Fall Meeting Poster Development of small molecular tools for the cellular study of adenosine A1 receptors 04.09.2015 Lausanne, Switzerland Lochner Martin;
Challenges in Chemical Biology (ISACS16) Poster Synthesis of photo-crosslinking probes and their application for the site-selective chemical modification of the 5-HT3 receptor 15.06.2015 Zürich, Switzerland Lochner Martin; Thompson Andrew J.; Jonathan Simonin;
Challenges in Chemical Biology (ISACS16) Talk given at a conference Development of Small Molecular Tools for the Cellular Study of Adenosine Receptors 15.06.2015 Zürich, Switzerland Lochner Martin;
3rd Swiss-Japanese Chemical Biology Symposium Talk given at a conference Studying Ion Channels and Receptors Using Synthetic Molecular Probes 02.10.2014 Bern, Switzerland Lochner Martin;
Swiss Chemical Society Fall Meeting Poster Fluorescent probes for the cellular study of the 5-HT3A receptor-development of binding assays and in-vivo imaging 11.09.2014 Zürich, Switzerland Lochner Martin; Thompson Andrew J.; Jonathan Simonin;
Swiss Chemical Society Fall Meeting Poster Novel fluorescent agonists for the A1 adenosine receptor 11.09.2014 Zürich, Switzerland Lochner Martin;
Swiss Chemical Society Fall Meeting Poster Site-selective chemical modification of the 5-HT3 receptor with newly developed photo-crosslinking probes 11.09.2014 Zürich, Switzerland Thompson Andrew J.; Lochner Martin;
Swiss Chemical Society Fall Meeting Poster Synthesis of inhibitors and probes for the cellular study of glutamate transporters 11.09.2014 Zürich, Switzerland Lochner Martin;
EMBO Conference Series: Chemical Biology Poster Synthesis of Molecular Tools for the Cellular Study of Ion Channels and Receptors 20.08.2014 Heidelberg, Germany Lochner Martin;
12th Swiss Snow Symposium Talk given at a conference Toolomics - Molecular Probes for Studying and Modifying Ion Channels and Receptors 24.01.2014 Saas-Fee, Switzerland Lochner Martin;


Self-organised

Title Date Place
Summer School in Chemical Biology 01.09.2014 Villars-sur-Ollon, Switzerland

Awards

Title Year
Springer Poster Prize at the GDCh/SCS conference Frontiers in Medicinal Chemistry 2017
Runner-up Poster Prize in Medicinal Chemistry/Chemical Biology at the Swiss Chemical Society Fall Meeting 2015 2015

Associated projects

Number Title Start Funding scheme
173409 Synthesis of alpha7 nACh receptor modulators and purification and pharmacology of the alpha7 nACh receptor 01.01.2017 International short research visits
147933 Synthesis of fluorescent alpha 7 nicotinic acetylcholine receptor agonists 01.04.2013 International short research visits
123536 Synthetic Neurochemistry - Introduction of Biophysical Tools into Ion Channels Using Chemical Approaches 01.01.2010 SNSF Professorships

Abstract

The aim of this proposal is the design and synthesis of molecular tools which will allow the study of ligand- and voltage-gated ion channels (LGICs and VGICs) and G-protein coupled receptors (GPCRs) in cells, and thus aid our understanding of the function of these multi-subunit, transmembrane proteins. In addition to probes that monitor the function and cellular localisation of the target proteins by using fluorescence we also propose to develop probes which will facilitate their site-specific covalent modification. We anticipate that such modification probes provide the means to specifically modify the wild-type target receptors and ion channels and endow them with new properties (e.g. fluorescence) which in turn can be utilised to study their function in the cellular environment. What is more, we believe that such fluorescent probes and chemically modified receptors or ion channels can be used in binding assays for small molecules. The flux of ions across the cell membrane in both directions is vital for numerous physiological processes but cells need to control these events tightly. Charged particles like ions are not able to diffuse freely through the lipid bilayer which forms the plasma membrane and therefore ion channels represent transmembrane gates which open and close in response to external or internal stimuli. Ligand-gated ion channels (LGICs) are mostly located at nerve terminals, also called synapses, and are responsible for the fast transmission of action potentials between neurons. LGICs are assembled from several subunits and activated by small compounds (e.g. neurotransmitters) that bind to particular clefts which induces a conformational change from a non-conducting «closed» state to a conducting «open» state. Voltage-gated ion channels (VGICs) are opened and closed due to changes in membrane potential. LGICs and VGICs often work in accord in neurons: high influx of cations through opened LGICs for instance will change the membrane potential of the cell (i.e. depolarise the cell) to a certain threshold which in turn will cause VGICs to open and this is ultimately leading to the generation of an new action potential. G-protein coupled receptors (GPCRs) are also mostly activated by an external stimulus (e.g. small molecules) but they don’t possess a pore for ions to pass. Instead, activation of GPCRs triggers intracellular signalling cascades which are initiated by the associated G-proteins.Numerous mutations in ion channels genes are known to cause human diseases (so-called channelopathies). This not only demonstrates the crucial physiological importance of ion channels but it also makes them formidable drug targets. Small molecules can activate (agonists), block (antagonists) or enhance (positive allosteric modulators) ion channel function. Despite considerable achievements in the past ten or so years to solve high-resolution crystal structures of homologous prokaryotic and eukaryotic proteins, in some cases in complex with small molecules, we still have a poor understanding as to where most of the small compounds bind and how they are able to activate these complex macromolecular machines. A high-resolution crystal structure of the 5-HT3 receptor is not known for instance. Structures which have been determined for homologous proteins have allowed construction of 5-HT3R homology models with ambiguous accuracy. It should be noted that, albeit informative, crystal structures only reveal snap shots of a highly dynamic system and many structures in complex with small compounds represent thermodynamically stable states rather than real intermediates. We believe that small molecular probes that interact with these dynamic receptors and ion channels can deliver information about their function and to some extend their structure, and thus they can complement current molecular biology and structural biology techniques. We have already contributed to the field by developing fluorescent ligands that specifically bind to the 5 HT3 receptor on the surface of live cells.With regards to the discovery of new drugs it is still very challenging to design compounds with desired properties (antagonist, (partial) agonist, positive allosteric modulator) from scratch and one ends up with synthesising large compound libraries that are tested using low- to medium-throughput biological assays.This present proposal is an extension of the current SNSF Professorship and we will focus on the projects that in our mind have the most solid basis and greatest prospect of success. More precisely, (i) we shall develop fluorescent probes for the in vivo imaging of the serotonin 5-HT3 receptor, a LGIC, in cells, native tissue or whole organisms. In addition, (ii) the fluorescent ligands should also be used in fluorescence-based binding assays (fluorescence polarisation) for small compounds that target this receptor. Furthermore, (iii) multi-functional probes containing the ligand moiety, a photo-inducible crosslinking tag and a fluorophore will be synthesised and used to covalently attach a fluorophore near the ligand binding site of the 5-HT3 receptor. This strategy is novel in that it avoids the necessity of introducing mutations in the target receptor in order to achieve conjugation with fluorophores. We propose to apply the same methodology and (iv) use our multi-functional probes to chemically label hERG, a cardiac voltage-gated potassium channel, with a small organic fluorophore. Finally, (v) fluorescent agonists acting on the adenosine A1 receptor (AA1R), a GPCR that regulates numerous physiological processes in the central nervous system, will be synthesised and used to study the activation and subsequent cellular internalisation of the AA1R.
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