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Functional Validation of Virtual Screening for Novel Agents with General Anesthetic Action at Ligand-Gated Ion Channelss
Swiss Federal Institute of Technology, Switzerland.
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2013 (English)In: Molecular Pharmacology, ISSN 0026-895X, E-ISSN 1521-0111, Vol. 84, no 5, p. 670-678Article in journal (Refereed) Published
Abstract [en]

GABA(A) receptors play a crucial role in the actions of general anesthetics. The recently published crystal structure of the general anesthetic propofol bound to Gloeobacter violaceus ligand-gated ion channel (GLIC), a bacterial homolog of GABA(A) receptors, provided an opportunity to explore structure-based ligand discovery for pentameric ligand-gated ion channels (pLGICs). We used molecular docking of 153,000 commercially available compounds to identify molecules that interact with the propofol binding site in GLIC. In total, 29 compounds were selected for functional testing on recombinant GLIC, and 16 of these compounds modulated GLIC function. Active compounds were also tested on recombinant GABA(A) receptors, and point mutations around the presumed binding pocket were introduced into GLIC and GABA(A) receptors to test for binding specificity. The potency of active compounds was only weakly correlated with properties such as lipophilicity or molecular weight. One compound was found to mimic the actions of propofol on GLIC and GABA(A), and to be sensitive to mutations that reduce the action of propofol in both receptors. Mutant receptors also provided insight about the position of the binding sites and the relevance of the receptor's conformation for anesthetic actions. Overall, the findings support the feasibility of the use of virtual screening to discover allosteric modulators of pLGICs, and suggest that GLIC is a valid model system to identify novel GABA(A) receptor ligands.

Place, publisher, year, edition, pages
2013. Vol. 84, no 5, p. 670-678
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-96087DOI: 10.1124/mol.113.087692ISI: 000325534800002OAI: oai:DiVA.org:su-96087DiVA, id: diva2:664248
Note

AuthorCount:9

Available from: 2013-11-14 Created: 2013-11-11 Last updated: 2018-03-27Bibliographically approved
In thesis
1. Allosteric modulation of pentameric ligand-gated ion channels by general anesthetics
Open this publication in new window or tab >>Allosteric modulation of pentameric ligand-gated ion channels by general anesthetics
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Pentameric ligand-gated ion channels (pLGICs) are key components of fast synaptic transmission and are targets of neuroactive drugs such as benzodiazepines, alcohol and muscle relaxants. Although early theories of general anesthesia suggested non-specific lipid interaction as the mechanism of anesthetic action, it has now become evident that they too bind to pLGICs. While general anesthetics act as positive allosteric modulators on most anion-conducting pLGICs, they inhibit cation-conducting channels. A detailed structural mechanism of how such opposite allosteric effects emerge has yet to be presented.

This thesis investigates the structure-function relationship underlying the dynamics of channel activation and explores the mechanisms behind allosteric modulation by general anesthetics. Key model systems include the glutamate-gated chloride channel of C. elegans (GluCl) and the G. violaceus ligand-gated ion channel (GLIC), that show considerable structural homology to mammalian channel but with the added simplicity of homomeric assembly and accessibility to crystallization. Functional assessment is performed through recombinant expression of the channels in Xenopus oocytes, which are then used for two-electrode voltage clamp electrophysiology. These measurements are combined with recent advances in structure determination and computational simulations to propose structural mechanisms behind the functional effects.

In this thesis I present the exploration and validation of the crystallographic construct GluCl as a model system to explore fundamental questions of mammalian pLGIC function. Further studies contribute to the understanding of the basis of allosteric modulation by identifying responsible binding sites for both potentiation and inhibition by general anesthetics in GLIC and substantiate a structural mechanism for these effects. The studies also offer a link between receptor- and lipid-based theories of anesthesia, and demonstrate successful discovery of new lead compounds with general anesthetic properties using virtual screening. The thesis therefore makes a contribution to the fundamental understanding of allosteric modulation in pLGICs and builds on the basis for rational drug discovery.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2018
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-154106 (URN)978-91-7797-151-1 (ISBN)978-91-7797-152-8 (ISBN)
Public defence
2018-05-04, Magnéli Hall, Chemical Practice Laboratory, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
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Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Accepted. Paper 4: Manuscript.

Available from: 2018-04-11 Created: 2018-03-15 Last updated: 2018-04-09

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