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  Computational studies on GABAA receptors
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 Ophav:
Sander, Tommy1, Forfatter
Balle, Thomas2, Vejleder
Tilknytninger:
1Det Farmaceutiske Fakultet, Københavns Universitet, København, Danmark, diskurs:7016              
2Institut for Medicinalkemi, Det Farmaceutiske Fakultet, Københavns Universitet, København, Danmark, diskurs:7019              
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 Abstract: γ-Aminobutyric acid (GABA) is the overall inhibitory neurotransmitter in the central nervous
system (CNS). It exerts its actions in the neuronal synapse primarily by binding to and activating
the GABAA receptor (GABAAR), a chloride ion channel and member of the structurally and
functionally related class of pentameric ligand-gated ion channels known Cys-loop receptors.
The nicotinic acetylcholine receptors (nAChRs), glycine receptors (GlyRs), and 5-HT3 receptors
are the other members of the Cys-loop family. The GABAARs are involved in a wide range of
physiological processes and, hence, also in several neurological disorders such as anxiety,
schizophrenia, stress, sleep disorders, and epilepsy. They are therefore considered important drug
targets; however, because no experimentally determined structure has yet been reported, the lack
of structural insights limits the possibilities of structure-based ligand design. With crystal
structures of the distantly homologous acetylcholine-binding protein (AChBP) isolated from
freshwater snails, the mouse nAChR α1 subunit, and bacterial ion channels (ELIC and GLIC), a
firm structural basis exists for creating computational models the structure of the GABAAR
based on these homologues.
The main focus of the present PhD project was to create a homology model of the GABAAR
ligand-binding domain, i.e. the extracellular (EC) part of the receptor. The model was based on
multiple templates and on incorporation of experimental data directly into the model building
process. The thorough approach taken to optimize and refine the model proved to increase its
reliability. Through computational ligand docking, and by exploiting extensive structure-activity
relationships that have been established for at series of antagonists, the model was used to create
a hypothesis for ligand binding to the orthosteric site. The combined receptor and ligand binding
models convincingly rationalized key experimental findings, both for the receptor and for the
ligands. The model was used in designing new ligands with the purpose of further exploring the
size and shape of the binding site, the results of which have so far supported the model.
Together, the results have helped increase the structural understanding of the GABAARs and
ligand binding properties. Future research in the field will be able to both benefit directly from
the findings, as well as to further build on the proposed model to improve its quality as more and
more experimental data become available.
In addition, a model of the GlyR transmembrane domain was created to investigate possibilities
and limitations in modeling this part of the Cys-loop receptors. The model was used to
rationalize the binding of ginkgolides and picrotoxin, which block the GlyR and GABAAR ion
channels, and to suggest further mutational studies in an ongoing molecular pharmacology
research project.
Because GABAARs, and Cys-loop receptors in general, are flexible, a study was undertaken to
address flexibility observed to significantly impact the shape and size of binding pockets and
hence also impact the outcome of ligand docking and virtual screening experiments. Using two
model systems (AChBP and the ionotropic glutamate receptor GluA2), ensembles of
conformationally distinct protein structures were generated by either normal mode analysis
(NMA) or by a targeted molecular mechanics protocol. Ligand docking to the generated
ensembles showed that it was possible with reasonable success to correctly predict the receptor
conformation induced by a given ligand. However, the necessity of detailed setup and a priori
knowledge of the systems were found to limit the broader applicability of these methods in e.g.
virtual screening protocols.
 Abstract: γ-Aminosmørsyre (GABA) fungerer som hjernens primære inhibitoriske neurotransmitter ved at
binde sig til og aktivere GABAA receptoren, en chloridionkanal tilhørende klassen af Cys-loop
receptorer. Cys-loop familien består af ligandaktiverede ionkanaler og tæller desuden de nikotine
acetylcholinreceptorer (nAChR), glycinreceptorerne (GlyR), og 5-HT3 receptorerne. GABAA
receptoren er involveret i en lang række fysiologiske processer samt neurologiske sygdomme,
bl.a. angst, skizofreni, stres, søvnforstyrrelser, og epilepsi. Af samme grund er den et eftertragtet
target i lægemiddeludviklingen, men samtidig er den svær at målrette nye lægemidler mod, fordi
man endnu ikke kender dens tredimensionelle struktur. Til gengæld findes sådanne strukturer for
visse andre proteiner i eller beslægtet med Cys-loop familien. Disse proteiner/receptorer er
isoleret fra så forskellige organismer som ferskvandssnegle, musen, en elektrisk ål, og to
bakteriestammer, og med dem i hånden kan man lave computerbaserede homologimodeller.
Hovedformålet med dette ph.d.-projekt var netop, ved at bruge strukturerne fra de nævnte
organismer som templates, at skabe en homologimodel af GABAA receptorens ligandbindende
domæne, der er placeret på den ekstracellulære side af den cellemembran, som receptoren sidder
forankret i. Ved at bruge information fra alle templatestrukturerne, samt kendte eksperimentelle
data fra GABAA receptoren selv, lykkedes det at producere en model af målbart høj kvalitet og
pålidelighed. Ved at kombinere computerbaseret docking med kendskab, via en farmakoformodel,
til struktur-aktivitetsforhold for en serie af orthosteriske antagonister, blev receptormodellen
anvendt til at foreslå en detaljeret bindingsmodel for disse ligander. Den foreslåede
model syntes at kunne forklare og bekræfte vigtige eksperimentelle data, både for receptoren og
for liganderne. Nye ligander blev desuden designet på baggrund af modellen, og efter syntese og
farmakologisk test heraf viste de sig at bekræfte modellens forudsigelser. Samlet har disse
modelleringsstudier bidraget til en øget strukturel forståelse af GABAA receptoren og dens
ligandbindende egenskaber.
Som et yderligere studie blev også den transmembrane del af en af Cys-loop receptorerne – GlyR
– modelleret på tilsvarende måde. Dette gav en række informationer om hvilke muligheder og
begrænsninger, der ligger i at modellere denne del af Cys-loop receptor strukturen, hvor der på
visse områder er større usikkerhed om validiteten af modellerne. Modellen blev brugt til at rationalisere hvordan ginkgolider og picrotoxin (begge naturstoffer) binder til ionkanalen og
derved blokerer for strøm gennem kanalen.
Endelig blev receptorfleksibilitet også undersøgt ved at benytte to computermetoder til at
beskrive og modellere den fleksibilitet, som bl.a. Cys-loop receptorerne viser ved at kunne ændre
konformation og tilpasse sig en ligand. Konformationelle ensembler af to modelsystemer (det
acetylcholinbindende protein (AChBP) fra de ovennævnte ferskvandssnegle, samt den ionotrope
glutamatreceptor) blev genereret vha. hhv. normalkoordinatanalyse samt en molekylmekanikbaseret
minimeringsprotokol. Vha. docking af kendte ligander til de genererede ensembler blev
det vist, at det med en vis succes var muligt at forudsige, hvilken receptor-konformation en given
ligand inducerer. På trods af, at metoderne kræver yderligere undersøgelser for at kunne bruge
dem mere bredt, kan fremgangsmåden potentielt udnyttes i fx virtuel screening efter nye
lægemiddelstoffer.
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Tommy_Sander_phd.pdf (Hovedtekst)
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Copyright dato:
2010
Copyright information:
De fulde rettigheder til dette materiale tilhører forfatteren.
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Bogmærk denne post: https://diskurs.kb.dk/item/diskurs:12154:4
 Type: Ph.D.
Alternativ titel: modeling the receptor structure, flexibility, and orthosteric ligand binding modes
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Detaljer

Sprog: English - eng
 Datoer: 2010-09-28
 Sider: xii, 95 s.
 Publiceringsinfo: København : Københavns Universitet
 Indholdsfortegnelse: -
 Note: -
 Type: Ph.D.
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