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D. Siebert, M. Wieder, L. Schlener, M. Ernst, T. Langer, G. Ecker, M. Schnürch, L. Richter:
"Post-Docking Derivatization Reveals Pyrazoloquinolinone Binding Mode At The α+/ γ- Gabaa Receptor Interface";
Vortrag: VSS 2017 - Vienna young Scientists Symposium, Wien; 01.06.2017 - 02.06.2017; in: "Vienna Young Scientist Symposium 2017", (2017), ISBN: 978-3-9504017-5-2.

gamma - Aminobutyric acid (GABA) is a wide-spread transmitter which binds to two pharmacologically diverging GABA receptors, GABAA and GABAB. GABAA receptors (GABAARs) are transmembrane pentameric ligand-gated chloride ion channels and represent an important target of many clinically relevant drugs (e.g. benzodiazepines, barbiturates, etc.). These receptors consist of different subunits (e.g. α,β,γ, etc.), which are drawn from nineteen subunit isoforms that are grouped into classes (e.g. α1-α6, β1- β3, etc.). Each GABAA receptor possesses several extracellular and transmembrane small molecule binding sites - such as the high affinity benzodiazepine binding site (BZ site) at the α+/γ- interface. This interface represents the allosteric binding site of the eponymous compound class of the benzodiazepines which exert anxiolytic, muscle-relaxant, sedative- hypnotic and anticonvulsant effects. Until today, benzodiazepines are widely used for medical treatments as well as in pharmaceutical research. To antagonize their effects various chemotypes have been introduced, e.g. the pyrazoloquinolinones (PQs). However, all chemotypes possess partly overlapping and partly distinct in vitro and in vivo effects. This ambiguous behaviour might be based on the lack of knowledge regarding their binding sites and modes respectively.
In order to determine the binding mode of pyrazoloquinolinones at the extracellular α+/γ- interface (BZ site) a homology model based on the crystal structure of the human GABAA β3 homopentamer was created consisting of two α1, two β3 and one γ2 subunits. Flexible Molecular Docking was then performed using the PQ CGS8216 to get 100 different binding poses. For each pose an array of seven selected derivatives were built using the initial coordinates of CGS8216 to expand the dataset up to 800 poses in total. From the 7 analogues, four were classified as active (pKi > 8) while three were classified as inactive (pKi < 7). To validate the poses, the derivative
analogue placements were analyzed for steric clashes and their compliance with Structure Activity Relationships (Experimental Scoring). The evaluation led to two reasonable binding modes which were further differentiated by Molecular Dynamic Simulations. Analysis of the variation of RMSD (root-mean-square deviation) and the occurring protein - ligand interactions resulted in one consistent binding hypothesis.
We were able to identify a binding hypothesis for the pyrazoloquinolinones at the benzodiazepine binding site (α+/γ- interface) that is in great accordance with known SAR data. The filtering of potential binding poses was undertaken with a novel methodology termed "post-docking derivati-zation". This methodology makes use of SAR information for validation of docking poses to narrow down reasonable binding modes. On top of this analysis we performed Molecular Dynamic Simulations that corroborated the binding hypothesis with the greatest SAR accordance.
The binding hypothesis of the pyrazoloquinolinones will simplify the structure guided optimisation in the retrieval of required antagonists for the BZ site of the GABAA receptor. Furthermore, the binding hypothesis will positively influence ongoing research at homologous binding site of the GABAA receptor, e.g. the α+/β- interface (low affinity allosteric binding site of PQs). In addition, the post-docking derivatization methodology represents a useful tool to easily cluster reasonable docking poses including important biological information.