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Mol Pharmacol. 2019 Oct 3. pii: mol.119.116871. doi: 10.1124/mol.119.116871. [Epub ahead of print]

Mutational analysis and modeling of negative allosteric modulator binding sites in AMPA receptors.

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University of Copenhagen.
University of Oxford.
University of Copenhagen;


The α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) constitute a subclass of the ionotropic glutamate receptor (iGluR) superfamily, which functions as glutamate-gated cation channels to mediate the majority of excitatory neurotransmission in the central nervous system (CNS). AMPARs are therapeutic targets in a range of brain disorders associated with abnormal glutamate hyperactivity. Multiple classes of AMPAR inhibitors have been developed during the past decades, including competitive antagonists, ion channel blockers and negative allosteric modulators (NAMs). At present, NAMs is the only class of AMPAR ligands that has been developed into safe and useful drugs in humans in the form of perampanel (Fycompa) for treatment of epilepsy. Yet, compared to the detailed understanding of other AMPAR ligand classes, surprisingly little information has been available regarding the molecular mechanism of perampanel and other classes of NAMs at AMPARs; including the location and structure of NAM binding pockets in the receptor complex. However, structures of the AMPAR GluA2 in complex with NAMs were recently reported that unambiguously identified the NAM binding sites. In parallel with this work, our aim with the present study was to identify specific residues involved in the formation of the NAM binding site for three prototypical AMPAR NAMs. Hence, we have performed a mutational analysis of the AMPAR region that link the four extracellular ligand binding domains (LBDs) to the central ion channel in the transmembrane domain (TMD) region. We furthermore perform computational ligand-docking of the NAMs into structural models of the homomeric GluA2 receptor and optimize side chain conformations around the NAMs to model how NAMs bind in this specific site. The new insights provide potentially important input for structure-based drug design of new NAMs. SIGNIFICANCE STATEMENT: Negative allosteric modulators (NAMs) of the AMPA receptor are clinically important for treatment of brain diseases, but their molecular pharmacology has historically been poorly understood. The present work characterizes the binding mode and sites for prototypical NAMs at the GluA2 AMPA receptor. The results provided new details of the molecular basis of NAM binding and mechanism of action that is of potential use in future drug development.


AMPA receptors; Anticonvulsants; Computational models; Electrophysiology; Epilepsy; Glutamate receptors; L-glutamate; Ligand docking; Ligand-gated ion channels; Receptor structure

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