Probing the Allosteric Modulator Binding Site of GluR2 with Thiazide Derivatives

Abstract

Ionotropic glutamate receptors mediate the majority of vertebrate excitatory synaptic transmission and are therapeutic targets for cognitive enhancement and treatment of schizophrenia. The binding domains of these tetrameric receptors consist of two dimers, and the dissociation of the dimer interface of the ligand-binding domain leads to desensitization in the continued presence of agonist. Positive allosteric modulators act by strengthening the dimer interface and reducing desensitization, thereby increasing steady-state activation. Removing the desensitized state for simplified analysis of receptor activation is commonly achieved using cyclothiazide (CTZ), the most potent modulator of the benzothiadiazide class, with the flip form of the AMPA receptor subtype. IDRA-21, the first benzothiadiazide to have an effect in behavioral tests, is an important lead compound in clinical trials for cognitive enhancement as it can cross the blood-brain barrier. Intermediate structures between CTZ and IDRA-21 show reduced potency suggesting that these two compounds have different contact points associated with binding. To understand how benzothiadiazides bind to the pocket bridging the dimer interface, we generated a series of crystal structures of the GluR2 ligand-binding domain complexed with benzothiadiazide derivatives (IDRA-21, hydroflumethiazide, hydrochlorothiazide, chlorothiazide, trichlormethiazide, and althiazide) for comparison with an existing structure for cyclothiazide. The structures detail how changes in the substituents in the 3- and 7-positions of the hydrobenzothiadiazide ring shift the orientation of the drug in the binding site and, in some cases, change the stoichiometry of binding. All derivatives maintain a hydrogen bond with the Ser754 hydroxyl, affirming the partial selectivity of the benzothiadiazides for the flip form of AMPA receptors.

Mechanisms of Modal Activation of GluA3 Receptors

“…In all cases, the LBD dimer interface, seen in the structure of the tetrameric protein (Sobolevsky et al, 2009), was observed. The dimer interface is essentially the same for each of the agonists bound to the LBD, and it is similar to the dimer interface observed with cyclothiazide derivatives bound (Ptak et al, 2009). FW bound GluA3 is 2.6°more open relative to glutamate (Protein Data Bank code 3DP6, B protomer, glutamate bound to GluA2 (Ahmed et al, 2009b; Table 4).…”

Structural and Functional Analysis of Two New Positive Allosteric Modulators of GluA2 Desensitization and Deactivation

“…7). This may suggest that modulator binding in subsite B can influence isoform selectivity through direct interaction or by destabilizing interactions through subsite C. Consistent with this, CTZ preferentially blocks desensitization of the flip isoform through the interaction of its N4 substituent with the serine/ asparagine site on helix J (Sun et al, 2002;Ptak et al, 2009). Although Ser/Asn750 lies between the B and C subsites, the asparagine limits access to the C subsite.…”

“…It has been proposed that the allosteric modulatory site can be delineated into five overlapping subsites at the dimer interface between subunits (Ptak et al, 2009). The central subsite, A, lies parallel to the axis of symmetry between the protomers and is occupied by the planar rings of aniracetam, CX614, and the biaryl compounds.…”

A Charge-inverting Mutation in the “Linker” Region of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors Alters Agonist Binding and Gating Kinetics Independently of Allosteric Modulators

“…These two modulators bind to a common pocket between LBD dimers (33). However, although CTZ reduces desensitization putatively through modulation of ␦ and/or ␥ (27), CX614 additionally slows deactivation (14,35).…”

“…These drugs were chosen because of their contrasting isoform and deactivation/desensitization efficacies. CTZ has been shown to be a flip-selective modulator of GluA2 desensitization with little effect on deactivation kinetics (27,(31)(32)(33), whereas CX614 is more selective for GluA2 flop, modulating both deactivation and desensitization of that isoform (16,32).…”