Molecular Mechanism of Flop Selectivity and Subsite Recognition for an AMPA Receptor Allosteric Modulator: Structures of GluA2 and GluA3 in Complexes with PEPA

Abstract

Glutamate receptors are important potential drug targets for cognitive enhancement and the treatment of schizophrenia in part because they are the most prevalent excitatory neurotransmitter receptors in the vertebrate central nervous system. One approach to the application of therapeutic agents to the AMPA subtype of glutamate receptors is the use of allosteric modulators, which promote dimerization by binding to a dimer interface thereby reducing desensitization and deactivation. AMPA receptors exist in two alternatively spliced variants (flip and flop) that differ in desensitization and receptor activation profiles. Most of the structural information on modulators of the AMPA receptor target the flip subtype. We report here the crystal structure of the flop-selective allosteric modulator, PEPA, bound to the binding domains of the GluA2 and GluA3 flop isoforms of AMPA receptors. Specific hydrogen bonding patterns can explain the preference for the flop isoform. This includes a bidentate hydrogen bonding pattern between PEPA and N754 of the flop isoforms of GluA2 and GluA3 (the corresponding position in the flip isoform is S754). Comparison with other allosteric modulators provides a framework for the development of new allosteric modulators with preferences for either the flip or flop isoforms. In addition to interactions with N/S754, specific interactions of the sulfonamide with conserved residues in the binding site are characteristics of a number of allosteric modulators. These, in combination, with variable interactions with five subsites on the binding surface lead to different stoichiometries, orientations within the binding pockets, and functional outcomes.

AMPA receptor potentiators: from drug design to cognitive enhancement

“…Most AMPAR positive allosteric modulators to fall into four major classifications: the benzamides (including aniracetam [8] and its derivatives, the CX ampakines such as CX516 [9], CX614 [10], CX717 [11], CX929 [12] and Org 26576 [13]); the benzothiadiazines (including cyclothiazide [14], IDRA-21 [15], S 18986 [16], and BPAM-97 [17,18]); the biaryl propylsulfonamides (including LY404187 [19], LY451395 [20], (R,R)-2a, and -2b, or PIMSD [21], PEPA [22], and CMPDA and CMPDB [23]), which recapitulate structural features of the first two classes; and the 3-trifluoromethylpyrazoles (including a series of compounds developed by Ward et al [24,25] and Jamieson et al [26–28] (Figure 1). Pirotte et al [29] document, from their review of patents filed between 2008–2012, that two smaller classes, including pyrrole/thiophenecarboxylic acids described by Lilly and phenyliminothiazoles described by GlaxoSmithKline may be further developed but currently do not seem to represent a major discovery target.…”

Dynamics of Cleft Closure of the GluA2 Ligand-binding Domain in the Presence of Full and Partial Agonists Revealed by Hydrogen-Deuterium Exchange

“…The strain associated with this process apparently leads to the dissociation of the dimer interface and desensitization (5). The dimer interface can be stabilized by allosteric modulators such as cyclothiazide (5,6) and 4-[2-(phenylsulphonylamino)ethylthio]-2,6,-difluorophenoxy acetamide (7,8), which in turn slow desensitization. The strain placed on the dimer interface is likely related to the time that the LBD exists in the fully closed state, which is related to the energetics associated with the equilibrium distribution of possible lobe orientations and the stability of the contacts across the cleft between the two lobes of the LBD.…”

Functional insight into development of positive allosteric modulators of AMPA receptors

“…The functional differences are striking, in light of the structural similarity shared by crystal structures of the flip and flop isoforms (Ahmed et al, 2009). The molecular basis of these differences is unclear and is believed to be largely governed by the S/N754 residue (Ahmed et al, 2010; Kessler et al, 2000; Partin et al, 1996); however, why there are differences in the thermodynamics of the flip and flop receptors that might impact compound efficacy or affinity remains poorly understood.…”

“…Some discovery approaches have focused on the possibility of teasing apart the biophysical process of deactivation from that of desensitization. However, in no case has a positive allosteric modulator of deactivation been identified which does not also modulate desensitization (Ahmed et al, 2010; Arai et al, 2000; Mitchell and Fleck, 2007; Quirk and Nisenbaum, 2002; Sun et al, 2002; Timm et al, 2011). …”