Structural Determinants and Mechanism of Action of a GluN2C-selective NMDA Receptor Positive Allosteric Modulator

Abstract

NMDA receptors are tetrameric complexes of GluN1, GluN2A-D, and GluN3A-B subunits and are involved in normal brain function and neurologic disorders. We identified a novel class of stereoselective pyrrolidinone (PYD) positive allosteric modulators for GluN2C-containing NMDA receptors, exemplified by methyl 4-(3-acetyl-4-hydroxy-1-[2-(2-methyl-1H-indol-3-yl)ethyl]-5-oxo-2,5-dihydro-1H-pyrrol-2-yl)benzoate. Here we explore the site and mechanism of action of a prototypical analog, PYD-106, which at 30 mM does not alter responses of NMDA receptors containing GluN2A, GluN2B, and GluN2D and has no effect on AMPA [a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid] and kainate receptors. Coapplication of 50 mM PYD-106 with a maximally effective concentration of glutamate and glycine increases the response of GluN1/GluN2C NMDA receptors in HEK-293 cells to 221% of that obtained in the absence of PYD (taken as 100%). Evaluation of the concentration dependence of this enhancement revealed an EC 50 value for PYD of 13 mM. PYD-106 increased opening frequency and open time of single channel currents activated by maximally effective concentrations of agonist but only had modest effects on glutamate and glycine EC 50 . PYD-106 selectively enhanced the responses of diheteromeric GluN1/GluN2C receptors but not triheteromeric GluN1/GluN2A/GluN2C receptors. Inclusion of residues encoded by GluN1-exon 5 attenuated the effects of PYD. Three GluN2C residues (Arg194, Ser470, Lys470), at which mutagenesis virtually eliminated PYD function, line a cavity at the interface of the ligand binding and the amino terminal domains in a homology model of GluN1/GluN2C built from crystallographic data on GluN1/GluN2B. We propose that this domain interface constitutes a new allosteric modulatory site on the NMDA receptor.

Emerging structural insights into the function of ionotropic glutamate receptors

“…Unfortunately, this method has not been successfully implemented on iGluR proteins, perhaps due to their large extracellular domains which exceed the predicted size limitation for the LCP matrix. Nevertheless, the crystal structures provide the long-awaited molecular templates to locate known pharmacological sites in the context of the heterotetramers and better predict potential binding sites for novel compounds, an example of which is the recently work on PYD-106 (Figure 6) [70]. …”

“…Co-agonists glycine [76] and glutamate bind at the LBD of the GluN1 and GluN2 subunits, respectively [77]. The small molecule PYD-106 is a GluN2C-specific allosteric activator which binds at the ATD/LBD interface [70]. Polyamines such as spermine and spermidine bind at the interface of two ATDs in GluN2B-containing receptors and potentiate channel activity [78], but only in GluN1 isoforms lacking Exon5 [79].…”

Structural basis of subunit selectivity for competitive NMDA receptor antagonists with preference for GluN2A over GluN2B subunits

“…Considerable progress has been made in the development of subunit-selective allosteric modulators (7)(8)(9)(10)(11)(12)(13), but the development of subtype-selective competitive NMDA receptor antagonists has been less successful. The competitive glutamate-site antagonist NVP-AAM077 (hereafter NVP) was originally reported to have 100-fold preference for GluN1/2A over GluN1/2B (14).…”

Identification of novel allosteric modulator binding sites in NMDA receptors: A molecular modeling study

“…Furthermore, the electropositive chemical nature of the guanidium side chain is critical for providing interaction energy to facilitate modulator binding. In addition, R392 has been found to be crucial for GluN2C specific potentiator binding as identified by a recent experimental study [55]. Lastly, the position of Y109 at MBS-4 is found to be critical because the aromatic sidechain of Y109 creates a pi-stacking interaction with the phenanthrene ring of UBP710, which is imperative for the modulator binding.…”

“…Then in the LBD, β1 forms the base; while the loop before β3 in LBD, the loop between β3 and β4, and β5 form the sides and ceiling. Remarkably, this binding site has been experimentally validated recently by others for binding with one of their GluN2C selective modulators[55].…”