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

Abstract

NMDA-type glutamate receptors are ligand-gated ion channels that contribute to excitatory neurotransmission in the central nervous system (CNS). Most NMDA receptors comprise two glycine-binding GluN1 and two glutamate-binding GluN2 subunits (GluN2A–D). We describe highly potent (S)-5-[(R)-2-amino-2-carboxyethyl]-4,5-dihydro-1H-pyrazole-3-carboxylic acid (ACEPC) competitive GluN2 antagonists, of which ST3 has a binding affinity of 52 nM at GluN1/2A and 782 nM at GluN1/2B receptors. This 15-fold preference of ST3 for GluN1/2A over GluN1/2B is improved compared with NVP-AAM077, a widely used GluN2A-selective antagonist, which we show has 11-fold preference for GluN1/2A over GluN1/2B. Crystal structures of the GluN1/2A agonist binding domain (ABD) heterodimer with bound ACEPC antagonists reveal a binding mode in which the ligands occupy a cavity that extends toward the subunit interface between GluN1 and GluN2A ABDs. Mutational analyses show that the GluN2A preference of ST3 is primarily mediated by four nonconserved residues that are not directly contacting the ligand, but positioned within 12 Å of the glutamate binding site. Two of these residues influence the cavity occupied by ST3 in a manner that results in favorable binding to GluN2A, but occludes binding to GluN2B. Thus, we reveal opportunities for the design of subunit-selective competitive NMDA receptor antagonists by identifying a cavity for ligand binding in which variations exist between GluN2A and GluN2B subunits. This structural insight suggests that subunit selectivity of glutamate-site antagonists can be mediated by mechanisms in addition to direct contributions of contact residues to binding affinity.

Structural basis of subtype-selective competitive antagonism for GluN2C/2D-containing NMDA receptors

“…The asymmetric unit contained one GluN1-GluN2A-4m LBD heterodimer assembled in the back-to-back orientation ( Fig. 4a) nearly identical to that observed in the previous GluN1-GluN2A LBD structures 12,[40][41][42][43] . GluN2A-4m and GluN2D LBDs had nearly identical overall fold with a minor difference in the Loop1 region ( Fig.…”

“…While crystal structures of GluN2D LBD in complex with agonists and partial agonists have been successfully obtained 38,39 , an antagonist-bound GluN2D LBD structure has been technically difficult to capture. The only competitive antagonist-bound crystal structures of NMDAR GluN2 LBDs to date are the ones complexed to GluN1-GluN2A LBDs [40][41][42] , which have been obtained by soaking Gly-and L-glutamate-bound GluN1-GluN2A LBD crystals against the crystallization buffer containing Gly and GluN2 antagonists to substitute L-glutamate with the antagonists within the GluN2A LBD (see "Methods"). Similarly soaking the agonist-bound GluN2D LBD crystals 38 or co-crystallization with antagonists did not result in antagonist-bound GluN2D LBD structures.…”

Physiological activation of mGlu5 receptors supports the ion channel function of NMDA receptors in hippocampal LTD induction in vivo

“…injection in vivo 47 . NVP is a competitive antagonist with an ~11-fold preference for GluN1/2 A over GluN1/2B receptors, which will likely only maintain its selectivity to block only GluN2A diheteromeric or GluN2A/B triheteromeric NMDAR-mediated synaptic responses when used at relatively low, non-saturating concentrations 77 .…”

Recent progress in allosteric modulators for GluN2A subunit and development of GluN2A‐selective nuclear imaging probes

“…However, its high hydrophilicity with a log D 7.4 value of −1.1 resulted in poor BBB permeability and consequently impeded its use as a PET tracer. Iodine‐124/125 and carbon‐11 were incorporated into several analogues of compound 3 to develop GluN2A‐targeting radiotracers, and dicarbomethoxy analogous were generated as prodrugs to overcome the low brain penetration caused by the carboxylic acids . A comparative study between [ 125 I] 14 (estimated K i of 250 and 270 nM to GluN2A and GluN2B, respectively; cLogP=−3.0) and its corresponding prodrug [ 125 I] 15 (cLogP=1.1 with no reports on affinity) was carried out in ex vivo autoradiography.…”

“…Recently, another series of competitive GluN2A antagonists were reported by Lind et al based on the core structure of ( S )‐5‐[( R )‐2‐amino‐2‐carboxyethyl]‐4,5‐dihy‐dro‐1 H ‐pyrazole‐3‐carboxylic acid (ACEPC). Among them, ST3 ( 3 ) was identified as the most potent compound with a K i value of 52 nM for the GluN2A subunits and a 15‐fold preference over GluN2B subunits . Crystal structures of GluN1/2A LBD complexed with ACEPC ligands indicate that the two nitrogen atoms of pyrazole form polar interactions with the hydroxyl group of GluN2A Thr690.…”