Elsevier

Neuropharmacology

Volume 75, December 2013, Pages 324-336
Neuropharmacology

Structure-based discovery of antagonists for GluN3-containing N-methyl-d-aspartate receptors

https://doi.org/10.1016/j.neuropharm.2013.08.003Get rights and content

Highlights

  • A method to study pharmacology of GluN3 subunit in GluN1/N3 receptors was developed.

  • A virtual screen of the orthosteric binding site of GluN3A was performed.

  • 99 selected compounds were evaluated for GluN3 activity and selectivity.

  • We identified a novel competitive antagonist with preference for the GluN3B subunit.

  • Two non-competitive antagonists with preference for GluN3 subunits were identified.

Abstract

NMDA receptors are ligand-gated ion channels that assemble into tetrameric receptor complexes composed of glycine-binding GluN1 and GluN3 subunits (GluN3A-B) and glutamate-binding GluN2 subunits (GluN2A-D). NMDA receptors can assemble as GluN1/N2 receptors and as GluN3-containing NMDA receptors, which are either glutamate/glycine-activated triheteromeric GluN1/N2/N3 receptors or glycine-activated diheteromeric GluN1/N3 receptors. The glycine-binding GluN1 and GluN3 subunits display strikingly different pharmacological selectivity profiles. However, the pharmacological characterization of GluN3-containing receptors has been hampered by the lack of methods and pharmacological tools to study GluN3 subunit pharmacology in isolation. Here, we have developed a method to study the pharmacology of GluN3 subunits in recombinant diheteromeric GluN1/N3 receptors by mutating the orthosteric ligand-binding pocket in GluN1. This method is suitable for performing compound screening and characterization of structure–activity relationship studies on GluN3 ligands. We have performed a virtual screen of the orthosteric binding site of GluN3A in the search for antagonists with selectivity for GluN3 subunits. In the subsequent pharmacological evaluation of 99 selected compounds, we identified 6-hydroxy-[1,2,5]oxadiazolo[3,4-b]pyrazin-5(4H)-one (TK80) a novel competitive antagonist with preference for the GluN3B subunit. Serendipitously, we also identified [2-hydroxy-5-((4-(pyridin-3-yl)thiazol-2-yl)amino]benzoic acid (TK13) and 4-(2,4-dichlorobenzoyl)-1H-pyrrole-2-carboxylic acid (TK30), two novel non-competitive GluN3 antagonists. These findings demonstrate that structural differences between the orthosteric binding site of GluN3 and GluN1 can be exploited to generate selective ligands.

Introduction

N-methyl-d-aspartate (NMDA) receptors are ligand-gated cation-selective channels that belong to the family of ionotropic glutamate receptors (iGluRs) (Traynelis et al., 2010). Most native NMDA receptors are composed of two GluN1 subunits and two GluN2 subunits and are activated by simultaneous binding of glycine and glutamate to GluN1 and GluN2, respectively (Laube et al., 1998, Ulbrich and Isacoff, 2007). GluN3-containing NMDA receptors are either glutamate/glycine-activated triheteromeric receptors composed of GluN1, GluN2, and GluN3 subunits or glycine-activated diheteromeric receptors composed of GluN1 and GluN3 subunits (Chatterton et al., 2002, Das et al., 1998, Pérez-Otaño et al., 2001, Sasaki et al., 2002, Smothers and Woodward, 2007, Smothers and Woodward, 2009, Ulbrich and Isacoff, 2007, Ulbrich and Isacoff, 2008).

Although the GluN3A subunit was cloned almost two decades ago (Ciabarra et al., 1995, Sucher et al., 1995) followed by cloning of the GluN3B subunit in the beginning of this century (Andersson et al., 2001, Chatterton et al., 2002, Matsuda et al., 2002, Nishi et al., 2001), many aspects of the relationship between structure and function for both GluN3A and GluN3B subunits remain elusive (Cavara and Hollmann, 2008, Henson et al., 2010, Low and Wee, 2010, Pachernegg et al., 2012). The GluN3 subunits appears to function as modulatory subunits that reduce the susceptibility of NMDA receptors to Mg2+-blockage and reduce Ca2+-permeability (Cavara et al., 2010, Chatterton et al., 2002, Pérez-Otaño et al., 2001, Sasaki et al., 2002). Furthermore, GluN3 subunits are involved in synapse maturation (Das et al., 1998, Henson et al., 2012, Pérez-Otaño et al., 2006, Roberts et al., 2009), synaptic plasticity (Larsen et al., 2011), and are neuroprotective in various cells (Káradóttir et al., 2005, Martínez-Turrillas et al., 2012, Micu et al., 2006, Nakanishi et al., 2009, Salter and Fern, 2005). Consequently, the GluN3 subunits could be promising new targets for therapeutic intervention in neuropathological conditions that include excitotoxicity and cognitive impairment (Cavara and Hollmann, 2008, Henson et al., 2010, Stys and Lipton, 2007). In this regard, agonists and antagonists that are selective for the GluN3 subunits are of crucial importance in order to evaluate the physiological roles and therapeutic potential of GluN3-containing NMDA receptors.

It appears feasible to develop GluN3-selective agonists and antagonists due to the observed structural and pharmacological differences between the glycine-binding NMDA receptor subunits GluN1 and GluN3. Studies using soluble ligand-binding domains (LBDs) of GluN1 and GluN3A have revealed that glycine binds with a 650-fold higher affinity at GluN3A over GluN1, indicating that the glycine binding site of GluN3 is different from that of GluN1 (Yao and Mayer, 2006). Furthermore, GluN3A is reported to have a selectivity profile strikingly different from that of GluN1 with respect to binding affinities of known glycine-site antagonist; 5,7-dichlorokynurenic acid (DCKA), L-689,560 and other high-affinity glycine-site antagonists exhibit strong selectivity for binding GluN1 over GluN3A (Yao and Mayer, 2006). Crystal structures of GluN1, GluN3A, and GluN3B LBDs in complex with glycine or d-serine have also revealed differences in the agonist binding pocket that could be exploited to achieve selectivity (Yao et al., 2008).

Advances in our understanding of the functional properties and pharmacology of GluN3-containing NMDA receptors have been hampered by the fundamental problem of establishing expression systems that allows GluN3 function to be studied in isolation. Recombinant co-expression of GluN1, GluN2, and GluN3 subunits generates multiple receptor populations (i.e. GluN1/N2 and GluN1/N2/N3), confounding the study of GluN3-containing receptors (Das et al., 1998, Pérez-Otaño et al., 2001, Sasaki et al., 2002). Expression of recombinant GluN1/N3 receptors in Xenopus oocytes provides a convenient solution to this problem (Awobuluyi et al., 2007, Chatterton et al., 2002, Madry et al., 2007). However, glycine has dual action at these GluN1/N3 receptors, in that glycine appears to act agonistically at the GluN3 subunit and inhibitory through binding to the GluN1 subunit (Awobuluyi et al., 2007, Madry et al., 2007) resulting in bell-shaped concentration-response curves.

In this study, we establish a method suitable for evaluation of compounds at recombinantly expressed GluN3-containing NMDA receptors by mutating the orthosteric ligand-binding pocket in GluN1. Based on virtual screening of the orthosteric binding site in the LBD of GluN3A subunit followed by pharmacological evaluation of 99 compounds, we identify antagonists with preference for GluN3-containing NMDA receptors. The novel antagonists have been characterized and the pharmacological mechanism of inhibition has been described. This discovery provides evidence that structural differences between GluN1 and GluN3 subunits can be exploited to generate GluN3-selective ligands that could be useful tools to study the physiological roles of GluN3 subunits.

Section snippets

Molecular biology

cDNAs encoding the GluN1-1a (GenBank: U11418; hereafter GluN1), GluN2A (GenBank: D13211), GluN2B (GenBank: M91562), GluN2C (GenBank: D13212), and GluN2D (GenBank: D13214) subunits were generously provided by Dr. S. Nakanishi (Osaka Bioscience Institute, Osaka, Japan). cDNAs encoding GluA1 (GenBank: X17184) and GluK2 (GenBank: Z11548) subunits were generously provided by Dr. S. Heinemann (Salk Institute for Biological Studies, San Diego, CA). cDNAs encoding the short variant GluN3A-1 (GenBank: U29873

Electrophysiological evaluation of the agonist binding site in GluN3

To enable evaluation of GluN3-selective antagonists, we aimed to establish a straightforward system suitable for screening of a compound library for GluN3 activity. Recombinant diheteromeric GluN1/N3 receptors can be functionally expressed in Xenopus oocytes. However, glycine binds to both GluN1 and GluN3, and previous studies have found that GluN1/N3 receptors have a bell-shaped glycine concentration–response relationship, where the receptor current is diminished at high glycine concentration (

Discussion

In the present study, we have identified a GluN1 LBD mutant (GluN1(F484A/T518L)) that completely eliminates the inhibitory action of glycine at GluN1/N3 receptors. Using this GluN1 LBD mutant, we have established a method to evaluate compounds for activity at the GluN3 subunit in GluN3-containing receptors. We performed a virtual screen of the GluN3A LBD in the search for antagonists with selectivity for GluN3-containing receptors. By exploiting this structure-based approach, we have succeeded

Conclusion

To date, the physiological role of GluN3-containing receptors remains largely unknown. The lack of useful pharmacological tools, such as GluN3-selective agonists or antagonists, has slowed down our progress to understand the physiological roles of GluN3-containing receptors. Generation of selective GluN3 ligands together with studies on GluN3 pharmacology and functional properties have largely been hampered by the fundamental problem of investigating GluN3 function in isolation. By the

Acknowledgments

This work was supported by the GluTarget Programme of Excellence at the University of Copenhagen, the Danish Ministry of Science, Innovation and Higher Education's EliteForsk Programme and NIH-NINS (N036654, NS065371 SFT).

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