Stroebel D et al. (2016), A Novel Binding Mode Reveals Two Distinct Class...

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Mol Pharmacol 2016 May 01;895:541-51. doi: 10.1124/mol.115.103036.

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A Novel Binding Mode Reveals Two Distinct Classes of NMDA Receptor GluN2B-selective Antagonists.

Stroebel D , Buhl DL , Knafels JD , Chanda PK , Green M , Sciabola S , Mony L , Paoletti P , Pandit J .

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N-methyl-d-aspartate receptors (NMDARs) are glutamate-gated ion channels that play key roles in brain physiology and pathology. Because numerous pathologic conditions involve NMDAR overactivation, subunit-selective antagonists hold strong therapeutic potential, although clinical successes remain limited. Among the most promising NMDAR-targeting drugs are allosteric inhibitors of GluN2B-containing receptors. Since the discovery of ifenprodil, a range of GluN2B-selective compounds with strikingly different structural motifs have been identified. This molecular diversity raises the possibility of distinct binding sites, although supporting data are lacking. Using X-ray crystallography, we show that EVT-101, a GluN2B antagonist structurally unrelated to the classic phenylethanolamine pharmacophore, binds at the same GluN1/GluN2B dimer interface as ifenprodil but adopts a remarkably different binding mode involving a distinct subcavity and receptor interactions. Mutagenesis experiments demonstrate that this novel binding site is physiologically relevant. Moreover, in silico docking unveils that GluN2B-selective antagonists broadly divide into two distinct classes according to binding pose. These data widen the allosteric and pharmacological landscape of NMDARs and offer a renewed structural framework for designing next-generation GluN2B antagonists with therapeutic value for brain disorders.

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Species referenced: Xenopus
Genes referenced: ttn

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	Graphical Abstract
	Fig. 1. X-ray crystal structure of the GluN1/GluN2B NTD dimer in complex with EVT-101. (A) Structure of the tetrameric GluN1/GluN2B receptors (Karakas and Furukawa, 2014; Lee et al., 2014). GluN1 subunits are in dark gray and GluN2B subunits in pale gray. One NTD heterodimer is highlighted (GluN1 in green, GluN2B in blue). NTD, N-terminal domain; ABD, agonist-binding domain; TMD, transmembrane domain. (B) Structure of the GluN1/GluN2B NTD heterodimer in complex with EVT-101. For comparison purposes, the ifenprodil molecule as seen in the GluN1/GluN2B NTD-ifenprodil complex is superimposed. The two ligands shown in sphere representation (ifenprodil in orange, EVT-101 in purple) sit at the heterodimer interface. (C) Side view (rotated 90°) with the GluN1 NTD removed and the ligands shown in stick representation. (D–F) Difference electron density maps (mFo-DFc) for ifenprodil, MK-22, and EVT-101 contoured at 3.0 σ.
	Fig. 2. Comparison of the EVT-101, MK-22, and ifenprodil binding sites. (A) Views of the binding pockets of MK-22, ifenprodil, and EVT-101 at the GluN1/GluN2B NTD dimer interface. Lateral views as seen from the GluN2B subunit. Ligands are represented in stick with carbons colored in gold. The color code for the surface of the binding cavities is the following: green for carbons, blue for amines of basic residues, cyan for amines of backbone or polar residues, red for carboxylate groups, and salmon for oxygens of noncarboxylate groups. The amide group shown in stick corresponds to that of residue GluN2B-Q110, which delineates the two subcavities. (B) Contact maps showing residues that interact with MK-22, ifenprodil, and EVT-101. GluN1 and GluN2B residues are shown in gray and yellow, respectively. Amino acids shown in circles are making direct contacts with the ligand. Residues below the dashed line locate in the lower lobe of the GluN2B NTD.
	Fig. 3. Effects of GluN1 and GluN2B NTD mutations on EVT-101 and ifenprodil sensitivity. Dose-response inhibition curves of GluN1/GluN2B mutant receptors (♦). The dashed curves are the fits of the ifenprodil or EVT-101 dose-response data points obtained on wild-type (wt) receptors (○). The number of cells and the estimated values of IC50 and maximal inhibition for each mutant receptor are listed in Table 1. Estimated values of nH are comprised in the range 0.6–1.3 for ifenprodil and 0.7–1.4 for EVT-101. (A) GluN1-F114S/GluN2Bwt. (B) GluN1-Y109C/GluN2Bwt. (C) GluN1wt/GluN2B-Q110G. (D) GluN1wt/GluN2B-A135P. (E) GluN1-L135W/GluN2Bwt. (F) GluN1-L135H/GluN2Bwt. Note that although certain mutations affect the sensitivity to ifenprodil and EVT-101 indiscriminately, others have specific effects for either one of the two ligands. Error bars represent S.D.
	Fig. 4. In silico docking analysis of structurally-diverse GluN2B antagonists. (A) Protein-ligand fingerprints ("heatmaps") based on the EVT-101 protein cocrystal structure and displaying computed amino acid-ligand distances. Amino acids are organized according to interaction distance (color code indicates minimal distance to ligands, in Å); numbers on the x-axis represent ligands listed in Table 2. Data show two distinct groups of ligands (see arborization on top) indicating at least two main modalities of binding. (B) Pose overlay for the two groups of compounds. Green: EVT-101; orange: ifenprodil. MK-22 depicted as overlapping with ifenprodil on the left in cyan.

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