Regan MC et al. (2019), Structural elements of a pH-sensitive inhibitor...

XB-ART-55699

Nat Commun 2019 Jan 18;101:321. doi: 10.1038/s41467-019-08291-1.

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Structural elements of a pH-sensitive inhibitor binding site in NMDA receptors.

Regan MC , Zhu Z , Yuan H , Myers SJ , Menaldino DS , Tahirovic YA , Liotta DC , Traynelis SF , Furukawa H .

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Context-dependent inhibition of N-methyl-D-aspartate (NMDA) receptors has important therapeutic implications for the treatment of neurological diseases that are associated with altered neuronal firing and signaling. This is especially true in stroke, where the proton concentration in the afflicted area can increase by an order of magnitude. A class of allosteric inhibitors, the 93-series, shows greater potency against GluN1-GluN2B NMDA receptors in such low pH environments, allowing targeted therapy only within the ischemic region. Here we map the 93-series compound binding site in the GluN1-GluN2B NMDA receptor amino terminal domain and show that the interaction of the N-alkyl group with a hydrophobic cage of the binding site is critical for pH-dependent inhibition. Mutation of residues in the hydrophobic cage alters pH-dependent potency, and remarkably, can convert inhibitors into potentiators. Our study provides a foundation for the development of highly specific neuroprotective compounds for the treatment of neurological diseases.

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	Fig. 1. pH-sensitive negative allosteric modulators of GluN2B-containing NMDA receptors. a The 93-series compounds28 are shown together with the phenylethanolamine ifenprodil and EVT-101. b Representative TEVC recordings of wild-type GluN1-4a/GluN2B NMDA receptors in Xenopus oocytes are shown in response to maximally effective concentration of glutamate and glycine (100 and 30 μM, respectively). When normalized to the maximal response, recordings at pH 6.9 showed substantially higher potency of 93-31 than at pH 7.6. c Concentration–response curves from TEVC experiments at pH 7.6 (gray) and 6.9 (black) for inhibition of wild-type GluN1-4a/GluN2B NMDA receptor by 93-31 (also see Table 2). Symbols and error bars represent mean ± S.E.M.; the number of replicates is listed in Table 2
	Fig. 2. ITC titrations of the GluN1/GluN2B ATD with different ligands at pH 7.6 and 6.5. Representative ITC isotherms of the isolated GluN1-GluN2B ATD titrated with 93-31 at pH 7.6 (a) and 6.5 (b). The average dissociation constant (KD) for 93-31 at pH 7.6 was 121 ± 5.1 nM, which drops to 78.4 ± 2.97 nM at pH 6.5 (Table 1). c Dissociation constants for various 93-series ligands at pH 7.6 (black bar) and pH 6.5 (gray bar). d The ratio of the KD at pH 7.6 to the KD at 6.5 (pH-boost) is shown as a function of N-substituent volume. For all panels, error bars represent mean ± S.E.M. All experiments were performed in triplicate
	Fig. 3. Structure of the 93-series binding site. a The intact heterotetrameric GluN1b/GluN2B NMDA receptor34 is composed of three structured domains, with the ATD furthest from the cell membrane (PDB code: 6CNA). b, c The crystal structure of the isolated GluN1b/GluN2B ATD heterodimer bound to the pH-sensitive 93-31 reveals the ligand-binding site at the heterodimer interface, as viewed from two angles. d, e The key residues surrounding the N-alkyl chain of 93-31 are primarily hydrophobic, shown here in stereo view. f Overlay of 93-31 and ifenprodil25 (yellow, PDB code: 3QEL). g Overlay of 93-31 and EVT-101 (ref. 29) (green, PDB code: 5EWM)
	Fig. 4. TEVC concentration–response curves for GluN1-4a–GluN2B mutants. a The N-alkyl group of 93-31 is accommodated by the “hydrophobic cage” as shown by the hydrophobic (red) to non-hydrophobic (white) surface presentation. b Key residues at the GluN1/GluN2B ATD dimer interface that interact with the N-alkyl chain of 93-31 were mutated and evaluated for effects on pH sensitivity of 93-31. c–i Current responses to maximally effective concentration of glutamate and glycine (100 μM glutamate, 30 μM glycine) are shown in the presence of varying concentrations of 93-31 as a proportion of maximal response. Experiments conducted at pH 7.6 are shown in gray; experiments at pH 6.9 are shown in black. h, j Two mutations, GluN1-4a(His134Ala) and GluN1-4a(Tyr109Trp), convert 93-31 into a potentiator. Experiments here were performed at pH 7.6 (gray), with the wild-type curve displayed for comparison (black). Error bars represent mean ± S.E.M. See Tables 2 and 3 for fitted IC50 values and the number of independent replicates

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Braun, Dynamic brain network reconfiguration as a potential schizophrenia genetic risk mechanism modulated by NMDA receptor function. 2016, Pubmed