Mechanism for non-competitive inhibition by novel GluN2C/D NMDA receptor subunit-selective modulators

The compound 4-(5-(4-bromophenyl)-3-(6-methyl-2-oxo-4-phenyl-1,2-dihydroquinolin-3-yl)-4,5-dihydro-1 H -pyrazol-1-yl)-4-oxobutanoic acid (DQP-1105) is a representative member of a new class of NMDA receptor antagonists. DQP-1105 inhibited GluN2C- and GluN2D-containing receptors with IC 50 values that were at least 50-fold lower than those for recombinant GluN2A-, GluN2B-, GluA1-, or GluK2-containing receptors. Inhibition was voltage-independent could not be surmounted by increasing concentrations of either co-agonists glutamate or glycine, consistent with a non-competitive mechanism of action. DQP-1105 inhibited single channel currents in excised outside-out patches without significantly changing mean open time or single channel conductance, suggesting that DQP inhibits a pre-gating step without changing the stability of the open pore conformation and thus channel closing rate. Evaluation of DQP-1105 inhibition of chimeric NMDA receptors identified two key residues in the lower lobe of the GluN2 agonist binding domain that control the selectivity of DQP-1105. These data suggest a mechanism for this new class of inhibitors and demonstrate that ligands can access, in a subunit-selective manner, a new site located in the lower, membrane-proximal portion of the agonist binding domain. 4-(5-(4-bromophenyl)-3-(6-methyl-2-oxo-4-phenyl-1,2-dihydroquinolin-3-yl)-4,5-dihydro-1 H -pyrazol-1-yl)-4-oxobutanoic acid (DQP-1105), which is approximately 50-fold selective for NMDA receptors containing GluN2C or GluN2D subunits over GluN2A- or GluN2B-containing receptors. We determined the mechanism of action and identified the structural determinants of subunit-selectivity for DQP-1105. Identification of the structure-activity glycine with various concentrations of DQP-1105 (from 0.3 M to 30 for 3 seconds, and a subsequent wash-out of antagonist with 100 μ M glutamate and 100 μ M glycine. All electrophysiological data from HEK cells in this study were collected at room temperature (23°C). Two-electrode voltage-clamp in Xenopus laevis oocytes recombinant GluN1/GluN2A, GluN1/GluN2B, μ M glutamate and 100 μ M glycine. DQP-1105 (30 μ M) inhibited the steady-state current response upon co-application with glutamate and glycine, with a final steady-state response of 0.2 ± 0.1% (n = 3) compared to the glutamate and glycine control. Data are from 6-8 cells. D, DQP-1105 composite concentration-response curves of the steady-state current responses activated by glutamate and glycine from dialyzed whole cell patch recordings give a fitted IC 50 value of 3.2 μ M GluN1/GluN2D receptors and 12 μ M for GluN1/GluN2A receptors (data are from 6 cells). E, Composite concentration-response a final steady-state response of 3.4 ± 1.1% (n=8) at 30 μ M DQP-1105 compared to control. B, The composite concentration-response relationship of the steady-state current responses to 1000 μ M NMDA plus 30 μ M glycine shows that DQP-1105 inhibits GluN1/GluN2D receptors with an IC 50 value of 1.9 μ M. C, There is a linear relationship between 1/ τ ONSET and DQP-1105 concentration, suggesting that the time-dependent inhibition of NMDA-activated GluN1/GluN2D reflects the association of DQP-1105 with its binding site. From this linear relationship, glycine in 6-8 cells. From HEK whole-cell experiments using conventional dialyzed patches, data are from 6-8 cells activated by 100 μ M glutamate or 1000 μ M NMDA plus 30 μ M glycine. Mean fitted Hill slopes for GluN1/GluN2C and GluN1/GluN2D were between 0.87-1.14; Hill slope was fixed to 1.0 for GluN1/GluN2A, GluN1/Glun2B, GluA1, and GluK2. NE indicates that there was no detectable effect at the concentrations evaluated; -- indicates that conditions were not assayed. Amino acid numbering and composition for chimeric GluN2A-GluN2D subunits. Amino acid numbering is according to the full-length GluN2 subunits, including the signal peptide (initiating methionine is 1).