Structure‐activity analysis of binding kinetics for NMDA receptor competitive antagonists: the influence of conformational restriction

Abstract

1 The kinetics of action of 17 structurally related NMDA receptor competitive antagonists were measured under voltage clamp in mouse hippocampal neurones. Analysis of the response to rapid changes in antagonist concentration during constant application of agonist was used to estimate microscopic association (k0.) and dissociation (k0ff) rate constants for antagonist binding, assuming a two-equivalent site model for competitive antagonism. Dose-inhibition curves were analysed to estimate antagonist equilibrium dissociation constants.2 For a series of 11 co-phosphono, a-amino acids k03 and k0ff varied 26 and 107 fold respectively. Rapid association and dissociation rate constants were obtained for flexible antagonist molecules such as D-2-amino-7-phosphonoheptanoic acid (D-AP7): k03 1.4 x 17 M-1 -I 1; kff 20.3 s -. For conformationally restrained molecules such as 3S,4aR,6S,8aR-6-phosphonomethyl-decahydroisoquinoline-3-carboxylic acid (LY 235959), association and dissociation rate constants were much slower: k01 1.1 x 106M-1s-1; koff 0.2 s'-. For the D-and L-isomers of 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) estimates for k0. were similar, but for the L-isomer koff was 10 fold faster than for the D-isomer.3 For 2-amino-5-phosphonopentanoic acid (AP5) and its piperidine derivative cis-44phosphonomethyl) piperidine-2-carboxylic acid (CGS 19755), an increase in chain length of two methylene groups between the co-phosphono and oc-carboxylate moieties caused a 1.6 to 1.8 fold decrease in k0. with little change in koffI In contrast, for AP5, CPP and its co-carboxylate analogue, addition of a double bond close to the phosphonate moiety caused a 1.3 to 1.6 fold increase in k0.. 0 4 For antagonists with an co-tetrazole moiety, k0. and koff were 2.8-4.6 times faster than for the parent co-phosphono compounds. A similar, but smaller increase in k0. and k0ff was observed for antagonists with an co-carboxylate moiety. S The slow kinetics of action of potent NMDA receptor antagonists were not an artefact of buffered diffusion. In neurones equilibrated with 200piM D-AP7, 2puM LY 235959 and 10pM NMDA, a transient agonist response was recorded following a rapid switch to D-AP7-free solution. This can only be explained by differences in the binding kinetics of AP7 and LY 235959, since at equilibrium, with these concentrations, either antagonist essentially eliminates the agonist response to 10puM NMDA. 6 For all antagonists studied, the ratio koff/k0. was agonist binding assays showed only 1.4 fold higher affinity. 7 The insights gained from our experiments may be of use for predicting the structural features required to generate more potent NMDA receptor antagonists, and suggest that novel acyclic compounds will have greater potential for high potency than derivatives of conformationally rigid compounds with piperazine, piperidine or bicyclic ring structures.

Equilibrium Constants for (R)-[(S)-1-(4-Bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-methyl]-phosphonic Acid (NVP-AAM077) Acting at Recombinant NR1/NR2A and NR1/NR2BN-Methyl-d-aspartate Receptors: Implications for Studies of Synaptic Transmission

“…Thus in our simulation, we have assumed an association rate (k ϩB ) for NVP-AAM077 at both NR2A and NR2B subunits of 1 ϫ 10 7 M Ϫ1 s Ϫ1 . Such a value is consistent with published values for this parameter obtained from studies that have examined a variety of competitive antagonists at NMDA receptors (Benveniste et al, 1990;Benveniste and Mayer, 1991). Thus, to agree with our K B values, we fixed the dissociation rate constants (k ϪB ) for NVP-AAM077 at 0.15 and 0.78 s Ϫ1 for NR1/NR2A and NR1/NR2B NMDA receptors, respectively.…”

Identification of Molecular Determinants That Are Important in the Assembly of N-Methyl-d-aspartate Receptors

“…However, the biochemical and functional studies reported to date are ambiguous with regard to NMDA receptor subunit stoichiometry. Functional studies indicate that binding of at least two molecules of both glutamate and glycine is required for NMDA receptor activation, suggesting that at least four subunits must co-assemble (11)(12)(13). The molecular size of native NMDA receptors, as determined by both gel filtration and native polyacrylamide gel electrophoresis, is in the range of 605-850 kDa, which is consistent with the coassembly of between four to five subunits (14 -16).…”

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

“…3A; see Table S2 for rate constants), the receptor contains two equivalent glutamate binding sites (i.e., GluN2 subunits) and requires agonist occupancy at both of these sites for activation. We assume that the association rate (k +B ) of the antagonist ST3 is 10 μM −1 ·s −1 , consistent with values for other competitive NMDA receptor antagonists (28,29). The antagonist dissociation rates (k -B ) for ST3 are therefore 0.52 s −1 at GluN1/2A and 7.8 s −1 at GluN1/2B, according to the experimentally determined…”

“…The presence of competitive antagonist will reduce the fraction of NMDA receptors that activate during synaptic release of glutamate. That is, the antagonist will bind a subset of NMDA receptors, depending on antagonist concentration and binding affinity, and prevent agonist binding and subsequent gating of these receptors after synaptic release of glutamate; the mean lifetimes of antagonist-receptor complexes are typically much longer than the few milliseconds that glutamate is available for binding (28,29). Frizelle et al (15) used kinetic modeling to predict that, under nonequilibrium conditions relevant to those encountered during rapid release and removal of synaptic glutamate, NVP is incapable of fully inhibiting Glutamate concentration-response data in the absence or presence of antagonist were analyzed by simultaneously fitting all data to both the Schild and Hill equations using global nonlinear regression (SI Materials and Methods).…”