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

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 ( k on ) and dissociation ( k off ) 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 ω‐phosphono, α‐amino acids k on and k off 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): k on 1.4 × 10 7 m −1 s −1 ; k off 20.3s −1 . 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: k on 1.1 × 10 6 m −1 s −1 ; k off 0.2s −1 . For the d ‐ and l ‐isomers of 3‐(2‐carboxypiperazin‐4‐yl)‐propyl‐1‐phosphonic acid (CPP) estimates for k on were similar, but for the l ‐isomer k off was 10 fold faster than for the d ‐isomer. 3 For 2‐amino‐5‐phosphonopentanoic acid (AP5) and its piperidine derivative cis ‐4‐(phosphonomethyl) piperidine‐2‐carboxylic acid (CGS 19755), an increase in chain length of two methylene groups between the ω‐phosphono and α‐carboxylate moieties caused a 1.6 to 1.8 fold decrease in k on with little change in k off . In contrast, for AP5, CPP and its ω‐carboxylate analogue, addition of a double bond close to the phosphonate moiety caused a 1.3 to 1.6 fold increase in k on . 4 For antagonists with an ω‐tetrazole moiety, k on and k off were 2.8–4.6 times faster than for the parent ω‐phosphono compounds. A similar, but smaller increase in k on and k off was observed for antagonists with an ω‐carboxylate moiety. 5 The slow kinetics of action of potent NMDA receptor antagonists were not an artefact of buffered diffusion. In neurones equilibrated with 200 μ m d ‐AP7, 2μ m LY 235959 and 10 μ m 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 10 μ m NMDA. 6 For all antagonists studied, the ratio k off / k on was consistent with equilibrium K i values obtained under similar experimental conditions, over a 40 fold range of potency. Comparison of these values with K i estimates determined from both agonist ([ 3 H]‐glutamate), and antagonist ([ 3 H]‐CGS 19755 and [ 3 H]‐CPP) radioligand competition studies revealed good correlation between data from voltage clamp and binding experiments. However, K i values obtained in antagonist binding assays showed on average 6.5 fold higher affinity than those obtained in voltage clamp experiments; in contrast K i values obtained in 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.