Stereoselectivity for the (R)‐Enantiomer of HA‐966 (l‐Hydroxy‐3‐Aminopyrrolidone‐2) at the Glycine Site of the N‐Methyl‐d‐Aspartate Receptor Complex

Abstract

HA-966 (1-hydroxy-3-aminopyrrolidone-2) is an antagonist at the glycine allosteric site of the N-methyl-D-aspartate receptor ionophore complex. Unlike presently known glycine antagonists, HA-966 is chiral. We report stereoselectivity for the (R)-enantiomer at the glycine antagonist site. In [3H]glycine binding, the (R)-enantiomer has an IC50 of 4.1 +/- 0.6 microM. The racemic mixture has an IC50 of 11.2 +/- 0.5 microM, whereas (S)-HA-966 has an IC50 greater than 900 microM. In glycine-stimulated [3H]1-[1-(2- thienyl)cyclohexyl]piperidine binding, the (R)-enantiomer inhibits with an IC50 of 121 +/- 61 microM, whereas the racemic mixture has an IC50 of 216 +/- 113 microM and (S)-HA-966 is inactive. The inhibition by (R)-HA-966 can be prevented by the addition of glycine. (R)-HA-966 and racemic HA-966, but not (S)-HA-966, also prevent N-methyl-D-aspartate cytotoxicity in cortical cultures. The (R)-enantiomer and, less potently, the (S)-enantiomer inhibit N-methyl-D-aspartate-evoked [3H]norepinephrine release from rat hippocampal slices (IC50 values of about 0.3 mM and 1.6 mM, respectively), but only the inhibition by (R)-HA-966 is reversed by added glycine. In glutamate-evoked contractions of the guinea pig ileum, (R)-HA-966 causes a glycine-reversible inhibition (IC50 of about 150 microM), whereas (S)-HA-966 is much less potent (IC50 of greater than 1 mM). These results demonstrate stereoselectivity of the glycine antagonist site of the N-methyl-D-aspartate receptor complex in a variety of tissues and assays. The stereoselectivity also confirms the specificity of N-methyl-D-aspartate receptors in glutamate-evoked contractions of the guinea pig ileum, and supports their similarity to central N-methyl-D-aspartate receptors.

The glycine antagonist (+)-HA-966 injected into the nucleus accumbens stimulates locomotion in mice

“…The failure of (-)-HA-966, which does not interact with the strychnineinsensitive glycine receptor (Pullan et al, 1990), to stimulate locomotion suggests that the locomotor response to (+)-HA-966 specifically involves the NMDA receptor-coupled glycine site. Again, though, other glycine site antagonists should be tested to support this interpretation.…”

“…To that end we injected the glycine site antagonist (+)-HA-966 into the nucleus accumbens of normal mice. The effects of (-)-HA-966, which does not interact with the strychnine-insensitive glycine receptor (Pullan et al, 1990), were also studied for comparative purposes.…”

Neurochemistry and pharmacology of the major hippocampal transmitter systems: Synaptic and nonsynaptic interactions

“…Since the glycine receptor is strychnine-sensitive, the results indicated that HA-966, similarly to kynurenic acid, acts at the modulatory glycine site of the NMDA receptor. The stereoselectivity of HA-966 at the glycine site was also demonstrated on NMDAevoked NA release (Pullan et al, 1990). It was observed that weaker agonists of the glycine site may act as antagonists when the glycine concentration is high (Clos et al, 1996).…”

Glycine Site Agonists of the NMDA Receptor: A Review

“…Compared to D-cycloserine, HA-966 has low intrinsic activity as a partial glycine site agonist: it is about 10% as effective as glycine (192). Consistent with the stereoselectivity of the glycine site, only the (D)-(-) enantiomer of HA-966 has activity at the site (133). The ( S ) -( + ) does not have activity at the glycine site.…”