Studies on the subtype selectivity of CP-101,606: evidence for two classes of NR2B-selective NMDA receptor antagonists
Introduction
The N-methyl-D-aspartate (NMDA) subtype of glutamate receptor is a major therapeutic target for a wide range of disparate clinical indications such as stroke, analgesia, epilepsy and many degenerative diseases (Chazot and Hawkins, 1999). The NMDA receptor is a hetero-oligomeric cation channel complex comprising two major subunit types, termed NR1 and NR2A-D. Based on protein biochemical, pharmacological and physiological studies, four major defined subtypes comprising NR1/NR2A, NR1/NR2B, NR1/NR2C and NR1/NR2D exist in the adult mammalian CNS (reviewed in Dingledine et al., 1999; Chazot and Hawkins, 1999). However, there is growing evidence for the existence of more complex NMDA receptor subtypes in the CNS comprising multiple NR2 subunit types (Blahos and Wenthold, 1996; Chazot and Stephenson, 1997; Chazot et al., 1994; Luo et al., 1996; Sheng et al., 1994). Therapeutic implications for understanding the heterogeniety of NMDA receptors in the brain and the development of subtype-selective compounds are clear (Chazot and Hawkins, 1999; Chazot, 2000; Chenard and Menniti, 1999). In previous studies, we have shown that Ro-25,6981 binds both NR1/NR2B and NR1/NR2A/NR2B receptors with similar high affinities, suggesting that Ro-25,6981 is an NR2B-selective compound, irrespective whether complexed with other NR2 subunit types (Hawkins et al., 1999). Preliminary indirect studies have indicated that the related compound CP-101,606, although clearly NR2B-selective, displays distinct pharmacological selectivity to Ro-25,6981, i.e. may be influenced by the presence of other NR2 subunit types within the NMDA receptor complex (Brimbcome et al., 1997; Chazot, 2000). Furthermore, recent in vitro mutagenesis studies have shown that Ro-25,6981 and CP-101,606 require distinct amino acid residues within the NR2B subunit (reviewed in Chenard and Menniti, 1999). We proposed to delineate directly the subtype-selectivity of racemic [3H]CP-101,606, a novel high-affinity NMDA receptor radiolabelled form of CP-101,606 using defined recombinant NMDA receptor subunits expressed in HEK 293 cells (radioligand kindly donated by Dr F. Menniti (Pfizer Inc., Groton, USA) (Chenard and Menniti, 1999).
Section snippets
Materials
CP-101,606 was synthesised as described by Chenard et al. (1995). Racemic [3H]CP-101,606 was prepared as in Menniti et al. (1997) (specific activity=22 Ci/mmol, stored at −20 °C). [3H]Ro-25,6981 was a kind gift from Dr. John Kemp and colleagues (Hoffman–LaRoche, Switzerland (specific activity=25 Ci/mmol, stored at -20 °C). Cell culture reagents were purchased from Sigma, UK. All other reagents were purchased from commercial sources.
Generation and characterisation of NMDA receptor subunit antibodies
A new panel of anti-NR1 (17–35Cys), anti-NR2A (1381–1394),
Radioligand binding studies in native brain preparations
A series of preliminary radioligand binding experiments were performed using adult rat forebrain membranes to determine the appropriate assay conditions and binding isotherms for [3H]CP-101,606, and to establish the inhibition constants for unlabelled CP-101,606 and spermidine (Table 1, Fig. 1(A)). 10 mM spermidine was established as the appropriate concentration to define specific [3H]CP-101,606 binding. This yielded >80% specific [3H]CP-101,606 binding at a concentration of 2 nM radioligand.
Discussion
[3H]CP-101,606 is a novel radioligand for studying native and recombinant NMDA receptors. In this study, we showed that there was no significant difference between the equilibrium constant for [3H]CP-101,606 and the affinity constants determined for unlabelled CP-101,606 and spermidine for NR1/NR2B receptors expressed in HEK 293 cells and adult rodent forebrain membranes (Table 1). Furthermore, these values corresponded well with previous reports using a range of NR2B-directed radioligands (
Conclusions
In conclusion, these results demonstrated that [3H]CP-101, 606 binds with high affinity to NR1/NR2B, significantly lower affinity to NR1/NR2A and NR1/NR2A/NR2B receptors. Therefore, this provides further evidence for two distinct classes of NR2B antagonists, which may have important therapeutic implications. Notably, previous work has suggested that distinct native cortical receptor populations which may contain NR2A and NR2B subunits, were blocked by ifenprodil but not by haloperidol (Kew
Acknowledgements
We wish to thank the Wellcome trust (PLC and CLT) for financial support. S Lawrence was a Wellcome Trust Vacation Scholar. We wish also to thank V Hann for technical assistance, Professor FA Stephenson for the NMDA receptor subunit cDNAs and Dr. F Menniti (Pfizer Inc, Groton, USA) for the [3H]CP-101,606 and unlabelled CP-101,606, and Dr. John Kemp and colleagues (Hoffman–LaRoche, Switzerland) for the [3H]Ro-25,6981.
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