Chopra, Bela; (2000) Characterisation of the binding of two novel glycine site antagonists to cloned NMDA receptors. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The focus of this thesis was to investigate the N-methyl-D-aspartate (NMDA) receptor subtype-selectivity of 3-[2-(phenylaminocarbonyl)ethy1] -4,6-dicloroindole-2-carboxylie acid sodium salt (GV150,526A) and E-4,6-dichloro-3-(2-oxo-1phenyl-pyrrolidin-3-ylidenemethyl)-1H-indole-2-carboxylic acid (GV 196,771 A) by assaying their ability to inhibit the radioligand binding of [3H] (E)-3-(2-pheny1-2-carboxyethenyl)-4,6-dichloro-1H-indole-2-carboxylic acid (MDL105,519), to human embryonic kidney (HEK) 293 cells transfected with various combinations of NMDA receptor clones. Competition binding between [3H] MDL105,519 and GV150,526A to both NR1-1a and NR1-2a subunits was best fit to a one-site model and there were no significant difference in affinity of GV150,526A for the different splice forms. In contrast, GV 196,771A binding to each of the NR1 splice variants were best fit by a two-site model with the same affinity for both NR1-1a and NR1-2a splice forms. Interestingly, the competition profiles of both GV150,526A and GV196,771A to heteromeric NR1-1a/NR2s were best fit to a two-site compared to a one-site model. A small but significant selectivity was observed for both GV150,526A and GV196,771A where the rank order of decreasing affinity for GV150,526A was NR1-1a/NR2D>NR1-1a/NR2A > NR1-1a/NR2B>NR1-1a/NR2C. In contrast to GV150,526A, GV196,771A displayed a four-fold lower affinity for NR1- 1a/NR2A compared to NR1-1a/NR2B, NR1-1a/NR2C and NR1-1a/NR2D receptors. The biphasic competition curves of [3H] MDL105,519 radioligand binding to heteromeric NMDA receptors by both GV150,526A and GV196,771A were investigated by various approaches including studying the competition profiles for the inhibition [3H] MDL105,519 radioligand binding to NR1-1a/NR2A receptors by a series of glycine site ligands with diverse chemical structures. Of the five compounds investigated L689,560 exhibited similar behaviour to GV150,526A with respect to inhibition binding of [3H] MDL105,519 to single A NR1-1a subunits and NR1-1a/NR2A receptors. Secondly, intact cell surface radioligand binding was performed to investigate the displacement by both GV150,526A and GV196,771A of [3H] MDL105,519 radioligand binding to cell surface NR1-1a/NR2A receptors versus total receptor populations i.e. NR1-1a and NR1-1a/NR2A. However, both the intact cell surface radioligand binding and control experiments indicated that the NR1-1a subunit alone may be expressed at the cell surface. Furthermore, allosteric modulation by glutamate on the inhibition binding of of [3H] MDL105,519 by GV150,526A for native membranes under non-equilibrium conditions suggested that the biphasic competition curves may be due to allosteric interactions within a receptor complex. On the basis of these results presented in this thesis it is proposed that glycine site antagonists may be subdivided into two pharmacological classes, i.e. those that show simple competitive behaviour with regard to inhibition of [3H] MDL105,519 radioligand binding and those that result in complex inhibition curves.

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