Binding characterization of N-(2-chloro-5-thiomethylphenyl)-N'-(3-[3 H]3 methoxy phenyl)-N'-methylguanidine ([3 H]GMOM), a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist

Abstract

Labeled with carbon-11, N-(2-chloro-5-thiomethylphenyl)-N'-(3-methoxyphenyl)-N'-methylguanidine ([¹¹ C]GMOM) is currently the only positron emission tomography (PET) tracer that has shown selectivity for the ion-channel site of N-methyl-D-aspartate (NMDA) receptors in human imaging studies. The present study reports on the selectivity profile and in vitro binding properties of GMOM. The compound was screened on a panel of 80 targets, and labeled with tritium ([³ H]GMOM). The binding properties of [³ H]GMOM were compared to those of the reference ion-channel ligand [³ H](+)-dizocilpine maleate ([³ H]MK-801), in a set of concentration-response, homologous and heterologous inhibition, and association kinetics assays, performed with repeatedly washed rat forebrain preparations. GMOM was at least 70-fold more selective for NMDA receptors compared to all other targets examined. In homologous inhibition and concentration-response assays, the binding of [³ H]GMOM was regulated by NMDA receptor agonists, albeit in a less prominent manner compared to [³ H]MK-801. Scatchard transformation of homologous inhibition data produced concave upward curves for [³ H]GMOM and [³ H]MK-801. The radioligands showed bi-exponential association kinetics in the presence of 100 μmol L^-1 l-glutamate/30 μmol L^-1 glycine. [³ H]GMOM (3 nmol L^-1 and 10 nmol L^-1 ) was inhibited with dual affinity by (+)-MK-801, (R,S)-ketamine and memantine, in both presence and absence of agonists. [³ H]MK-801 (2 nmol L^-1 ) was inhibited in a monophasic manner by GMOM under baseline and combined agonist conditions, with an IC₅₀ value of ~19 nmol L^-1 . The non-linear Scatchard plots, biphasic inhibition by open channel blockers, and bi-exponential kinetics of [³ H]GMOM indicate a complex mechanism of interaction with the NMDA receptor ionophore. The implications for quantifying the PET signal of [¹¹ C]GMOM are discussed.

Keywords: N; NMDA receptor; N′-diaryl-N-methylguanidine; [3H]GMOM; [3H]MK-801; binding; ion-channel.

Figure 1

Structures and affinity values of NMDA receptor PET tracers used in human imaging studies

Scheme 1

Synthesis of [³H]GMOM. (1) [³H]methyl nosylate. (2) N‐(2‐chloro‐5‐thiomethylphenyl)‐N΄‐(3‐hydroxyphenyl)‐N΄‐methylguanidine. (3) N‐(2‐chloro‐5‐thiomethylphenyl)‐N΄‐(3‐[³H]₃ methoxyphenyl)‐N΄‐methylguanidine ([³H]GMOM). i: DMF, NaOH (5 mol L⁻¹), 80°C, 30 minutes

Figure 2

Analytical HPLC chromatogram of [³H]GMOM. The upper panel shows the radioactivity channel, the lower panel is the UV channel, spiked with unlabeled GMOM. Radiochemical and chemical purity was >99%

Figure 3

Evaluation of optimal binding conditions. A, Pretreatment of GF/B filters in 0.15% PEI for 1 hours reduces non‐specific [³H]GMOM binding. B, The binding of [³H]GMOM and [³H]MK‐801 is stable for at least 24 hours at 25°C. C, Tissue dilution curves were constructed to avoid radioligand depletion in subsequent studies. D, The binding of [³H]GMOM and [³H]MK‐801 is heat‐labile. E, For [³H]GMOM, maximum, pH‐corrected specific binding occurs at pH 7.4. Percentages in black and blue correspond to the proportion of non‐protonated and protonated species of GMOM at different pH values, respectively

Figure 4

Regulation of radioligand binding by orthosteric NMDA receptor agonists/antagonists. Repeatedly washed, crude synaptic membranes were prepared from rat brain and immediately incubated with [³H]GMOM and [³H]MK‐801 for ~20 hours, in the presence and absence of NMDA receptor agonists/antagonists. A, Maximal l‐glutamate‐induced increases over baseline were 3‐fold lower for the binding of [³H]GMOM compared to [³H]MK‐801. B, [³H]GMOM binding was increased in a linear, rather than exponential manner by increasing concentrations of glycine. C, NMDA and (RS)‐(Tetrazol‐5‐yl)glycine were used at concentrations spanning ~1, 2 and 10× their respective affinity values for the GluN2 subunit of NMDA receptors, to confirm the differential sensitivity of [³H]GMOM and [³H]MK‐801 to NMDA receptor agonism. D, The GluN1 subunit antagonist CGP 78608 and the GluN2 subunit antagonist D‐AP5 decreased the binding of [³H]GMOM, albeit less than [³H]MK‐801. Results are the mean ± SEM of 3‐4 experiments, each conducted in triplicate. *P < 0.05; **P < 0.01; ***P < 0.001 vs [³H]GMOM, LSD post‐hoc tests

Figure 5

Homologous inhibition curves. Inhibition of the binding of [³H]GMOM (A and B, SA: 84.5 Ci/mmol) and [³H]MK‐801 (C and D; SA: 22.5 Ci/mmol) by increasing concentrations of unlabeled GMOM and MK‐801, respectively, under baseline (A and C) and combined agonist conditions (B and D). The inserts show Scatchard plots of [³H]GMOM and [³H]MK‐801, obtained by transformation of homologous binding data by using 100 μmol L⁻¹ of unlabeled ligand to define NSB. Repeatedly washed preparations from the rat cortex and hippocampus were incubated with the radioligands for a period of 20 hours. Mean radioactivity counts added were 32 196 ± 1316 for 1 nmol L⁻¹ [³H]GMOM and 21 610 ± 1516 for 2 nmol L⁻¹ [³H]MK‐801. Radioligand depletion was <10% for [³H]GMOM. For [³H]MK‐801, under combined agonist conditions, maximum observed radioligand depletion was 16%

Figure 6

Inhibition curves of [³H]GMOM and [³H]MK‐801 by NMDA receptor ion‐channel blockers. Best‐fit curves to one‐ or two‐site models for the inhibition of [³H]GMOM (A) and [³H]MK‐801 (B) are shown for baseline and agonist‐stimulated conditions. Model preference was confirmed by visual inspection of the inhibition curves, and by the Akaike method. Each point is the mean ± SEM of triplicate determinations from single experiments, repeated 3‐9 independent times. Mean total bound [³H]GMOM (3 nmol L⁻¹) and [³H]MK‐801 (2 nmol L⁻¹) under baseline conditions was 2216 ± 442 and 1137 ± 135 fmol per mg protein, respectively. In the presence of agonists, mean total bound [³H]GMOM (3 nmol L⁻¹) and [³H]MK‐801 (2 nmol L⁻¹) were 2733 ± 208 and 2615 ± 124 fmol per mg protein, respectively

Figure 7

Association kinetics of [³H]GMOM and [³H]MK‐801. Representative association curves and concentration‐induced changes in the observed kinetic rate constants (k _ob) of [³H]GMOM and [³H]MK‐801, derived by analysis of total (A and B) and specific (C and D) radioligand binding values. The fast and slow k _ob values of total radioligand binding changed in opposite directions by increasing the concentration of radioligand (B). Analysis of specific binding, determined in the presence of 500 μmol L⁻¹ ketamine, revealed concentration‐induced increases in the fast rather than slow association phase of radioligand binding (D)