The N-methyl-D-aspartate receptor (NMDA-R), a subtype of glutamate-activated receptors, is critical for several brain functions, including synaptic plasticity, learning, and memory (Collingridge, 1987; Kauer et al., 1988). Proper NMDA-R signaling is essential for various normal brain functions, but excessive activation can lead to excitotoxicity, resulting in conditions such as ischemic stroke, traumatic brain injury, and Alzheimer’s disease (Stanika et al., 2009; Mira and Cerpa, 2021; Companys-Alemany et al., 2022).

The NMDA-R is a heterotetramer composed of two GluN1 subunits and two GluN2 subunits (GluN2A, GluN2B, GluN2C, or GluN2D) arranged diagonally (Paoletti et al., 2013). The GluN1 subunit contains binding sites for glycine or D-serine, which act as co-agonists necessary for receptor activation (Furukawa and Gouaux, 2003). In contrast, the GluN2 subunits contain binding sites for glutamate, the primary excitatory neurotransmitter in the brain (Kuryatov et al., 1994). Additionally, the NMDA-R has binding sites for modulators such as zinc, magnesium, and polyamines (Erreger et al., 2005). This subunit configuration is crucial for the receptor’s ion channel function, contributing to its diverse ionotropic modulation, channel kinetics, mobility, and signal transduction capabilities.

NMDA-R activation by its co-agonists glutamate and glycine leads to ion channel opening, allowing Ca²⁺ and Na⁺ influx and limited K⁺ efflux (Dingledine et al., 1999). This ionic flux initiates various downstream signaling cascades. The NMDA-R channel exhibits voltage-dependent properties, requiring membrane depolarization to displace the Mg²⁺ ion that blocks the channel pore at resting membrane potential (Mayer et al., 1984; Nowak et al., 1984).

Emerging research highlights that NMDA-Rs are not just ligand-gated ion channels but also play versatile roles in cellular signaling, encompassing both ionotropic and non-ionotropic functions (Harney et al., 2006; Nabavi et al., 2013; Li et al., 2022). Non-ionotropic signaling is initiated by the binding of glutamate alone, which induces conformational changes in the receptor’s extracellular agonist-binding domain. These changes are transmitted across the cell membrane, altering the conformation of the intracellular C-terminal domain (Traynelis et al., 2010; Zhu et al., 2016). This conformational shift activates downstream signaling pathways through protein-protein interactions with various intracellular mediators associated with the NMDA-R complex.

The therapeutics targeting NMDA-R encompass both agonists and antagonists. For example, ketamine and its enantiomer esketamine demonstrate rapid antidepressant efficacy in treatment-resistant depression (Zarate et al., 2006). In moderate to severe Alzheimer’s disease, memantine is prescribed for symptom management and to delay disease progression (Reisberg et al., 2003). Additionally, amantadine serves as a therapeutic option for managing Parkinsonian motor symptoms and drug-induced adverse effects (Sawada et al., 2010). However, whether these drugs’ clinical benefits stem directly from their NMDA-R antagonism or involve alternative targets or non-ionotropic mechanisms remains to be fully elucidated.

In this study, we investigated the structure-activity relationships (SAR) targeting 30 NMDA-R ligands. Additionally, we examined the correlation between ligand binding affinity and the ERK activation through the metabotropic signaling pathways of NMDA-R.

Reagents

N-methyl-D-aspartate (NMDA), 4-Amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine (PP2), Gö6976, Gö6983, and wortmannin were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Antibodies to phospho-ERK1/2 and ERK2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and anti-mouse HRP-conjugated secondary antibodies were obtained from Jackson ImmunoResearch (West Grove, PA, USA). [³H]-TCP (Piperdyl-3,4-³H]-N-(1-[thienyl] cyclohexyl)piperidine) was purchased from PerkinElmer Life Sciences (Waltham, MA, USA). Thirty putative NMDA ligands were provided by the Korean Ministry of Food and Drug Safety (Cheongju, Korea).

Cell culture and transfection

Human embryonic kidney (HEK)-293 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in media supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) and antibiotics (100 μg/mL penicillin and 100 μg/mL streptomycin) at 37°C in a humidified 5% CO₂ incubator. Transfections were performed using polyethyleneimine (PEI, MW 25,000) which was obtained from Polysciences, Inc. (Warrington, PA, USA).

Determination of the binding affinity of NMDA ligands

HEK-293 cells were transfected with GluN2B subunits and were plated onto the 24-well plates coated with poly-l-lysine (Thermo Fisher Scientific). The next day, cells were incubated with 10 nM [³H]-TCP in serum-free media containing 0.3% BSA for 1 h at 20°C. For the non-specific binding, 10 μM MXE was added. After incubation, the cells were washed four times with warm serum-free media, lysed with 250 μL of 1% sodium dodecyl sulfate (SDS) per well, and counted using a liquid scintillation analyzer (1450 MicroBeta TriLux, PerkinElmer). The resulting dose-response curves were obtained by fitting nonlinear regression using GraphPad Prism, and IC₅₀ values were estimated from the fitting curves. Ki values were converted from IC₅₀ values according to the Cheng-Prusoff equation (Cheng and Prusoff, 1973), Ki=IC₅₀/(1+[S]/Kd), where Kd and [S] represent the equilibrium dissociation constant and the concentration of [³H]-TCP used.

ERK measurement

Cells expressing the GluN2B subunit of the NMDA-R were cultured in 6-well plates and starved overnight in a serum-free medium containing 0.1% bovine serum albumin (BSA). They were then treated with the specified concentration of putative NMDA-R ligands for 10 min, and the reaction was halted by adding 1% SDS directly to each well. ERK activation was determined using an antibody against phospho-ERK or ERK2.

Immunoblotting

The cell lysates were incubated for 20 min at 65°C and sonicated to shear genomic DNA. The cell lysates were incubated for 20 min at 65°C and sonicated to shear genomic DNA. Proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. The membranes were blocked for 1 h at room temperature in TBS-Tween 20 (TBS-T) containing 5% nonfat dry milk or 4% BSA, followed by a 1-h incubation with an antibody against phospho-ERK or ERK2 (1:1,000 dilution) and subsequent 1-h incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (1:3,000 dilution) in 2% nonfat dry milk. Protein bands were visualized using a chemiluminescent western blotting kit. The same samples were also processed to detect ERK, and signals were quantified using a ChemiDoc MP imaging system (Bio-Rad, Hercules, CA, USA).

Statistical analysis

The analysis was conducted using GraphPad Prism 8 software (GraphPad Software Inc., San Diego, CA, USA). Data were expressed as the mean ± SD and were not assessed for normality. Statistical significance was assessed using ANOVA with Tukey’s post hoc test for multiple groups. The post hoc tests were conducted only if the F value in ANOVA achieved a significance level of p<0.05 and there was no significant variance inhomogeneity.

Structure-activity relationship of NMDA receptor ligands

For the structure-activity relationship (SAR) study, the compounds were broadly classified into two groups based on their substituents: 1-(1-Phenylcyclohexyl)amines (Table 1, series A, 1-12) and α-amino-2-phenylcyclohexanones (Table 2, series B, 13-30). The ligand binding study was performed using cells expressing the GluN2B subunit. These cells were treated with [³H]-TCP along with increasing concentrations of potential NMDA-R ligands.

Table 1 Binding affinity of compounds 1-14 for NMDA receptors (Ki, nm)

Compd	Name	R1	R2	X	Ki (nM)	ERK activation
1	PCA	-NH2	-	C	205.5	No
2	PCE	-NH-CH2CH3	-	C	745.6	Yes
3	PCPy	Pyrrolidine	-	C	175.4	Yes
4	PCAz	Azepane	-	C	13.9	No
5	PCBn	Phenylmethanamine	-	C	84.5	No
6	3-HO-PCE	-NH-CH2CH3	3-OH	C	29.3	Yes
7	3-MeO-PCE	-NH-CH2CH3	3-OMe	C	56.4	No
8	4’-F-PCPy	Pyrrolidine	4-F	C	151.8	Yes
9	4’-F-PCP	Piperidine	4-F	C	127.7	Yes
10	4’-MeO-PCP	Piperidine	4-OMe	C	190.1	No
11	3-HO-PCP	Piperidine	3-OMe	C	164.1	Yes
12	3-MeO-PCMo	Morpholine	3-OMe	C	81.4	Yes
13	4-Keto-PCP	Piperidine	-	C=O	2,100	No
14	4-Keto-4’-MeO-PCP	Piperidine	4-OMe	C=O	29.9	No

*Compounds 13 and 14 belong to 4-phenyl-4-(piperidin-1-yl)cyclohexanones.

Table 2 Binding affinity of compounds 15-30 for NMDA receptors (Ki, nM)

Compd	Name	R1	R2	Ki (nM)	ERK activation
15	Deschloroketamine	-CH3	-	23.6	Yes
16	2-Oxo-PCE	-CH2CH3	-	1,690	No
17	Nor-2-MXE	H	2-OMe	1,820	No
18	Nor-MXE	H	3-OMe	674	No
19	Nor-4-MXE	H	4-OMe	30.5	Yes
20	Desmethyl-nor-MXE	H	3-OH	5.1	Yes
21	Desmethyl-nor-4-MXE	H	4-OH	5.0	Yes
22	Desmethyl-MXE	-CH2CH3	3-OH	205.5	No
23	Desmethyl-4-MXE	-CH2CH3	4-OH	219.5	Yes
24	2-MXE	-CH2CH3	2-OMe	4,390	No
25	MXE (methoxetamine)	-CH2CH3	3-OMe	191	Yes
26	4-MXE	-CH2CH3	4-OMe	2,020	No
27	Ketamine	-CH3	2-Cl	4,290	No
28	N-ethyl-norketamine	-CH2CH3	2-Cl	343.5	Yes
29	2-Fluorodeschloroketamine	-CH3	2-F	2,540	No
30	9b-ethlaminodibenzofuranol			1.2	No

In the case of the 1-(1-phenylcyclohexyl)amine series (Table 1), R1 and the phenyl ring are attached to the same carbon on the cyclohexyl ring, and R2 is attached to the 3 or 4 positions of the phenyl ring. When X=C and R2=H, a ligand exhibited three times lower affinity for the NMDA receptor (NMDA-R) when R1 was changed from NH₂ to NHCH₂CH₃ (from compound 2 to compound 1). Similarly, the affinity increased 12-fold and 6-fold when R1 was azepane (compound 4) compared to pyrrolidine (compound 3) and phenylmethanamine (compound 5), respectively. When X=C and R1=NHCH₂CH₃, the affinity for the NMDA-R increased approximately 13 times when R2 was changed from H (compound 2) to 3-OH (compound 6) and about 25 times when R2 was changed to 3-OMe (compound 7). Additionally, when R1=piperidine and R2=4-OMe, changing X from C (compound 10) to C=O (compound 14) resulted in an approximately 11-fold increase in affinity. When X was C=O and R1=piperidine, changing R2 from H (compound 13) to 4-OMe (compound 14) increased the affinity by approximately 70 times.

α-Amino-2-phenylcyclohexanone (Table 2), more commonly known as methoxetamine (MXE), is an analog of ketamine. While it shares a similar cyclohexanone core structure with ketamine, MXE differs by having a 3-methoxy group and a different positioning of the amine group. Structurally, α-amino-2-phenylcyclohexanone features a cyclohexanone ring with an NH-R1 group and a phenyl ring attached to the carbon adjacent to the carbonyl carbon. The R2 group is bonded to positions 2, 3, or 4 of the phenyl ring.

The highest binding affinity was observed for compounds with R1=H and R2=OH (compounds 20, 21). When R1 was CH3, adding 2-Cl (compound 27) or 2-F (compound 29) to the R2 group significantly decreased the affinity by about 180 times and 107 times, respectively, compared to the compound without an R2 group (compound 15). Additionally, when R1 was CH2CH3, the ligand generally exhibited low affinity.

The metabotropic signal transduction pathway of the NMDA receptor involved in the ERK activation

NMDA-Rs are tetrameric structures consisting of GluN1 and GluN2 subunits arranged in a dimer of dimers, with the subunits alternating around the ion pore (Furukawa et al., 2005; Kohr, 2006). This specific assembly of subunits into a tetramer is essential for the proper functioning of the ion channel.

Recent studies are expanding our understanding of NMDA-Rs beyond their traditional role as ligand-gated ion channels, highlighting their capabilities as complex signaling entities with both ionotropic and non-ionotropic functions. Non-ionotropic signaling of NMDA-Rs occurs when ligand binding to the extracellular agonist-binding domain induces conformational changes that are transmitted across the cell membrane, altering the conformation of the intracellular C-terminal domain (Nabavi et al., 2013; Dore et al., 2015). This emerging view positions NMDA-Rs as versatile macromolecules in various cellular signaling pathways.

Because it was reported that the GluN2B subunit plays an important role in the metabotropic signaling of NMDA-R (Kessels et al., 2013; Dore et al., 2015), we conducted experiments using cells expressing GluN2B, which binds NMDA ligands. Under these conditions, pore formation is impossible, so only non-ionotropic signaling pathways of NMDA-R are possible.

A time course study showed that the ERK activation mediated by the GluN2B subunit of NMDA-R became evident 2 min after NMDA treatment and kept increasing until 10 min (Fig. 1A). A dose-response study showed that the ERK activation became evident at 1 μM NMDA and was further increased with 10 μM NMDA (Fig. 1B).

Figure 1. Characterization of ERK activation via non-ionotropic NMDA-R signaling pathway. (A) HEK-293 cells were transfected with 2 µg of GluN2B in pCMV5. The cells were treated with 1 µM NMDA for various times (0 to 10 min). Cell lysates were analyzed by immunoblotting using antibodies against phosphorylated ERK2 (p-ERK2) and total ERK2. Statistical significance: *p<0.05, **p<0.01, ***p<0.001 compared to the ‘0 min’ group. ^#p<0.05, ^##p<0.01 when the ’10 min’ group was compared to the ‘5 min’ and ‘2 min’ groups, respectively (n=3). (B) HEK-293 cells expressing NR2B were treated with varying concentrations of NMDA (0 to 10 µM) for 10 min. Statistical significance: *p<0.05, ***p<0.001 compared to cells treated with 0 to 100 nM NMDA (n=3).

As a next step, we conducted research on the signal transduction pathway involved in non-ionotropic signaling of NMDA-R. Previous studies showed that Src kinases are involved in the NMDA-R regulation (Wang and Salter, 1994; Salter and Kalia, 2004) through the tyrosine phosphorylation of the GluN2B subunit (Moon et al., 1994; Nakazawa et al., 2001). In agreement with these reports, treatment with PP2, a Src inhibitor, blocked the ERK activation (Fig. 2A).

Figure 2. Determination of signaling components involved in the non-ionotropic signaling pathway of NMDA receptor. (A) HEK-293 cells expressing GluN2B were pretreated with either vehicle (Veh) or 1 μM PP2 for 30 min, followed by either vehicle or 1 μM NMDA for 10 min. ***p<0.001 compared to other groups (n=3). (B) Cells were pretreated with either vehicle, 1 μM Gö6983, or 1 μM Gö6976 for 30 min, followed by either vehicle or 1 μM NMDA for 10 min. **p<0.01, ***p<0.001 compared to Veh/Veh, Veh/Gö6987, and Gö6983 group (n=3). (C) Cells were pretreated with either vehicle or 1 μM wortmannin (Wort) for 30 min, followed by either vehicle or 1 μM NMDA for 10 min. **p<0.01 compared to other groups (n=3).

Along with Src, PKC is also known to be involved in the signaling of NMDA-R through intricate interaction with Src. For example, stimulation of endogenous PKC potentiates NMDA-R currents and the PKC-stimulated potentiation requires Src (Lu et al., 1999). In addition, PKC and Src family kinase activities were required for the surface expression of NMDA-R (Grosshans et al., 2002). As shown in Fig. 2B, treatment with a PKC inhibitor, Gö6983 not Gö6976 inhibited the ERK activation.

Phosphoinositide 3-kinase (PI3K) is also known to be induced by NMDA-R activation (Kim et al., 2011; Brennan-Minnella et al., 2013). As shown in Fig. 2C, pretreatment with wortmannin, a PI3K inhibitor, blocked the ERK activation. Fig. 3 illustrates the proposed metabotropic signaling pathways of GluN2B that result in ERK activation.

Figure 3. A proposed metabotropic signaling cascade of GluN2B that mediates ERK activation. Upon agonistic NMDA-R activation, Src family kinases phosphorylate specific tyrosine residues on the carboxyl terminal tail of GluN2B, including Y1252, Y1336, and Y1472. These phosphorylated tyrosines create docking sites for SH2 domain-containing proteins such as PLCγ and PI3K. Through these protein-protein interactions, GluN2B serves as a scaffold that orchestrates multiple signaling cascades converging on ERK activation. Direct signaling events are indicated by solid arrows, while indirect signaling relationships are shown with dotted arrows. The blue dotted arrows specifically denote calcium-dependent signaling pathways. Src, Rous Sarcoma Oncogene; PI3K, Phosphoinositide 3-Kinase; PLC, Phospholipase C; CaMKII, Calcium/Calmodulin-Dependent Protein Kinase II; PKC, Protein Kinase C; ERK, Extracellular Signal-Regulated Kinase.

Characterization of NMDA receptor ligands via their activities for the activation of ERK

The effect of putative NMDA-R ligands was tested for their effects on the ERK activation to determine the agonistic nature of each compound and to see whether the extent of activation is related to their affinity. To enhance the validity of the experimental results, the ERK assay was conducted using a combination of compounds with varying affinities. NMDA was employed as a positive control.

As depicted in Fig. 4A-4F, the ERK activation was observed in cells expressing GluN2B upon treatment with the listed compounds. The numbers in parentheses correspond to the compound IDs and Ki values provided in Tables 1 and 2. PCE (#2, 746 nM), PCPy (#3, 175 nM), 3-HO-PCE (#6, 6 nM), 4’-F-PCPy (#8, 151.8 nM), 4’-F-PCP (#9, 128 nM), 3-HO-PCP (#11, 164 nM), 3-MEO-PCMo (#12, 81 nM), Desmethylamine (#15, 23.6 nM), N-4-MXE (#19, 31 nM), DM-nor-MXE (#20, 5.1 nM), DM-nor-4-MXE (#21, 5 nM), Desmethyl-4-MXE (#23, 219.5 nM), MXE (#25, 191 nM), N-ethyl-nor-Ketamine (#28, 344 nM). The final column of Tables 1 and 2 specifies which compounds induce ERK activation and which do not.

Figure 4. Determination of the metabotropic signaling activities of the NMDA receptor ligands. In each group, compounds with high and low affinity for NMDA-R were combined to examine their activity in activating ERK through GluN2B. NMDA served as a positive control for the NMDA-R in each group. The cells were treated with 1 µM of the compounds for 10 min. *p<0.05, **p<0.01, ***p<0.001 compared to the vehicle group (n=3). The numbers in parentheses indicate the compound IDs and Ki values listed in Tables 1 and 2.

We examined whether these compounds affected any receptors endogenously expressed in HEK-293 cells, aside from NMDA-R, leading to ERK activation. As depicted in Fig. 4G, NMDA-induced ERK activation in cells expressing GluN2B. However, in cells with the Mock vector, none of the compounds that exhibited positive effects in Fig. 4A-4F, including NMDA, triggered ERK activation. These findings indicate that all of these compounds selectively activate NMDA-R, resulting in ERK activation.

In this study, the GluN2B subunit, which plays a crucial role in ligand binding and signaling to NMDA receptors (NMDA-Rs), was used to investigate the following: 1) The structural features of the ligand that are important for binding to NMDA-Rs; 2) The signal transduction mechanisms involved in the non-ionotropic functions of NMDA-Rs, which have recently gained significant attention; and 3) The effects of NMDA-R ligands, known as ionotropic antagonists, on the non-ionotropic functions.

In the 1-(1-phenylcyclohexyl)amine series, R1 and the phenyl ring are attached to the same carbon on the cyclohexyl ring, and R2 is located at positions 3 or 4 of the phenyl ring. Changes in R1 and R2 affect NMDA-R affinity. For example, when X=C and R2=H, switching R1 from NH2 to NHCH₂CH₃ reduced affinity threefold. Substituting R1 with azepane increased affinity 12-fold compared to pyrrolidine, and 6-fold compared to phenylmethanamine. When R1=NHCH₂CH₃, replacing R2 from H to 3-OH or 3-OMe enhanced affinity 13-fold and 25-fold, respectively. Changing X from C to C=O with R1=piperidine and R2=4-OMe increased affinity 11-fold. Additionally, switching R2 from H to 4-OMe in compounds with X=C=O and R1=piperidine increased affinity 70-fold.

Methoxetamine (MXE), an analog of ketamine, features a cyclohexanone ring with a 3-methoxy group and an amine group differently positioned. The highest binding affinity was seen in compounds with R1=H and R2=OH. Adding 2-Cl or 2-F to R2 significantly reduced affinity when R1 was CH₃. Generally, ligands with R1 as CH₂CH₃ showed low affinity.

A number of studies have highlighted the important role of GluN2B-containing NMDA-Rs in non-ionotropic signaling, which is essential for synaptic plasticity, particularly long-term depression (LTD) (Massey et al., 2004). Unlike ionotropic signaling, which relies on ion flow through the receptor, non-ionotropic signaling involves protein-protein interactions and intracellular signaling pathways, such as p38 MAPK, without requiring calcium influx (Kessels et al., 2013). Studies have shown that GluN2B-containing NMDA-Rs are crucial for LTD induction, and their inhibition prevents LTD from occurring. These receptors are also implicated in neuropsychiatric and neurodegenerative diseases, including Alzheimer’s disease, where dysfunction in NMDA-R-mediated signaling contributes to early synaptic damage (Zhang et al., 2014).

It has been proposed that the rapid antidepressant effects of ketamine are likely due to its antagonism of NMDA-Rs containing the GluN2B subunit (Gass et al., 2018). This mechanism may explain ketamine’s quick onset of action in treating depression, as blocking GluN2B-containing receptors can initiate changes in synaptic plasticity and intracellular signaling pathways that are associated with mood regulation (Rodriguez et al., 2000; Beaurain et al., 2024).

Thus, GluN2B-containing NMDA-Rs are not only crucial for ionotropic functions but also play significant roles in metabotropic signaling pathways that influence synaptic plasticity and disease pathology.

A series of compounds that induce ERK activation through the metabotropic pathway have all been reported to inhibit ionotropic NMDA-R signaling: PCE (Domino, 1992), PCPy (Cho et al., 1991), 4’-F-PCP (Ortiz et al., 2021), N-4-MXE (N-4-MXE (N-Ethyl-4-methoxyphencyclidine)) (Irie et al., 2022), Desmethyl-nor-MXE (Irie et al., 2022), Desmethyl-nor-4-MXE (DM-nor-4-MXE) (Irie et al., 2022), 3-HO-PCE (3-hydroxyphencyclidine)(Morris and Wallach, 2014), 3-HO-PCP (3-hydroxyphencyclidine) (Morris and Wallach, 2014), 3-MeO-PCMo (Abiero et al., 2020), Methoxetamine (MXE) (Abe et al., 2013), N-ethyl-nor-ketamine (Sayson et al., 2019), Desmethylamine and Desmethyl-4-MXE (Irie et al., 2022).

These findings indicate that the compounds tested in this study function as antagonists for the ionophore activity of NMDA-Rs while simultaneously acting as agonists for their metabotropic activity. It is fascinating how these seemingly contradictory effects can coexist. This dual functionality may help explain the pharmacological effects of these compounds, which cannot be fully understood by considering them solely as NMDA-R antagonists.

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (RS-2023-00239943) and by the Ministry of Food and Drug Safety of Korea (23212MFDS218).

The authors have no conflicts of interest to declare.