An NMDAR positive and negative allosteric modulator series share a binding site and are interconverted by methyl groups

Article abstract of DOI:10.7554/eLife.34711

N-methyl-_D-aspartate receptors (NMDARs) are an important receptor in the brain and have been implicated in multiple neurological disorders. Many non-selective NMDAR-targeting drugs are poorly tolerated, leading to efforts to target NMDAR subtyp

Full text of DOI:10.7554/eLife.34711

RESEARCH ARTICLE
An NMDAR positive and negative
allosteric modulator series share a binding
site and are interconverted by methyl
groups
Riley Perszyk1, Brooke M Katzman2, Hirofumi Kusumoto3, Steven A Kell2,
Matthew P Epplin2, Yesim A Tahirovic2, Rhonda L Moore2, David Menaldino2,
Pieter Burger2, Dennis C Liotta2, Stephen F Traynelis1*
1Department of Pharmacology, Emory University, Atlanta, United States;
3
2Chemistry, Emory University, Atlanta, United States; Pharmacology, Emory
University, Atlanta, United States
Abstract N-methyl-D-aspartate receptors (NMDARs) are an important receptor in the brain and
have been implicated in multiple neurological disorders. Many non-selective NMDAR-targeting
drugs are poorly tolerated, leading to efforts to target NMDAR subtypes to improve the
therapeutic index. We describe here a series of negative allosteric NMDAR modulators with
submaximal inhibition at saturating concentrations. Modest changes to the chemical structure
interconvert negative and positive modulation. All modulators share the ability to enhance agonist
potency and are use-dependent, requiring the binding of both agonists before modulators act with
high potency. Data suggest that these modulators, including both enantiomers, bind to the same
site on the receptor and share structural determinants of action. Due to the modulator properties,
submaximal negative modulators in this series may spare NMDAR at the synapse, while
augmenting the response of NMDAR in extrasynaptic spaces. These modulators could serve as
useful tools to probe the role of extrasynaptic NMDARs.
DOI: https://doi.org/10.7554/eLife.34711.001
*For correspondence:
strayne@emory.edu
Competing interest: See
Introduction
page 33
N-methyl-D-aspartate receptors (NMDARs) are a subtype of ionotropic glutamate receptors that are
broadly expressed in the brain and are important for normal development, neuronal plasticity and
memory formation (Traynelis et al., 2010; Paoletti et al., 2013). NMDARs contribute a slow, Ca2+
permeable component to fast excitatory neurotransmission that is voltage dependent by virtue of its
sensitivity to pore block by extracellular Mg2+ (Traynelis et al., 2010). The typical NMDAR is com-
prised of 2 GluN1 and 2 GluN2 subunits, creating the potential for diversity given that there are
eight splice variants of GluN1 and 4 independent genes encoding GluN2 subunits (A-D)
(Traynelis et al., 2010). The regulation and functional properties of the NMDAR are controlled by
the subunits incorporated into the receptor (Monyer et al., 1994; Vicini et al., 1998; Vance et al.,
2012). NMDARs are expressed at most excitatory synapses, and are also found in peri- and extrasy-
naptic locations (Sans et al., 2000; Steigerwald et al., 2000; Groc et al., 2006; Traynelis et al.,
2010). Although NMDARs have been studied extensively, there are still important questions about
the different roles that NMDARs may play given differences in subunit composition and synaptic
localization (Hardingham and Bading, 2010; Gladding and Raymond, 2011; Paoletti et al., 2013).
Pharmacological approaches can be useful for probing these questions, but tool compounds to dif-
ferentiate these NMDAR subtypes have been limited (Ogden and Traynelis, 2011;
Funding: See page 34
Received: 29 December 2017
Accepted: 28 March 2018
Published: 24 May 2018
Reviewing editor: Kenton J
Swartz, National Institutes of
Health, United States
Copyright Perszyk et al. This
article is distributed under the
terms of the Creative Commons
Attribution License, which
permits unrestricted use and
redistribution provided that the
original author and source are
credited.
Perszyk et al. eLife 2018;7:e34711. DOI: https://doi.org/10.7554/eLife.34711
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Research article
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eLife digest The neurons in the brain form networks that can change in response to experience,
causing new connections to form between certain neurons and breaking the connections between
others. This remodeling process underlies learning and memory. However, in certain neurological
disorders, such as schizophrenia and epilepsy, these networks are disrupted and no longer work
correctly.
A receptor protein called the NMDA receptor plays an important role in reshaping the networks
of neurons. Chemicals called neurotransmitters that are released by one neuron bind to and activate
NMDA receptors on a neighbouring neuron to communicate with it. This activation encourages new
connections to form between neurons.
Drugs that alter the activity of the NMDA receptor could potentially act as treatments for
neurological conditions that disrupt how the networks of neurons work. However, few have been
approved for use in patients because most of the potential drug compounds investigated so far
produce severe side effects.
Perszyk et al. have now identified a new group of compounds that can potentially alter the
activity of NMDA receptors without fully blocking the response, potentially eliminating the
unwanted side effects. The compounds were tested on frog egg cells and human embryonic kidney
cells that had been engineered to produce NMDA receptors. Small changes to the chemical
structure of these compounds could switch their effect from increasing to decreasing the activity of
the receptor. The compounds only interact with active receptors that have a neurotransmitter bound
to them, and compete with each other to bind to the receptors.
Perszyk et al. also found that some compounds behaved differently depending on how active the
NMDA receptor was. These compounds could potentially be used to sense the activity in a network
of neurons, which opens up new options for treating neurological conditions that affect the
networks.
Further experiments are now required to see how these compounds affect the activity of NMDA
receptors in neurons and living animals. The range of effects produced by the compounds studied
by Perszyk et al. suggests that other related compounds may have different effects on receptor
activity. Future work could investigate the properties of these compounds to see if they could treat
a different set of neurological disorders.
DOI: https://doi.org/10.7554/eLife.34711.002
Monaghan et al., 2012; Santangelo et al., 2012; Strong et al., 2014; Zhu and Paoletti, 2015).
Whereas NMDAR receptors have been implicated in many neurological diseases, there remains a
dearth of clinically-approved drugs that target NMDARs (Kalia et al., 2008; Traynelis et al., 2010;
Paoletti et al., 2013; Strong et al., 2014).
Multiple endogenous and exogenous modulatory sites in the NMDAR recently have been
described. In addition, nearly complete NMDAR structures obtained using crystallographic
approaches are now available (Karakas and Furukawa, 2014; Lee et al., 2014), and these data,
together with recent cryo-EM structures in the receptor super family (Twomey and Sobolevsky,
2018; Twomey et al., 2017a; Twomey et al., 2017b), illustrate the overall topography of the
NMDAR and suggest a mechanism for some allosteric modulators, such as GluN2B-selective ifenpro-
dil (Tajima et al., 2016; Zhu et al., 2016). However, a complete understanding of how other modu-
latory sites operate and can be exploited has been elusive. Advances in the understanding of
specific roles of particular NMDAR subunits have led to renewed interest in targeted therapeutic
intervention, and recent work has yielded a growing tool box of novel ligands that act on NMDARs
with diverse sites and mechanisms (Ogden and Traynelis, 2011; Monaghan et al., 2012;
Hackos and Hanson, 2017). To date, there are positive and negative modulators that bind to the
amino-terminal domain (ATD), agonist binding domain (ABD), or transmembrane domain (TMD).
Each new class of compounds discovered enriches our understanding of the function of NMDAR and
how these receptors can be modulated. The information gained from these modulators has the
potential to provide insight into both NMDAR function and therapeutic strategies to treat complex
neurological diseases.
Perszyk et al. eLife 2018;7:e34711. DOI: https://doi.org/10.7554/eLife.34711
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O
O
A
B
C
D
E
O
O
O
O
NH
NH
O
NH
NH
O
GluN2D
GluN2D
S
S
GluN2D
EU1794-2
Glu/Gly
300
200
100
0
300
200
100
0
S
S
N
H
N
H
EU1794-2
EU1794-4
EU1794-5
EU1794-19
EU1794-2
EU1794-5
O
O
O
100 nA
50 s
O
NH
NH
O
NH
O
S
O
S
NH
S
N
S
N
H
H
O
GluN2D
EU1794-5
Glu/Gly
EU1794-4
EU1794-27
O
O
O
O
NH
NH
O
NH
O
S
O
S
NH
EU1794-5
S
N
H
S
N
H
O
EU1794-27
EU1794-25
EU1794-29
EU1794-5
EU1794-25
O
O
O
O
NH
NH
O
NH
100 nA
50 s
O
S
O
S
NH
0.1
1
10 100
Concentration (µM)
0.1
1
10 100
S
N
H
S
N
H
O
Concentration (µM)
EU1794-19
EU1794-29
Figure 1. The EU1794 series of NMDAR NAMs can be converted to PAMs with subtle structural modifications. (A)
Representative concentration-response experiments for a negative (EU1794-2, above) and positive (EU1794-5,
below) allosteric modulator using two-electrode voltage-clamp recordings of GluN1/GluN2D NMDARs activated
by saturating concentrations of agonist. 100/30 mM glutamate/glycine application is represented by the grey bar
and increasing concentrations of modulator are shown by grey scale boxes (0.3, 1, 3, 10, 30 mM for EU1794-2 and
1, 3, 10, 30, 100 mM for EU1794-5). (B) Chemical structures of the NAMs EU1794-2, EU1794-4, EU1794-19 and
PAM EU1794-5 are given, which show all permutations of two methyl substitutions. This series illustrates how
subtle changes in chemical structure interconvert modulator activity. (C) The concentration-response curves for the
compounds shown in (B) acting on GluN1/GluN2D NMDARs activated by maximally effective concentrations of co-
agonists and fitted by the Hill equation. (D) Chemical structures of EU1794 PAMs. (E) Concentration-response
curves for EU1794 PAMs on the responses of GluN1/GluN2D NMDARs to maximally effective concentrations of
co-agonists. See Table 1 for fitted EC50 values at all diheteromeric NMDARs. Data represent 4–18 oocytes
recorded in at least two independent experiments.
DOI: https://doi.org/10.7554/eLife.34711.003
This study describes a series of compounds that includes both positive allosteric modulators
(PAMs) as well as negative allosteric modulators (NAMs) of NMDAR function (Katzman et al., 2015).
A set of aliphatic substitutions off an ester linkage to the scaffold interconverts these compounds
between positive and negative allosteric modulators. Remarkably, the difference between the posi-
tive and negative modulation to the same chemical scaffold was influenced by the addition or
removal of individual methyl groups. These modulators appear to bind to a shared site to bring
about opposing actions, and share mechanistic features, such as agonist dependence and enhance-
ment of agonist potency. The spectrum of properties in this series of modulators could serve as use-
ful tool compounds for probing the role of NMDARs in circuits in both healthy brain and in
neuropathological situations.
Results
Identification of a new class of positive allosteric modulators of
NMDAR function
We have previously described the structure-activity relationship (SAR) of a series of negative alloste-
ric pan-NMDAR modulators that contained an amidothiophene core, the most potent possessing a
tetrahydrobenzothiophene, with different alkyl and aryl substitutions connected via an ester linkage
at the 3-position (Katzman et al., 2015). These compounds inhibited the response to saturating con-
centration of co-agonists at NMDARs expressed in Xenopus laevis oocyte experiments, typified by
EU1794-2 (compound 4 in Katzman et al., 2015). We describe here a new set of closely related
analogues that can either potentiate or inhibit responses depending on subtle changes in structure
and agonist concentration. As shown in Figure 1 and Table 1, EU1794-4 contained an ethyl ester
similar to EU1794-2 and lacked a methyl substituent on the tetrahydrobenzothiophene core.
Perszyk et al. eLife 2018;7:e34711. DOI: https://doi.org/10.7554/eLife.34711
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Table 1. The effect of alkyl ester and tetrahydrobenzothiophene ring substitutions on the potency and efficacy of EU1794 analogues.
EC50 (mM) [conf. int.]*
Maximal Degree of Modulation (% of control)†
3
˚
Compound
R1
R1 volume (A )
R2
GluN2A
GluN2B
GluN2C
GluN2D
EU1794-4
Et
45.15
45.15
62.16
62.16
79.23
98.87
79.12
H
2.2 [1.8, 2.8]
32 3%
2.6 [1.4, 4.8]
67 4%
0.42 [0.28, 0.61]
52 2%
0.36 [0.29, 0.45]
51 3%
EU1794-2
EU1794-5
EU1794-19
EU1794-27
EU1794-25
EU1794-29
Et
Me
H
0.60 [0.44, 0.82]
1.2 [0.8, 1.9]
10 3%
0.26 [0.21, 0.31]
14 1%
0.20 [0.17, 0.25]
14 1%
6
2%
iPr
18 [9.0, 36]
36 5%
2.5 [1.4, 4.4]
71 4%
4.0 [3.5, 4.6]
140 3%
9.3 [8.1, 11]
169 7%
iPr
Me
H
1.1 [0.5, 2.3]
19 6%
1.1 [0.9, 1.3]
43 2%
0.74 [0.57, 0.96]
32 3%
0.43 [0.32, 0.59]
46 2%
tBu
Bn
7.4 [5.3, 10]
52 7%
1.4 [1.2, 1.7]
130 3%
2.8 [2.5, 3.2]
230 10%
2.4 [1.8, 3.3]
250 8%
H
-
1.0 [0.9, 1.2]
220 13%
0.77 [0.56, 1.0]
180 12%
1.0 [0.95, 1.1]
250 19%
n-Bu
H
3.7 [1.7, 8.0]
59 5%
1.4 [1.1, 1.8]
140 3%
3.8 [2.6, 5.7]
130 7%
5.7 [5.1, 6.4]
166 8%
*EC50 values for potentiation of responses to saturating glutamate and glycine (100 mM, 30 mM) were obtained by least-squares fitting of data from individ-
ual experiments by the Hill equation. EC50 values are given as the mean with the 95% confidence interval determined from log(EC50).
†The maximal degree of modulation is given as mean SEM. For all compounds, data are from 4 to 18 oocytes recorded in at least two independent
experiments. Data were not fit (shown as -) if the response recorded at 30 mM of test compound did not differ by more than 15% from control.
DOI: https://doi.org/10.7554/eLife.34711.004
EU1794-4 potently inhibited GluN1/GluN2D with a substantial residual current remaining at saturat-
ing concentrations (Figure 1B,C, Table 1). Interestingly, ester substitutions that had a larger calcu-
lated functional group volume than the ethyl ester in EU1794-4 potentiated NMDAR responses to
saturating concentrations of glutamate and glycine. For example, the isopropyl ester (EU1794-5)
potentiated GluN1/GluN2D responses to nearly 200% of control (Figure 1A–C). Restoration of the
methyl to the tetrahydrobenzothiophene core restored negative allosteric modulation (EU1794-19,
Figure 1B,C). Thus, the direction of modulation (positive or negative) could be determined by the
size of the alkyl ester and substitution to the tetrahydrobenzothiophene core.
The size of the ester-linked substituent controlled the extent and potency of positive modulation.
The t-butyl ester EU1794-27 potentiated GluN1/GluN2D NMDARs with greater efficacy and higher
potency, increasing responses to maximally effective glutamate and glycine to 250% of control with
an EC50 value of 2.4 mM (Figure 1D,E). The benzyl ester (EU1794-25) also strongly potentiated
responses with a potency similar to EU1794-27 (Figure 1D,E). However, EU1794-29, which had a
longer substitution, n-butyl, reduced both the maximal potentiation and potency, suggesting an
optimal substituent size and shape (Figure 1D,E).
Allosteric modulation of agonist potency by NAMs and PAMs
The tetrahydrobenzothiophene-containing NAMs reported here inhibit all diheteromeric NMDARs
without substantial subunit selectivity, similar to those that we previously described (Katzman et al.,
2015). By contrast, the novel PAMs described here show distinct GluN2 dependence (Table 1). All
PAMs are active at GluN1/GluN2C and GluN1/GluN2D, but do not enhance the response of GluN1/
GluN2A to maximally effective concentrations of agonist, in some cases resulted in inhibition of
these responses. Most PAMs were also capable of potentiating GluN1/GluN2B (Table 1,
Figure 2A). As seen with other series of NMDAR PAMs (Malayev et al., 2002; Horak et al., 2004;
Horak et al., 2006; Hackos et al., 2016; Wang et al., 2017), allosteric modulation can be depen-
dent on agonist concentrations. Thus, we assessed the ability of EU1794-27 to modulate NMDAR
responses activated by sub-saturating co-agonist concentrations, clearly exemplified by modulation
Perszyk et al. eLife 2018;7:e34711. DOI: https://doi.org/10.7554/eLife.34711
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A
Saturating AgonistB
C
GluN2C
EU1794-27
Glu/Gly
Sub-Saturating Agonist
GluN2A
GluN2B
GluN2C
GluN2D
600
400
200
0
600
400
200
200 nA
50 s
0
0.1
0.1
1 10 100
EU1794-27 (µM)
1
EU1794-27 (µM)
10 100
D
Saturating AgonistE
F
GluN2C
EU1794-4
Glu/Gly
Sub-Saturating Agonist
400
400
GluN2A
GluN2B
GluN2C
GluN2D
300
200
100
0
300
200
100
0
200 nA
50 s
0.1
1
10 100
EU1794-4 (µM)
0.1
1
10 100
EU1794-4 (µM)
Figure 2. Modulation of both EU1794 PAMs and NAMs is dependent on agonist concentration. (A) The
concentration-response relationship for EU1794-27 at diheteromeric NMDARs activated by 100/30 mM glutamate/
glycine (data for GluN1/GluN2D shown in Figure 1E is included here for clarity). GluN1/GluN2C and GluN1/
GluN2D (which are superimposed) are potentiated to a greater extent than GluN1/GluN2B; there appears to be
slight inhibition of GluN1/GluN2A. (B) A representative concentration-response recording of EU1794-27 at GluN1/
GluN2B activated by sub-saturating concentrations of agonist. The initial response was activated using saturating
100/30 mM glutamate/glycine (black box) followed by 1/0.3 mM glutamate/glycine (grey box, roughly resulting in
an activity state that was 20% of the maximal response) administered in the absence and presence of increasing
concentrations of EU1794-27 (0.3, 1, 3, 10, 30 mM). (C) Concentration-response relationship for EU1794-27 at
diheteromeric NMDARs activated by sub-saturating concentration of agonist (glutamate/glycine concentrations
used for GluN1/GluN2A were 2/0.6 mM, GluN1/GluN2B and GluN1/GluN2C were 1/0.3 mM, and GluN1/GluN2D
were 0.6/0.2 mM). Note potentiation of all subunits. (D–F) Similar experiments as in A-C, but using the NAM
EU1794-4 at saturating (D) or sub-saturating (E,F) concentrations of agonist (glutamate/glycine concentrations
used for GluN1/GluN2A, GluN1/GluN2B and GluN1/GluN2C were 1/0.3 mM, and GluN1/GluN2D were 0.3/0.09
mM); data for GluN1/GluN2D presented in Figure 1C are included here for comparison. Note the inhibition by the
NAM at saturating agonist concentration (D) compared to potentiation in submaximal concentrations of agonist in
(E,F). Data represent 4–13 oocytes recorded in two independent experiments.
DOI: https://doi.org/10.7554/eLife.34711.005
The following figure supplement is available for figure 2:
Figure supplement 1. EU1794-2 actions possess modest agonist concentration-dependence.
DOI: https://doi.org/10.7554/eLife.34711.006
of GluN1/GluN2C (Figure 2B, Table 2). In these conditions, EU1794-27 positively modulated all
NMDAR subtypes and enhanced previously potentiated subtypes to a greater extent than in saturat-
ing agonist (compare Figure 2A and C). We hypothesized that this agonist-dependence was due to
EU1794-27 altering the agonist potency. Therefore, we determined the glutamate and glycine EC50
values in the absence and presence of EU1794-27 (Figure 3A,B, Table 3, Figure 3—figure supple-
ment 1). EU1794-27 produced modest but significant decreases (higher potency) in the EC50 values
for both glutamate and glycine at all NMDARs (Table 3, Figure 3—figure supplement 1). Given this
effect by the PAMs in this series, we subsequently considered whether the NAMs in the series
shared this mechanism. We selected EU1794-4 for use in evaluating actions on agonist potency
since it retains a large steady-state current even for receptors that have bound EU1794-4 (saturated
inhibition is between 40–70% of control, Figure 2D). We co-applied EU1794-4 during responses
stimulated by sub-saturating concentrations of agonist (glutamate and glycine concentrations that
resulted approximately in a EC20 response), which resulted in positive modulation (Figure 2E,F,
Table 2). Furthermore, the negative modulator EU1794-4 enhanced the glutamate and glycine
potencies (Figure 3C,D, Table 4, Figure 3—figure supplement 1). Interestingly, positive modula-
tion elicited by 10 mM EU1794-4 is dependent on the sub-saturating agonist concentrations used
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Table 2. Effect of EU1794-4 and EU1794-27 at sub-saturated NMDAR responses.
EC50 (mM) [conf. int.]*
Maximal Degree of Modulation (% of control)†
GluN2A
GluN2B
GluN2C
GluN2D
EU1794-4
3.9 [0.78, 20]
500 32%
4.3 [1.3, 14]
210 6.8%
15 [6.5, 35]
220 20%
14 [6.5, 29]
540 190%
EU1794-27
8.1 [5.7, 12]
340 32%
6.3 [5.0, 8.0]
400 50%
5.0 [3.7, 6.6]
680 50%
8.3 [5.3, 13]
580 86%
*EC50 values of modulator action on responses to sub-saturating glutamate/glycine concentrations were obtained by
least-squares fitting of data from individual experiments by the Hill equation. For EU1794-4 modulation of GluN1/
GluN2A, GluN1/GluN2B and GluN1/GluN2C, glutamate/glycine concentrations were 1/0.3 mM; for GluN1/GluN2D
glutamate/glycine were 0.3/0.09 mM. For EU1794-27 potentiation, glutamate/glycine concentrations were 2/0.6 mM
(GluN1/GluN2A), 1/0.3 mM (GluN1/GluN2B, GluN1/GluN2C), and 0.6/0.2 mM (GluN1/GluN2D). EC50 for potentiation
values are given as the mean with the 95% confidence interval determined from log(EC50).
†The extent of modulation is given as a percent of the control response in the absence of test compound. The maxi-
mal degree of modulation is given as mean SEM. For all compounds, data are from 4 to 13 oocytes recorded in
two independent experiments.
DOI: https://doi.org/10.7554/eLife.34711.009
(Table 5). EU1794-4 inhibited GluN1/GluN2C responses to saturating concentrations of agonist to
54% of control, and positively modulated equivalent EC30 responses (equal effective concentrations
of glutamate and glycine concentrations that when applied resulted in a 30% of a maximal response)
to 140% of control, and positively modulated average equivalent EC3 responses by 660% (Table 5).
We did not observe augmentation of sub-saturating agonist responses by EU1794-2, most likely
due to its greater extent of inhibition (Figure 2—figure supplement 1). However, the degree of
inhibition produced by EU1794-2 was reduced on sub-saturating responses at GluN1/GluN2C from
16% to 45% of control (Table 5).
PAMs and NAMs display both glutamate and glycine dependence
We studied NMDARs expressed in HEK293 cells to investigate the time course of modulator action.
Concentration-dependent association of EU1794-2, EU1794-4 and EU1794-27 and concentration-
independent disassociation were evaluated by co-applying modulator with glutamate and glycine
(Figure 4A,B). These modulators were tested on GluN1/GluN2A and GluN1/GluN2D to see whether
there were differences in modulation given the distinct properties of these NMDAR subtypes. We
analyzed the concentration-dependence of the exponential time course describing the onset of
action during co-application with saturating concentrations of glutamate plus glycine to determine
modulator association and dissociation rates. From these we calculated the kinetically-determined
affinity constant (Kd), which we found to be similar to the EC50 values determined from concentra-
tion-response experiments. EU1794-2 Kd was 1.1 mM at GluN1/GluN2D (Figure 4A,B). Complex
actions of EU1794-4 and EU1794-27 were observed, with two temporally-distinct phases of modula-
tion evident for the association of these modulators (Figure 4A). Both compounds produced a rapid
inhibition followed by a slowly developing potentiating phase for EU1794-27. The rapid association
rate determined during the rapid inhibition produced by EU1794-4 was approximately three times
faster than EU1794-2; similarly, the dissociation rate was also faster for EU1794-4 than EU1794-2.
Quantitative analysis of the slower phases was challenging due to its lower signal-to-noise ratio. Like-
wise, the rapid inhibitory phase was also difficult to measure for EU1794-27 because its rapid time
course was convolved with the potentiation time course. Additionally, the depotentiation time
course is preceded by a transient enhancement of the current response, followed by a relaxation to
the pre-modulation level. The time course for potentiation and depotentiation were best fit with an
exponential function summed with an additional linear component after the rapid phase subsided.
Kd determined from the association and dissociation rates of EU1794-4 was 4.2 mM and EU1794-27
was 4.3 mM (Figure 4B). All modulatory effects were independent of voltage (Figure 4—figure sup-
plement 1A). The steady state modulator responses from HEK293 cells approximately match the
concentration-response relationship determined from TEVC recordings from X. laevis oocytes
(Figure 4A, inset graphs). The time-course of modulator binding of EU1794-2, EU1794-4 and
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A
GluN2C
GluN2C
EU1794-27
Gly
Glu
EU1794-27
Gly
Glu
400 nA
50 s
200 nA
50 s
B
100
80
60
40
20
0
100
80
60
40
20
0
GluN2C
w/EU1794-27
0.01 0.1 1 10 100
[Glu] (µM)
0.01 0.1 1 10
[Gly] (µM)
C
GluN2C
GluN2C
EU1794-4
Gly
EU1794-4
Gly
Glu
Glu
400 nA
50 s
100 nA
50 s
D
100
80
60
40
20
0
100
80
60
40
20
0
GluN2C
w/EU1794-4
0.01 0.1 1 10 100
[Glu] (µM)
0.01 0.1 1 10
[Gly] (µM)
Figure 3. EU1794 PAMs and NAMs enhance glutamate and glycine potency. (A) Representative glutamate (left)
and glycine (right) concentration-response curves of oocytes expressing GluN1/GluN2C with and without EU1794-
27 (10 mM, grey box). The glutamate concentration-response curve (0.1, 0.3, 1, 3, 10, 30 mM) was generated with 30
mM glycine and the glycine concentration-response curve (0.03, 0.1, 0.3, 1, 3, 10 mM) was recorded in 100 mM
glutamate. (B) Mean concentration-response data for glutamate (left) and glycine (right) at GluN1/GluN2C (see
Figure 2—figure supplement 1 for other subunits). Each curve was normalized to the maximal response in the
absence of EU1794-27 to illustrate the actions of EU1794-27 on agonist EC50. (C,D) A similar set of experiments as
in (A,B), but with EU1794-4 are shown (10 mM, grey box). Note the variation of the order of EU1794-4 application
in experimental traces, which was selected randomly. The glutamate concentration-response curve (glutamate
concentration are 0.03, 0.1, 0.3, 1, 3, and 100 mM and glycine concentrations are 10 and 30 mM) and the glycine
concentration-response curve (glycine concentrations are 0.01, 0.03, 0.1, 0.3, 1, 30 mM, and glutamate
concentration are 30 and 100 mM). Each curve was normalized to the maximal response from each oocyte in the
absence of EU1794-4. Data are from 4 to 12 paired oocytes recordings from at least two independent
experiments.
DOI: https://doi.org/10.7554/eLife.34711.007
Figure 3 continued on next page
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Figure 3 continued
The following figure supplement is available for figure 3:
Figure supplement 1. The effects of EU1794-27 and EU1794-4 on agonist potency for all diheteromeric
NMDARs.
DOI: https://doi.org/10.7554/eLife.34711.008
EU1794-27 was also determined for GluN1/GluN2A (Figure 4C,D). Higher Kd values (lower potency)
were determined for each molecule at GluN1/GluN2A as compared to GluN1/GluN2D that parallel
EC50 values determined using X. laevis oocytes. Similar to oocyte data, robust inhibition was pro-
duced by EU1794-2, modest inhibition was produced by EU1794-4, and transient inhibition and
recovery was observed by the application of EU1794-27. Interestingly, the extent of steady-state
modulation for this series appeared to be dependent on the level of desensitization of the receptors
at the time of modulator application. When classifying the cells as either having high or low levels of
desensitization (using 35% steady-state/peak response as a cut-off), the extent of modulation by 10
mM was significantly different for EU1794-27 (p=0.02, unpaired t-test, N = 4,2, respectively). The
low desensitized group (40% average desensitization) was modulated by 107%, whereas the high
desensitized group (10% average desensitization) was modulated to 247% of control by 10 mM
EU1794-27 (Figure 4C and Figure 4—figure supplement 1F).
Given that other NMDAR modulators have displayed agonist-dependence (Petrovic et al., 2005;
Acker et al., 2011; Hansen and Traynelis, 2011; Borovska et al., 2012; Vyklicky et al., 2015;
Wang et al., 2017), we performed rapid solution exchange experiments to examine if these modula-
tors had different affinities at agonist-bound and apo receptors (Figure 5A,B). The instantaneous
current response to a rapid step into glutamate plus glycine (from glycine alone) gives an estimate
of whether a modulator was pre-bound to receptors with only glycine (but not glutamate) bound.
The immediate NMDAR activation after a rapid switch to a solution containing glutamate and glycine
should occur faster than modulator binding. We chose to test for agonist-dependence using GluN1/
GluN2D since they lack desensitization, which would complicate interpretation of this modulator
property. We found that the peak current was similar when cells were preincubated in glycine with
or without 3 mM EU1794-2, suggesting that EU1794-2 does not bind appreciably to the receptor in
the absence of glutamate (Figure 5A,C). After the rapid step into glutamate where glycine and
modulator were pre-exposed, we observed a relaxation to a new response level that was similar in
amplitude to that observed with steady state co-application of glutamate plus glycine and modulator
(Figure 5A,C). Interestingly, we also observed a similar effect for the converse experiment, in which
we pre-applied glutamate with or without EU1794-2, followed by a rapid step into glutamate plus
Table 3. EU1794-27 effects on glutamate and glycine EC50 values.
EC50 (mM) [conf. int.]*
EU1794-27
Glutamate
Fold
Difference
Control
Glycine
EU1794-27
Glycine
Fold
Difference
Control Glutamate
GluN2A
GluN2B
GluN2C
GluN2D
2.9
[2.2, 4.0]
1.1
[0.88, 2.1]
2.3†
0.57
[0.34, 0.96]
0.38
[0.23, 0.64]
1.6
1.2
[1.1, 1.3]
0.65
[0.49, 0.85]
2.0†
3.3†
5.3†
0.28
[0.22, 0.36]
0.14
[0.08, 0.24]
2.9†
2.4
0.67
[0.61, 0.73]
0.22
[0.16, 0.31]
0.23
[0.13, 0.40]
0.10
[0.07, 0.15]
0.21
0.054
0.086
0.020
4.9†
[0.17,0.27]
[0.028, 0.11]
[0.077, 0.095]
[0.013, 0.029]
*Glutamate EC50 values (in the presence of 30 mM glycine) and glycine EC50 values (in the presence of 100 mM glutamate) were obtained by least-squares
fitting of data from independent oocyte recordings by the Hill equation. EC50 values are given as the mean with the 95% confidence interval determined
from log(EC50). Data in the absence and presence of EU1794-27 were obtained from the same oocyte. Data are from 5 to 12 paired oocytes recordings
from at least two independent experiments.
†indicates paired measurements with non-overlapping confidence intervals.
DOI: https://doi.org/10.7554/eLife.34711.010
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Table 4. EU1794-4 effects on glutamate and glycine EC50 values.
EC50 (mM) [conf. int.]*
Control
Glutamate
EU1794-4
Glutamate
Fold
Difference
Control
Glycine
EU1794-4
Glycine
Fold
Difference
GluN2A
GluN2B
GluN2C
GluN2D
3.6
[3.3, 4.0]
1.5
[1.2, 2.1]
2.4†
2.0†
3.2†
3.9†
0.89
[0.72, 1.1]
0.35
[0.17, 0.70]
2.8†
3.3†
4.6†
8.9†
1.5
[1.2, 2.0]
0.8
[0.48, 1.3]
0.46
[0.37, 0.56]
0.14
[0.12, 0.17]
0.84
[0.57, 1.2]
0.29
[0.15, 0.55]
0.31
[0.15, 0.62]
0.08
[0.024, 0.25]
0.63
0.17
0.15
0.016
[0.41, 0.95]
[0.065, 0.43]
[0.089, 0.24]
[0.007, 0.038]
*Glutamate EC50 values (in the presence of 10 mM glycine) and glycine EC50 values (in the presence of 30 mM glutamate) were obtained by least-squares
fitting of data by the Hill equation. EC50 values are given as the mean with the 95% confidence interval determined from log(EC50). Data in the absence
and presence of EU1794-4 were obtained from the same oocyte. Data are from 4 to 8 paired oocytes recordings from two independent experiments.
†indicates paired measurements with non-overlapping confidence intervals.
DOI: https://doi.org/10.7554/eLife.34711.011
glycine and EU1794-2 (Figure 5B,C). We interpret these data to suggest that EU1794-2 associates
with the receptor with higher affinity after glutamate and glycine binding.
We repeated these use-dependent experiments with EU1794-27, which yielded a similar result,
although a slightly different experimental design was required for consistent responses. The pre-
application of glycine with EU1794-27 produced a similar level of immediate activation following
glutamate application (Figure 5D,E). This was then followed by the complex actions observed when
EU1794-27 was applied to steady-state responses of NMDARs (Figure 5D, right panel). Using the
same protocol to pre-apply glutamate became problematic for some cells due the degree to which
EU1794-27 enhances agonist potency, a feature exemplified by the prolongation of deactivation.
This action of EU1794-27 was able to render nanomolar contaminate levels of glycine (when pres-
ent) more active, which necessitated a different experimental design (Figure 5—figure supplement
1A). When EU1794-2 was used in this experimental paradigm, inhibition of the contaminate level of
activity was observed upon modulator application (Figure 5—figure supplement 1B). Nevertheless,
we still observed a similar result with pre-application of glycine and EU1794-27, with the instanta-
neous response reaching the control level, followed by a slow relaxation to a new potentiated level
that reflected the time course for association of EU1794-27 after binding of both glutamate and
Table 5. Comparison of EU1794-27, EU1794-4, and EU1794-2 effects at GluN1/GluN2C NMDAR
responses to sub-saturating agonist.
Glu/Gly
Relative agonist Response*
(% of maximal response)
Modulation by 10 mM†
(% of control)
GluN2C
(mM)
EU1794-27
100/30
1/0.3
-
190 10%
480 31%‡
20 1.6
EU1794-4
EU1794-2
100/30
1/0.3
0.3/0.09
-
54 1.6%
27.7 3.3
2.8 0.7
140 12%‡
660 110%‡
100/30
0.6/0.2
-
16 1.0%
43 2.1%‡
21.5 6.8
The data presented are mean SEM.
*Reported average responses to sub-saturating agonist were normalized to a 100/30 mM glutamate/glycine
response.
†The degree modulation is reported as a percent of the control response to sub-saturating agonist. Some of these
data points correspond to those used to calculated EC50 values for each modulator in Table 1, Table 2 or
Supplementary file 1. Data are mean SEM.
‡p<0.05 as compared to 100/30 mM glutamate/glycine response, determined by an unpaired t-test, n = 4–10 cells.
DOI: https://doi.org/10.7554/eLife.34711.012
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A
GluN2D
GluN2D
GluN2D
EU1794-2
Glu+Gly
EU1794-4
Glu+Gly
EU1794-27
Glu+Gly
1 µM
3 µM
10 µM
10 µM
3 µM
1 µM
10 µM
3 µM
1 µM
10 s
10 s
200
100
0
200
100
0
200
100
0
1
10
1
10
1
10
10 s
[EU1794-2]
[EU1794-4]
[EU1794-27]
B
0.025
0.020
0.015
0.010
0.005
0.000
0.004
0.003
0.002
0.001
0.000
EU1794-2 GluN2D
kon=4.30x105 M-1s-1
koff=4.95x105 s-1
EU1794-4 GluN2D
kon=1.12x106 M-1s-1
koff=4.66x106 s-1
EU1794-27 GluN2D
kon=1.87x105 M-1s-1
koff=8.02x105 s-1
0.006
0.004
0.002
0.000
Kd=4.16μM
Kd=1.14 μM
Kd=4.30 μM
0
5
[EU1794-2] (µM)
10
0
5
[EU1794-27] (µM)
10
0
5
[EU1794-27] (µM)
10
C
D
GluN2A
GluN2A
GluN2A
EU1794-2
Glu+Gly
EU1794-4
Glu+Gly
EU1794-27
Glu+Gly
200 pA
10 s
200 pA
10 s
200 pA
10 s
EU1794-4 GluN2A
kon=5.02x105 M-1s-1
0.010
EU1794-2 GluN2A
kon=3.90x105 M-1s-1
koff=9.38x105 s-1
EU1794-27 GluN2A
kon=5.92x104 M-1s-1
koff=4.26x105 s-1
0.006
0.004
0.002
0.000
0.0020
0.0015
0.0010
0.0005
0.0000
0.008 koff=3.94x106 s-1
0.006
0.004
Kd=2.41 μM
Kd=7.85 μM
0.002
0.000
Kd=7.20 μM
0
5
10
0
5
10
0
5
[EU1794-2] (µM)
10
[EU1794-4] (µM)
[EU1794-27] (µM)
Figure 4. The association and dissociation time course of EU1794-2, EU1794-4 and EU1794-27 differ at GluN1/GluN2A and GluN1/GluN2D. (A)
Representative whole-cell current recordings of GluN1/GluN2D NMDARs exemplifying the modulation time course of EU1794-2, EU1794-4 and
EU1794-27 (1, 3, 10 mM). Responses were evoked by 100/30 mM glutamate/glycine and were normalized for display. Insets show the steady state
modulation extent from the HEK cell experiments fit by the Hill equation (Hill slope fixed to 1). (B) Analysis of the time course for the onset of PAM and
NAM action as a function of concentrations for EU1794-2 (left), EU1794-4 (left) and EU1794-27 (right) used to determine the association rate kON (black
circles) and dissociation rate kOFF (grey squares), from which we can calculate Kd for each modulator. Data are from at least 3 cells for each
concentration of modulator. For EU1794-27 the association and dissociation rate linear component was also found to be concentration dependent
(mon = À0.98 pA/(ms*mM), moff = 0.62 pA/(ms*mM)). (C,D) Similar set of panels as A,B but examining EU1794-2, EU1794-4 and EU1794-27 modulation
of GluN1/GluN2A. In example recordings, all modulator concentrations are 10 mM. Data are from at least 3 cells for each concentration of modulator.
For EU1794-27 time-course was fitted with the sum of an exponential and linear function, and both the time constant and slope were concentration
dependent (mon = À0.33 pA/(s*mM), moff = 0.16 pA/(s*mM)).
DOI: https://doi.org/10.7554/eLife.34711.013
The following figure supplement is available for figure 4:
Figure supplement 1. The actions of EU1794 modulators are voltage-independent and are influenced by the desensitization level.
DOI: https://doi.org/10.7554/eLife.34711.014
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Glycine
Pre-apply
Glutamate
Pre-apply
A
B
Glu
Gly
EU1794-2
C
Gly
Glu
EU1794-2
*
*
150
100
50
3µM EU1794-2
40 pA
5 s
100 pA
5 s
40 pA
0.2 s
Control
EU1794-2
3µM
Control
EU1794-2
3µM
100 pA
0.2 s
0
Peak Steady Peak Steady
Amp. State Amp. State
D
E
Glycine
Pre-apply
Glutamate
Pre-apply
Glu
Gly
EU1794-27
*
*
300
200
100
0
100 pA
0.4 s
100 pA
10 s
Control
EU1794-27
3µM
3µM EU1794-27
Peak Steady Peak Steady
Amp. State Amp. State
Figure 5. The EU1794 series has agonist-dependence that alters the response time-course. (A) Representative whole-cell recordings of GluN1/GluN2D
NMDARs exemplifying the EU1794-2 dependence on glutamate binding. The cell was exposed continuously to 30 mM glycine with/without 3 mM
EU1794-2, and then was rapidly stepped into a solution additionally containing 100 mM glutamate. The right panel shows an expanded view of the rise
of the receptor response and the rapid time course of EU1794-2 action. (B) Representative whole-cell current recordings of GluN1/GluN2D NMDARs
exemplifying the EU1794-2 dependence on glycine binding. The cell was exposed continuously to 100 mM glutamate with/without 3 mM EU1794-2, and
then was rapidly stepped into a solution additionally containing 30 mM glycine. The right panel shows an expanded view of the rise of the receptor
response and the rapid time course of EU1794-2 action. (C) Quantification of the modulation of the immediate peak response and steady state data
(shown as a percentage EU1794-2/control for each phase) from agonist-dependence experiments for EU1794-2 (3 mM, Figure 5A,B and S4B). *
signifies p<0.05 as determined by a Bonferroni’s multiple comparison test where only EU1794-2 vs control was compared for both peak and steady
state conditions after a significant repeat measures ANOVA (F(3,3) = 7.66 for pre-applied glycine and F(3,7) = 12.4 for pre-applied glutamate). (D)
Representative whole-cell current recordings of GluN1/GluN2D NMDARs exemplifying the EU1794-27 dependence on glutamate binding. The cell was
exposed continuously to 30 mM glycine with/without 3 mM EU1794-27, and the solution was rapidly changed to one additionally containing 100 mM
glutamate. The right panel is an expanded view of the activation kinetics of the responses and the onset of modulation. (E) Quantification of the
modulation of the immediate peak and steady state response (shown as a percentage EU1794-27/control for each phase) from agonist-dependence
experiments for EU1794-27 (3 mM, Figure 5D and S4A). * signifies p<0.05 from the post hoc Bonferroni’s multiple comparison test (F(3,3) = 17.8 for
pre-applied glycine and F(3,2) = 13.5 for pre-applied glutamate).
DOI: https://doi.org/10.7554/eLife.34711.015
The following figure supplement is available for figure 5:
Figure supplement 1. EU1794 modulators are dependent on co-agonist binding.
DOI: https://doi.org/10.7554/eLife.34711.016
glycine (Figure 5E). This result illustrates a requirement for glycine to be bound to the receptor for
high-affinity binding of EU1794-27. To circumvent any ambiguity associated with glycine contamina-
tion, we utilized another experimental design described by Vyklicky et al. (2015). Glutamate was
pre-applied with 7-CKA (100 mM) to antagonize the glycine site, blocking any occupancy by contami-
nant glycine. The receptor was activated by switching to a solution that lacked 7-CKA and contained
glycine plus glutamate (with or without modulator, Figure 5—figure supplement 1C). To test the
ability of EU1794-27 to bind to the receptor with the GluN1 ABD bound to antagonist, EU1794-27
(3 mM) was added to the solution containing 7-CKA. Upon the switch from the 7-CKA/EU1794-27
solution containing glutamate to a solution just containing glutamate and glycine, no detectable
change in the response rise time or peak amplitude was observed. If EU1794-27 bound during the
7-CKA phase prior to agonist binding, we would have expected an increased instantaneous peak
current upon switching to glutamate plus glycine given that the dissociation of EU1794-27 is slower
Perszyk et al. eLife 2018;7:e34711. DOI: https://doi.org/10.7554/eLife.34711
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Magenta- loss of activity
Yellow- augments activity
Green - inverts activity
B
A
GluN1
Pocket
GluN2
Pocket
EU1794-2
(5 μM, % Modulation)
WT Response
EU1794-27
(10 μM, % Modulation)
WT Response
1
5
0 0
1
0
M3
1
1
2
3
5
1
2
3
4
0
5
Ion
Pore
4
8
2
5
5
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
M3
M4
1a-R548A
1a-S549A
1a-T550A
1a-S553A
1a-F554A
1a-Q556A
1a-P557A
1a-S646A
1a-Y647A
1a-N650A
1a-F654A
1a-L655A
1a-L655Y
1a-N803A
1a-T809A
1a-N812A
2D-N566A
2D-T568A
2D-S572A
2D-F574A
2D-E576A
2D-P577A
2D-P580A
2D-S669A
2D-Y670A
2D-N673A
2D-F677A
2D-M678A
2D-M678Y
2D-S835A
2D-K836A
2D-N841A
M3
Pre-M1
M4
Pre-M1
Pre-
M1
90°
30°
GluN2
Pocket
GluN1
Pocket
M3
M3
M3
GluN1
NT
NT
M4
Pre-M1
M3
M4
Pre-M1
M4
C
EU1794-2
EU1794-27
Pre-
M1
30°
GluN2
M3
M4
NT
NT
Figure 6. Distinct and overlapping pattern of residues contribute to the actions of EU1794 modulators with opposing effects. (A) Ribbon representation
(left) of a GluN1/GluN2D model based on published crystal structures of GluN1/GluN2B and GluA2 (Karakas and Furukawa, 2014; Lee et al., 2014;
Yelshanskaya et al., 2016). The GluN1 subunits are grey and the GluN2D are purple; surface shell indicate residues of interest in GluN1 and GluN2D,
including residues that are homologous to those identified in recent studies investigating transmembrane domain interacting modulators of NMDAR
and AMPAR (Yelshanskaya et al., 2016; Wang et al., 2017). These GluN1 and GluN2D residues were changed to alanine (or tyrosine in two cases, as
previously reported) and tested for effects on the NMDAR sensitivity to EU1794-2 and EU1794-27. Top down (right top) and side (right bottom) view of
the model TMD and linker segments highlight two potential pockets with GluN1 and GluN2 M3 helices in the background. One pocket is primarily
associated with GluN1 and the other GluN2. (B) The responses (mean 99% CI) in EU1794-2 (5 mM, left) and EU1794-27 (10 mM, right) are shown as a %
of the mutant receptor response to 100 mM glutamate and 30 mM glycine in the absence of the test compound. The wild type mean response is shown
by the dashed line, surrounding dotted lines, which indicate the 99% confidence interval. Residues with non-overlapping confidence intervals with the
wild type are colored with magenta indicating apparent loss of activity, yellow indicating augmented activity, and green indicating robust inverted
modulation. Data for the mutations shown represent 4–18 oocytes from at least two independent experiments and the wild type response shown
represent 83 oocytes for EU1794-2 and 76 oocytes for EU1794-27, recorded each experimental day to ensure consistency. We were not able to test all
mutant receptors due to low expression for some (NT, not tested). (C) The residues (shown by the space-filling shell) identified in (B) that altered the
Figure 6 continued on next page
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Figure 6 continued
Neuroscience Structural Biology and Molecular Biophysics
effects of EU1794-2 (left) and/or EU1794-27 (right) were mapped onto the model with colors as described in (B). Views are the same as (A), with the all
residues probed shown as grey transparent shell representation.
DOI: https://doi.org/10.7554/eLife.34711.017
The following figure supplements are available for figure 6:
Figure supplement 1. EU1794 series effects are not altered by ATD perturbations.
DOI: https://doi.org/10.7554/eLife.34711.018
Figure supplement 2. Compound competition screen of the EU1794 series with NMDAR modulators highlights potential interactions with the ABD
and the TMD.
DOI: https://doi.org/10.7554/eLife.34711.019
Figure supplement 3. Residues in known modulator binding sites do not perturb actions of the EU1794 series.
DOI: https://doi.org/10.7554/eLife.34711.020
Figure supplement 4. Mutated residues that impact EU1794 actions have lower open probability.
DOI: https://doi.org/10.7554/eLife.34711.021
than 7-CKA (compare the steady state response and the 7-CKA response in Figure 5—figure sup-
plement 1C). Additionally, comparable experiments were performed with 100 mM APV with similar
results, as APV stabilizes the GluN2 agonist binding domain (ABD) open-cleft conformation, and
thereby prevents the pre-binding of EU1794-27 (Figure 5—figure supplement 1D). When EU1794-
27 (3 mM) was included in all solutions, potentiation was observed but APV unbinding was a required
prior to modulation (Figure 5—figure supplement 1D). Together these data suggest that the
EU1794 series is capable of high affinity binding only when glutamate and glycine are bound to the
receptor.
Evaluation of interactions with known modulatory sites
To identify the molecular determinants of action for the EU1794 series, we evaluated the ability of
GluN2 ATD deletion, GluN1 ATD splice variants, and co-application of known modulators to alter
the effects of the EU1794 modulators. The ATD harbors the binding site for the GluN2B-selective
negative allosteric modulator ifenprodil. Deletion of the ATD from GluN2A, GluN2B, or GluN2C had
no effect on the actions of the PAM EU1794-27; deletion of the ATD from GluN2D reduced but did
not eliminate potentiation (Figure 6—figure supplement 1A). Similarly, inclusion of 21 residues in
the ATD encoded by alternatively spliced GluN1 exon5 only slightly altered the extent of potentia-
tion of EU1794-27, but was without effect on EC50 for potentiation of GluN2B- and GluN2D-contain-
ing NMDARs activated by saturating agonist (Figure 6—figure supplement 1B). We previously
described a similar result for the negative allosteric modulator EU1794-2 (Katzman et al., 2015).
These data are consistent with minimal involvement of the GluN1 or GluN2 ATD in the actions of
EU1794 modulators.
We next screened for interaction with known modulators to focus our search for the molecular
determinants of EU1794 series modulation (Mullasseril et al., 2010; Acker et al., 2011;
Hansen and Traynelis, 2011; Hansen et al., 2012; Khatri et al., 2014; Ogden et al., 2014;
Hackos et al., 2016; Tajima et al., 2016; Yi et al., 2016) (Figure 6—figure supplement 2A). In
these experiments, a known positive or negative modulator was co-applied with either EU1794-2 or
EU1794-27, with each pair always containing one PAM and one NAM. If there is no interaction
between paired modulators, their combined activity should be predicted by multiplying the extent
of their independent actions. Co-application of the modulator pairs ifenprodil/EU1794-27, EU1794-
2/CIQ and EU1794-2/PYD-106 produced levels of modulation that largely could be predicted from
their independent actions (Figure 6—figure supplement 2B, top row). Modest differences from pre-
dictions were observed with modulators that bind to the ABD interface, TCN-201/EU1794-27,
EU1794-2/GNE-6901 and EU1794-2/GNE-0723 (Figure 6—figure supplement 2B, bottom left).
Co-application of the GluN2C/GluN2D-selective negative allosteric modulators QNZ46 and DQP-
1105 paired with EU1794-27 resulted in the greatest divergences from predictions, raising the possi-
bility that these modulators have partially overlapping binding sites (Figure 6—figure supplement
2B, bottom right) or similar downstream mechanisms.
We also examined the ability of EU1794-2 and EU1794-27 to modulate NMDARs harboring
mutations within the structural determinants for other known allosteric modulators. Mutations in
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GluN1 (I519A, R755A) and GluN2A (L780A, G786A) that block TCN-201 inhibition were evaluated
for effects on EU1794 series of modulators (Hansen et al., 2012). NMDARs that contained GluN1/
GluN2A(L780A), GluN1/GluN2A(G786A), GluN1(R755A)/GluN2D, GluN1(I519A)/GluN2D were
equally sensitive as wild type receptors to inhibition by EU1794-2 or potentiation by EU1794-27
(Figure 6—figure supplement 3A). GluN1/GluN2A(E530A), GluN1/GluN2A(V783W), GluN1
(Y535W)/GluN2A, GluN1(Y535V)/GluN2D and GluN1(Y535W)/GluN2D, which reduce the actions of
GNE-6901 and GNE-0723 (Hackos et al., 2016), produced no significant effects on inhibition by
EU1794-2 or potentiation by EU1794-27 (Figure 6—figure supplement 3B). Inhibition by EU1794-
2 of GluN1/GluN2C(K470G) and GluN1/GluN2C(S472T), which block PYD-106 potentiation of
GluN1/GluN2C (Khatri et al., 2014), was similar to wild type NMDARs (Figure 6—figure supple-
ment 3C). Interestingly, inhibition by EU1794-2 and potentiation by EU1794-27 was not significantly
changed by GluN1/GluN2D(Q701Y) and GluN1/GluN2D(L705F), mutations that appear to confer
subunit selectivity for GluN2C/D over GluN2A/B for QNZ-46 and DQP-1105 (Figure 6—figure sup-
plement 3D) (Acker et al., 2011; Hansen and Traynelis, 2011).
Mutagenesis suggests shared structural determinants of action for
PAMs and NAMs
The result obtained with the QNZ-46/DQP-1105 interaction test suggested that the two residues we
evaluated for QNZ-46 and DQP-1105 insufficiently probed the structural determinants of action for
these compounds. We therefore examined a GluA2 AMPA receptor structure bound to CP-465,022,
which shares a core scaffold with QNZ modulators (Yelshanskaya et al., 2016). Residues identified
as being important for CP-465,022 binding in Yelshanskaya et al., 2016 were aligned to the GluN1
and GluN2D subunits to map this modulatory site onto the NMDAR subunits, in addition to critical
residues for GNE-9278 in this same region (Figure 6A, Wang et al., 2017). This broader range of
residues were located on the pre-M1, M3, and pre-M4 regions of both GluN1 and GluN2D, which
have previously been suggested to cooperate to control gating (Ogden and Traynelis, 2013;
Alsaloum et al., 2016; Chen et al., 2017; Ogden et al., 2017; Yelshanskaya et al., 2017). These
residues were suggested by Yelshanskaya et al., 2016 to constitute a binding site in homomeric
GluA2 AMPARs. However, mapping the homologous residues onto NMDAR structures yields two
pockets given the multimeric subunit architecture, one of which consists of the residues of GluN1
and the other of residues of GluN2. Certain residues of M3, depending on their position on the
helix, could point towards either pocket, rendering the two pockets to be lined by a mixture of
GluN1 and GluN2 residues. 15 GluN1 and 15 GluN2D residues in these two regions that probed
these pockets were identified and mutated to allow a test of the contribution of each residue to
EU1794-2 inhibition and EU1794-27 potentiation. Inhibition by EU1794-2 was significantly altered
by substitutions at six residues in GluN1 and 2 GluN2D residues (Figure 6B), which were found on
pre-M1 and M3 regions of both GluN1 and GluN2D. Potentiation by EU1794-27 was more labile,
being altered in mutations at 9 GluN1 residues and 6 GluN2D residues (Figure 6B). Residues that
perturbed the actions of EU1794-27 were spread across all regions tested except for the GluN2D
pre-M1. Modulation was observed to be altered in three different ways by the mutations studied
here: activity could be reduced, increased, or inverted.
The distributions of residues that altered modulation by EU1794-2 and EU1794-27 shows clear
overlap (Figure 6C). Inhibition by EU1794-2 was altered primarily by mutations in GluN1, whereas
potentiation by EU1794-27 was perturbed by residues both in GluN1 and in the M3 helix of
GluN2D. Interestingly, the mutations that invert activity of EU1794-2 and EU1794-27 were distinct,
but in some cases were in close proximity. For example, EU1794-2 was inverted by GluN1-F654A,
whereas mutation of the adjacent residue GluN1-L655A/Y inverted the modulatory action of
EU1794-27 (Figure 6D). EU1794-27 potentiation was also converted to inhibition by four mutations
at residues residing on the 3 areas of GluN1 that were investigated (pre-M1, M1, M3) and also on
the M3 GluN2D helix, which were in close proximity to each other (Figure 6D). EU1794-27 actions
on GluN1-L655A/Y and GluN2D-M678Y, which are homologous residues immediately downstream
of the SYTANLAAF motif, resulted in opposite effects (GluN1-L655A/Y converts EU1794-27 to an
inhibitor and GluN2D-M678Y increases the potentiation of EU1794-27). We interpret these results
to suggest that the activity of both PAMs and NAMs of the EU1794 series is dependent on multiple
residues in the GluN1 subunit, some of which are overlapping. Furthermore, potentiation by
EU1794-27 is dependent on a wider range of GluN1 and GluN2 residues. One possible way to
Perszyk et al. eLife 2018;7:e34711. DOI: https://doi.org/10.7554/eLife.34711
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Dose Ratio Plot:
Model 1
Dose Ratio Plot:
Model 2
A
B
C
Model 1
Model 2
3
2
1
0
2
1
N
N
P
P
0
-1
-7
-7
-6
-5
-4
-6
-5
-4
[PAM]
[PAM]
D
E
F
GluN2D
Experimental Competition:
GluN2D
100
Experimental Data
Dose Ratio Plot: GluN2D
3
EU1794-27
EU1794-2
Glu/Gly
EU1794-27
80
60
40
20
0
0 µM
3 µM
10 µM
20 µM
2
1
0
500 nA
60 s
-7
-6
-5
EU1794-27 (µM)
-4
0.1
1 10 100
EU1794-2 (µM)
Figure 7. EU1794 PAMs and NAMs act in a competitive manner that matches receptor models with mutually
exclusive modulator binding. (A) Cartoon models show two potential modulator binding schemes. Model 1
possesses mutually exclusive modulator binding whereas Model 2 can bind either or both modulators. Ligands are
denoted by [N] (NAM) and [P] (PAM). (B,C) Dose-ratio plots are shown for simulated concentration-response
curves of the NAM in various concentration of the PAM for Model 1 (B) and Model 2 (C). IC50’ is the observed
NAM IC50 in the presence of a fixed concentration PAM. (D) A representative TEVC recording (fixed 3 mM EU1794-
27 and 0.3, 1, 3, 10, 30 mM EU1794-2) is shown for GluN1/GluN2D NMDARs (top). (E) The mean steady state
responses in (D) were fitted with the Hill equation. Concentration-response data in the absence of EU1794-27 are
included for comparison from Figure 1C. (F) Dose-ratio analysis of experimental data illustrates the shift in the
IC50 of the NAM as a function of PAM concentration. Data are from at least six oocytes evaluated from two
independent experiments.
DOI: https://doi.org/10.7554/eLife.34711.022
The following figure supplement is available for figure 7:
Figure supplement 1. EU1794 PAMs and NAMs act in a competitive manner that suggests mutually exclusive
modulator binding.
DOI: https://doi.org/10.7554/eLife.34711.023
account for this would be if both modulators bound near the GluN1 pre-M1 helix, with potentiator
actions dependent on the nearby GluN2 M3 residues associated with the GluN1 pocket (Figure 6A,
C).
To assess whether mutated residues impacted EU1794 series modulation through altering recep-
tor open probability, we estimated mutant open probability using MTSEA modification of an intro-
duced cysteine residue (Jones et al., 2002; Yuan et al., 2005). The open probability of GluN2D-
containing receptors is very low and may not allow for precise determination of the effects of muta-
tions, thus we used GluN2B-containing receptors for comparison. GluN1 mutations were expressed
with a GluN2B A7C construct (See Figure 6—figure supplement 4 legend) and GluN2 mutations
were expressed with a GluN1 A7C construct. Several residues were assayed that resulted in
reversed, enhanced, or blocked modulator activity (Figure 6—figure supplement 4A). GluN1-
P557A, which blocked activity of both EU1794-2 and EU1794-27, had an increased open probability
as compared to wild type. GluN1-F654A, which converted EU1794-2 into a potentiator and
enhanced EU1794-27 potentiation, had a similar open probability as compared to wild type (Fig-
ure 6—figure supplement 4A). Interestingly, mutation of the homologous GluN2 residue (GluN2-
F653A), which reduced EU1794-27 potentiation, also lowered open probability as compared to wild
type (Figure 6—figure supplement 4B). Figure 6—figure supplement 4 shows that GluN1-L665A
and GluN1-N803A, which both converted EU1794-27 into an inhibitor, had significantly lower open
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Table 6. The response to co-application of EU1794-2 and EU1794-27 suggest a common binding site.
EU1794-2 IC50 (mM) [conf. int.]* EU1794-27 EC50 (mM) [conf. int.]*
+EU1794-27
+EU1794-27
+EU1794-27
+EU1794-27
+EU1794-2
+EU1794-2
+EU1794-2
0 mM
3 mM
10 mM
20 mM
0 mM
1 mM
3 mM
§
§
§
GluN2A
GluN2B
GluN2C
GluN2D
0.60†
[0.44, 0.82]
2.5‡
[1.9, 3.1]
3.6‡
[2.4, 4.8]
8.0‡
[1.3, 15]
-
-
-
1.2†
[0.8, 1.9]
NR
3.5‡
[2.1, 4.9]
12
[~, 26]
1.4†
[1.2, 1.7]
6.0‡
[4.6, 7.4]
8.4
[~, 18]
0.21†
[0.18, 0.25]
1.2‡
[0.91, 1.4]
2.4‡
[1.8, 3.0]
6.0‡
[4.1, 7.8]
2.8†
[2.5, 3.2]
7.3‡
[5.9, 8.8]
7.3
[2.1, 11.74]
0.20†
1.4‡
2.7‡
7.3‡*
2.4†
7.5‡
7.8
[0.17, 0.25]
[1.2, 1.6]
[2.3, 3.2]
[4.6, 9.9]
[1.8, 3.3]
[5.6, 9.3]
[1.1, 14]
A five point concentration response curve was obtained for modulator effects on responses to maximally effective concentrations of glutamate and glycine
(100/30 mM) for EU1794-2 co-administered with increasing concentrations of EU1794-27, and for EU1794-27 in increasing concentrations of EU1794-2.
*IC50 and EC50 values were obtained by least-squares fitting of data by the Hill equation. IC50 and EC50 values are given as the mean with the 95% confi-
dence interval determined from log(IC50) or log(EC50). Data are from at least six oocytes evaluated from two independent experiments for the EU1794-2
IC50 determinations and from at least three oocytes evaluated from one experiment for the EU1794-27 EC50 determinations.
NR, Given the low potency for modulators at GluN2B, the EU1794-2 concentration-response curve with 3 mM EU1794-27 determination was not recorded.
† Data from Table 1 is included here for clarity.
‡indicates non-overlapping 95% confidence interval with the EU1794-2 IC50 without EU1794-27 or EU1794-27 EC50 without EU1794-2.
§EU1794-27 does not potentiate GluN2A responses to maximally effective concentrations of glutamate and glycine.
~indicates that the confidence interval reached the theoretical limit (EC50 = 0).
DOI: https://doi.org/10.7554/eLife.34711.024
probabilities as compared to wild type NMDARs (Figure 6—figure supplement 4A). These data
suggest that altered EU1794 modulator action by mutated residues is not directly dependent on
alterations in open probability.
PAMs and NAMs exert their opposing effects via a shared binding site
on NMDARs
Given the similarity in chemical structure between positive and negative modulators in this series, we
hypothesized that they might have overlapping binding sites. In order to conduct a detailed
Table 7. Enantiomeric preference of EU1794-27.
EC50 (mM) [conf. int.]*
Maximal Modulation Extent (% of control)
Compound
EU1794-27
(-)-EU1794-27
(+)-EU1794-27
GluN2A
7.4 [5.3, 10]†
52 7%
5.6 [2.7, 12]
67 5%
-
‡
‡
‡
‡
GluN2B
GluN2C
GluN2D
1.4 [1.2, 1.7]†
130 3%
2.8 [2.0, 3.8]
170 10%
-
2.8 [2.5, 3.2]†
230 10%
5.1 [4.1, 6.4]
260 24%
7.1 [3.6, 14]
160 6.2%
2.4 [1.8, 3.3]†
250 8%
8.2 [6.7, 9.9]
340 21%
7.4 [3.7, 15]
130 4.7%
*EC50 values were obtained by least-squares fitting of data from individual experiments by the Hill equation. EC50
values are given as the mean with the 95% confidence interval determined from log(EC50); the maximal degree of
modulation is given as mean SEM. Data are from 4 to 7 oocytes from at least two independent experiments. Data
were not fitted (shown as -) if the response recorded at 30 mM of test compound did not differ by more than 15%
from control.
†Data from Table 1 is included here for clarity.
‡indicates significant unpaired t-test between the percent modulation between 30 mM (-)-EU1794-27 and (+)-
EU1794-27.
DOI: https://doi.org/10.7554/eLife.34711.027
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A
GluN2D
(-)-EU1794-27
(+)-EU1794-27
Glu+Gly
10 s
B
GluN2D
(-)-EU1794-4
(+)-EU1794-4
Glu+Gly
10 s
GluN2D
GluN2D
300
200
100
0
300
C
*
*
200
100
0
(-)-EU1794-4 (10μM)
(-)-EU1794-27 (20μM)
(+)-EU1794-4 (10μM)
(-)-EU1794-27 (20μM)
+
-
-
+
+
+
+
-
-
+
+
+
GluN2A
GluN2A
100
80
60
40
20
100
80
60
40
20
*
*
0
(-)-EU1794-4 (20μM)
(-)-EU1794-27 (20μM)
0
(+)-EU1794-4 (20μM)
(-)-EU1794-27 (20μM)
+
-
-
+
+
+
+
-
-
+
+
+
Figure 8. The enantiomers of EU1794-4 and EU1794-27 have different action. (A) Representative modulation of
GluN1/GluN2D responses by the enantiomers of EU1794-27 (both 10 mM, 100/30 mM glutamate/glycine). Similar
results were observed in at least 3 cells to fresh modulator solution made less than one hour before experiment
conclusion. (B) Representative modulation of GluN1/GluN2D responses by the enantiomers of EU1794-4 (both 10
mM, 100/30 mM glutamate/glycine). Similar results were observed in at least 3 cells to fresh modulator solution
made less than one hour before experiment conclusion. (C) Single concentration competition experiments
illustrating an interaction between both enantiomers of EU1794-4 (+and -) and the positive modulating
enantiomer of EU1794-27 (-) at GluN1/GluN2D. The individual average effects of the pairs of modulators along
with the co-applied effect are plotted, and the predicted mean net effect of the modulators is displayed as a
dashed line (SEM, dotted lines, n = 10 from two independent experiments). * signifies p<0.05 as determined by a
paired t-test comparing co-applied modulators and predicted net effect of modulator actions.
DOI: https://doi.org/10.7554/eLife.34711.025
The following figure supplement is available for figure 8:
Figure supplement 1. The concentration response and stability of the enantiomers of EU1794-27 and EU1794-4.
DOI: https://doi.org/10.7554/eLife.34711.026
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functional analysis of competition between allosteric modulators, we first used modeling to examine
a receptor’s functional response to co-application of positive and negative modulators acting at the
same or different sites. We evaluated two models of modulator binding built into a scheme first pro-
posed by (Lester and Jahr, 1992). Model 1 describes receptors with a single binding site that can
accommodate either PAM or NAM, but not both. Model 2 describes a receptor to which both mod-
ulators can bind at the same time in different sites (Figure 7A, Figure 7—figure supplement 1). We
evaluated hypothetical responses from these models (see Materials and methods, Figure 7—figure
supplement 1A–C). Responses to maximally effective concentrations of agonist were simulated at
various concentrations of the positive and negative modulators. When the modulators compete for
the same binding site, they act similarly to stated theory about competitive antagonists
(Arunlakshana and Schild, 1959; Christopoulos and Kenakin, 2002), with the PAM causing a right-
ward shift of the NAM IC50 and generating a linear relationship in dose ratio analysis (Figure 7B, Fig-
ure 7—figure supplement 1D,E, Model 1). By contrast, there is no apparent EC50 shift when the
two modulators are capable of binding simultaneously (Figure 7C, Figure 7—figure supplement
1D,E, Model 2). Additional simulations showed the reciprocal effect of NAM on PAM EC50 (Fig-
ure 7—figure supplement 1F,G). We subsequently performed competition experiments to test the
hypothesis that this series of PAMs and NAMs compete for a mutually exclusive modulatory pocket
(Figure 7D). We observed that fixed concentrations of the PAM EU1794-27 caused parallel shifts in
the concentration-response curve of the NAM EU1794-2 (Figure 7E, Table 6). A similar phenome-
non was shown for the reverse, as fixed concentrations of EU1794-2 caused parallel shifts in the
EU1794-27 concentration-response curves (Table 6). These results closely match Model 1, where
PAMs and NAMs bind in a mutually exclusive fashion. This suggests that the opposing modulators in
the EU1794 series share overlapping binding sites instead of the coincidence that the subtle chemi-
cal differences confer unique binding sites.
The enantiomers of EU1794-27 were separated to determine whether the racemic activity
reflected the actions of only one enantiomer (see Supplemental methods). (-)-EU1794-27 potenti-
ated NMDAR responses similarly to the racemic mixture (Figure 8—figure supplement 1A, Table 7).
By contrast, (+)-EU1794-27 exhibited only weak potentiation of GluN1/GluN2C and GluN1/GluN2D,
which may be due to the purity achieved via chiral separation (Table 7). We previously reported
(Katzman et al., 2015) that purified enantiomers were likely to racemize in aqueous solutions, which
should proceed by a first-order reaction dependent on multiple factors such as ionic strength, pH,
buffer, etc. (Smith et al., 1978). Thus, to quantitatively determine if racemization would impact our
experiments using the enantiomers, we performed a functional assessment of this property in our
standard experimental solution. We observed racemization of the enantiomers of EU1794-27 with a
half-life of 197 min (Figure 8—figure supplement 1B, Table 7). Both enantiomers of EU1794-4
inhibited NMDAR responses, but with different potencies (Figure 8—figure supplement 1C,
Supplementary File 2). A similar rate of racemization was observed for the enantiomers of EU1794-
4 (half-life of 196 min, Figure 8—figure supplement 1D). All enantiomer studies, other than the
racemization time-course, were performed rapidly to minimize any racemization, being completed in
less than 80 min after making the aqueous solution of modulator. Similar to results in oocytes, when
the enantiomers of EU1794-27 were applied to steady-state GluN1/GluN2D responses in HEK cells,
(-)-EU1794-27 potentiated responses whereas (+)-EU1794-27 had
a slight inhibitory effect
(Figure 8A). In additional, 10 mM of both enantiomers of EU1794-4 had similar inhibitory actions on
GluN1/GluN2D responses to maximally effective concentrations of glutamate and glycine
(Figure 8B).
To assess whether both enantiomers bound to similar or distinct sites on the NMDAR, we per-
formed single concentration competition experiments. Co-application of each enantiomer of
EU1794-4 with the (-)-EU1794-27 resulted in a degree of modulation that was significantly different
than that predicted for independent sites of action at both GluN1/GluN2A and GluN1/GluN2D
(Figure 8C). Interestingly, the competition by EU1794-4 was not dependent on the direction of
modulation by (-)-EU1794-27, which had a potentiating action at GluN1/GluN2D (Figure 8C, top
panels) and an inhibitory effect at GluN1/GluN2A (Figure 8C, bottom panels). Although there
remain potential caveats, the evidence suggests that the co-application of (-)-EU1794-27 with either
enantiomer of EU1794-4 displays mutual exclusivity in modulator binding.
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Discussion
This study highlights how subtle chemical variations in a series of NMDAR allosteric modulators can
result in fundamentally different actions on NMDAR responses to maximally effective concentrations
of agonists. This series of small molecule allosteric modulators show a spectrum of effects ranging
from strong negative modulation to robust positive modulation. In addition, there are features that
are shared across this series, including agonist-dependence and the ability to enhance agonist
potency. Thorough analysis of the actions of this series illustrates a potential way forward in design-
ing new analogues to achieve a wide range of activities. Additionally, the novel features found in this
class of modulators suggest potential new strategies for targeting distinct populations of NMDARs,
such as extrasynaptic receptors that typically are not activated by high concentrations of glutamate
or distinct patterns of stimulation.
The site and mechanism of action for the EU1794 series
In the evaluation of the kinetic properties of modulator action, we observed complex actions of
EU1794-4 and EU1794-27, which could arise from multiple binding sites, enantiomers of these com-
pounds, or could reflect distinct modulator-dependent mechanistic actions from occupancy of a sin-
gle binding site. The modulation by EU1794-4 and EU1794-27 is voltage independent, eliminating
potential channel block within the ion channel pore by potential cationic species as a confounding
site that contributes to the observed effects. Given that there is high homology between GluN1 and
GluN2, especially in the ABD and the TMD, a reasonable hypothesis is that multiple binding sites
exist for EU1794 series modulators in homologous regions on GluN1 and GluN2 subunits. However,
the mutagenesis data argue against this idea, given that the residues at which mutations perturbed
the actions of EU1794-2 and EU1794-27 overlapped and clustered around the pre-M1, M3, and M4
of the GluN1 subunit. Whereas there were a few residues of the GluN2 M3 helix that influenced
EU1794-27 modulation, the structural NMDAR models suggest that these residues face the GluN1
M3 helix, and thus could interact with the pocket adjacent to the GluN1 pre-M1 helix. Additionally,
it’s likely the M3 helixes of both GluN1 and GluN2, which are in close contact, act in concert with
one another to control rapid pore opening or closing. If these residues identified by mutagenesis
controlled conformational changes downstream of the EU1794 binding site(s), it would limit poten-
tial binding site candidates to the interface between the GluN1 and the GluN2 ABDs, which would
then have only two identical binding sites in diheteromeric NMDARs. Therefore there would be less
potential for non-identical binding sites that contribute to the mixed actions that are observed for
EU1794-27 and EU1794-4. For these reasons, the idea that the positive and negative allosteric
actions reflect modulator specific mechanisms from occupancy of a single site seems the most plausi-
ble interpretation.
Evaluation of the effects of enantiomers provides further insight into the binding site of the
EU1794 series. EU1794-27 exhibits strong stereoselectivity, with the (-) enantiomer showing a typi-
cal potentiation time course and the (+) enantiomer producing inhibition. The enantiomers of
EU1794-4 are both capable of producing inhibition but with the (-) enantiomer being more potent
(3–10 fold) than the (+) enantiomer. Additionally, both enantiomers of EU1794-4 appear to compete
with EU1794-27 for access to the binding site, suggesting that enantiomers may interact differently
with the same binding site. An alternative explanation for lack of additivity of the effects of the two
compounds could be that there are shared residues downstream of the PAM and NAM binding sites
that mediate their effects; additional studies are required to evaluate this hypothesis. However, the
idea that the enantiomers of the EU1794 series act at the same site suggests their complex actions
on NMDARs expressed in HEK is a mixture of receptors bound to one or the other enantiomer of
racemic EU1794-27 and EU1794-4. The available enantiomeric data further support the idea that
positive and negative modulators within the EU1794 series share a single or overlapping binding
site.
EU1794 series links positive and negative allosteric modulators that act
at the TMD
An increasing number of NMDAR and AMPAR modulators have been identified with structural
determinants of action that reside in transmembrane linker regions and extracellular portions of the
transmembrane domain (Mullasseril et al., 2010; Acker et al., 2011; Hansen and Traynelis, 2011;
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Ogden and Traynelis, 2013; Yelshanskaya et al., 2016; Swanger et al., 2018; Wang et al., 2017).
Other cell surface receptor families have bi-directional modulator pockets, including multiple GPCRs
as well as the benzodiazepine binding site in GABA-A receptors (Barnard et al., 1998; Rudolph and
Knoflach, 2011; Wootten et al., 2013). Among all ionotropic glutamate receptor modulators inter-
acting with this region of the receptor, there are positive (CIQ, GNE-9278) and negative (DQP-1105,
QNZ-46, CP-465,022, GYKI 52466) modulators with diverse scaffolds. However, the EU1794 com-
pounds represent the first bidirectional NMDAR modulator series with structural determinants of
activity within this region, low micromolar potency and with clear rules to control modulation. The
similarity in structure of positive and negative modulators illustrates how subtle differences in the
ligand can interconvert functional actions between inhibition and potentiation. It is unclear precisely
how the different sized ester alkyl chain substitutions of these analogs bring about opposing actions,
further investigation of the structure activity relationship may elucidate these details. The ability to
potentiate NMDAR seems to be unique to (-)-EU1794-27, which may have a specific interaction with
the receptor achieved only by its stereoselective active pose in the binding pocket. Work with this
series may lead to an understanding of the mechanistic link between the PAMs and NAMs that inter-
act in this portion of the receptor.
Differential actions on synaptic and extrasynaptic receptors by
submaximal EU1794 analogues
The EU1794 series has a property of agonist-dependence, requiring both glutamate and glycine to
be bound before the modulator binding site adopts a high potency orientation for members of the
series. Relatedly, use-dependence is a property of open channel blockers (e.g. memantine, MK-801,
ketamine, etc.) that inhibit the receptor through interactions within the ion permeation path, and
thus rely on pore opening (Traynelis et al., 2010). Given that the EU1794 series are allosteric modu-
lators are not voltage-dependent, the mechanism of their agonist-dependence is unclear. Previously,
the NAMs QNZ-46, DQP-1105, and NAB-14 have been reported to show varying degrees of gluta-
mate- but not glycine-dependence (Acker et al., 2011; Hansen and Traynelis, 2011;
Swanger et al., 2018). Moreover, neurosteroid derivatives with NAM activity have also been shown
to have glutamate- and glycine-dependence (Vyklicky et al., 2015), and the PAM GNE-9278 was
reported to be glutamate-dependent (Wang et al., 2017). Thus, the EU1794 series is the first series
of positive and negative modulators that has been shown to possess the property of being both glu-
tamate- and glycine-dependent. Our working hypothesis is that the EU1794 series of modulators
requires conformational changes in both GluN1 and GluN2 subunits that reflect pre-gating or gating
transitions, after which its affinity for its binding site is increased. However, we cannot rule out at this
time the possibility that the pore must open to increase modulator binding. Resolving the specific
mechanism of the EU1794 may lead to a more complete understanding of the activation transitions
NMDAR.
We believe that EU1794-4 highlights a novel sub-class of NMDAR modulators that have the capa-
bility to selectively act at extrasynaptic NMDARs based on the combinations of its properties. Extra-
synaptic NMDAR are hypothesized to respond to glutamate spillover or glial release of glutamate
(Rusakov and Kullmann, 1998; Haydon and Carmignoto, 2006; Sahlender et al., 2014). The
potential ability to preferentially act at these non-synaptic sites arises from three mechanistic fea-
tures of the allosteric mechanism: (1) the submaximal inhibitory effects at saturating concentrations
of modulator and agonist, (2) the agonist-dependent and slow association rate, which might limit
activity at synaptic receptors, and (3) the ability to enhance NMDAR responses to low agonist con-
centrations. A similar property has been described for the GluN2B-selective agent ifenprodil
(Kew et al., 1996), although the extent to which EU1794-4 can enhance the response to low con-
centrations of agonist is amplified by the large degree of residual current at saturating levels of
EU1794-4 (30–60%) compared to ifenprodil (~10%). There are numerous studies that suggest the
importance of extrasynaptic NMDARs in normal biology but study of them requires complex experi-
mental paradigms (Harris and Pettit, 2008; Paoletti et al., 2013; Papouin and Oliet, 2014). Alter-
natively, the agonist-dependence of EU1794-4 may alter this capability in instances of repeated
stimulation. In either case, EU1794-4 is a unique compound that may act as a tool that could be
used to probe the contribution of distinct types of NMDARs or their activity in circuit function. Fur-
ther work is required to fully understand the utility of this modulator as a probe for extrasynaptic
NMDARs.
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Materials and methods
Key resources table
Reagent type (species)
or resource
Designation
Source or reference
U11418
Identifiers
Additional information
gene (Rattus norvegicus)
gene (Rattus norvegicus)
gene (Rattus norvegicus)
gene (Rattus norvegicus)
gene (Rattus norvegicus)
gene (Rattus norvegicus)
cell line (Xenopus laevis)
GluN1-1a
GluN1-1b
GluN2A
GluN2B
GluN2C
GluN2D
U08263
D13211
U11419
M91563
L31611
stage VI oocytes Ecocyte Biosciences
(Austin, TX);
NA; #10004
Xenopus 1 (Dexter, MI)
cell line (Homo sapiens)
HEK293
ATCC (Manassas, VA)
CRL-1573
200524
commercial assay or kit
QuikChange kit
Agilent Technologies
(Santa Clara, CA)
commercial assay or kit
mMessage
mMachine
SP6 or t7
Ambion/ThermoFisher
Scientific (Waltham, MA)
AM1340 or AM1344
chemical compound, drug EU1794-4
chemical compound, drug EU1794-2
chemical compound, drug EU1794-5
chemical compound, drug EU1794-19
PMID: 26525866
PMID: 26525866
this paper
SMILES: CCOC(=O)C1 = C(NC(=O)
CC2SC(=N)NC2 = O)SC2 = C1CCCC2
SMILES: CCOC(=O)C1 = C(NC(=O)
CC2SC(=N)NC2 = O)SC2 = C1CCC(C)C2
SMILES: CC(C)OC(=O)C1 = C(NC(=O)
CC2SC(=N)NC2 = O)SC2 = C1CCCC2
See sumplemental
information
this paper
SMILES: CC(C)OC(=O)C1 = C
(NC(=O)CC2SC(=N)NC2 = O)
SC2 = C1CCC(C)C2
See sumplemental
information
chemical compound, drug EU1794-25
chemical compound, drug EU1794-27
this paper
this paper
this paper
SMILES: CC1CCC2 = C(C1)SC(NC(=O)
CC1SC(=N)NC1 = O)=C2C(=O)
OC1 = CC = CC=C1
See sumplemental
information
SMILES: CC(C)(C)OC(=O)C1 = C
(NC(=O)CC2SC(=N)NC2 = O)
SC2 = C1CCCC2
See sumplemental
information
chemical compound, drug EU1794-29
chemical compound, drug bCD
chemical compound, drug APV
SMILES: CCCCOC(=O)C1 = C(NC(=O)
CC2SC(=N)NC2 = O)SC2 = C1CCC(C)C2 information
See sumplemental
Acros Organics or
ChemCenter
2-(hydroxypropyl)-b-cyclodextrin
(CAS# 128446-35-5)
Tocris (Minneapolis, MN)
(2)-amino-5-phosphonovaleric acid
(CAS# 76326-31-3)
chemical compound, drug 7-CKA
chemical compound, drug MTSEA
Tocris (Minneapolis, MN)
TRC Canada (Toronto, ON)
7-chlorokynurenic acid (CAS# 18000-24-3)
Methanethiosulfonate ethylammonium
(CAS# 16599-33-0)
chemical compound, drug CIQ
Tocris (Minneapolis, MN)
Tocris (Minneapolis, MN)
PMID: 25205677
CAS# 486427-17-2
CAS# 23210-56-2
chemical compound, drug Ifenprodil
chemical compound, drug PYD-106
chemical compound, drug TCN-201
chemical compound, drug GNE-6901
Tocris (Minneapolis, MN)
CAS# 852918-02-6
PMID: 26875626; Genentech
(San Francisco, CA)
generously provided by
Jesse Hanson and
Genentech
chemical compound, drug GNE-0723
PMID: 26919761; Genentech
(San Francisco, CA)
generously provided by
Jesse Hanson and
Genentech
chemical compound, drug DQP-1105
Tocris (Minneapolis, MN)
CAS# 380560-89-4
Continued on next page
Perszyk et al. eLife 2018;7:e34711. DOI: https://doi.org/10.7554/eLife.34711
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Continued
Reagent type (species)
or resource
Designation
Source or reference
Tocris (Minneapolis, MN)
this paper
Identifiers
CAS# 1237744-13-6
Additional information
chemical compound, drug QNZ-46
software, algorithm
Perszyk2018_
sim_Markov_
Modulator
Matlab code, see
sumplemental
information
Competition
software, algorithm
software, algorithm
Axopatch and
Clampfit
Molecular Devices
(San Jose, CA)
Easy Oocyte
Custom written Software
(Emory University, Atlanta, GA)
software, algorithm
software, algorithm
Prism
GraphPad (La Jolla, CA)
Mathworks (Natick, MA)
Matlab
Molecular Biology
cDNAs for rat wild type NMDAR subunits GluN1-1a (GenBank U11418, U08261; hereafter GluN1 or
GluN1a), GluN1-1b (U08263, hereafter GluN1b), GluN2A (D13211), GluN2B (U11419), GluN2C
(M91563), and GluN2D (L31611, modified as described in Monyer et al., 1994) were provided by
Drs. S. Heinemann (Salk Institute), S. Nakanishi (Kyoto University), and P. Seeburg (University of Hei-
delberg). Site-directed mutagenesis was conducted using the QuikChange kit (Agilent Technologies,
Santa Clara, CA) following the recommended protocol. DNA sequencing was used to verify all muta-
tions. The amino acid numbering system started with the initiating methionine as residue number
one. For expression in Xenopus laevis oocytes, cDNA constructs were linearized by restriction
enzymes, and subsequently used to synthesize in vitro cRNAs following the manufacturer’s protocol
(mMessage mMachine, Ambion, ThermoFisher Scientific, Waltham, MA).
Two-electrode voltage-clamp recordings
Xenopus laevis stage VI oocytes (Ecocyte Biosciences, Austin, TX) were injected as previously
described (Hansen et al., 2013) 2–7 days prior to recording with cRNA encoding the NMDAR subu-
nits and stored at 15 C in media containing (in mM) 88 NaCl, 2.4 NaHCO3, 1 KCl, 0.33 Ca(NO3)2,
˚
0.41 CaCl2, 0.82 MgSO4, 5 Tris-HCl; pH was adjusted to 7.4 with NaOH and the solution supple-
mented with 1 U/mL penicillin, 0.1 mg/mL gentamicin sulfate, and 1 mg/mL streptomycin. Two-elec-
trode voltage-clamp recordings were performed at room temperature (21–23 C) using different
˚
internal solutions for voltage and current electrodes (0.3 M and 3.0 M KCl, respectively). All extracel-
lular solutions were made from a solution containing (in mM) 90 NaCl, 1 KCl, 10 HEPES, 0.5 BaCl2,
0.01 EDTA (pH 7.4 with NaOH). Experiments were performed using an eight port Modular Valve
Positioner (Hamilton Company, Reno, NV) and controlled by custom software (Easy Oocyte, Emory
University, Atlanta, GA)to exchange solutions. Oocyte currents were recorded at a holding potential
of À40 mV using a two-electrode voltage-clamp amplifier (OC-725B or OC-725C, Warner Instru-
ments, Hamden, CT). For some recordings, 1 mM 2-(hydroxypropyl)-b-cyclodextrin was added to
wash solutions to ensure modulators did not adhere to the tubing, valves, or recording chamber. In
MTSEA experiments, fresh MTSEA solutions (0.2 mM) were prepared and utilized in less than 30 min
to limit compound degradation.
Whole cell patch-clamp recordings
HEK293 cells (CRL-1573, ATCC, Manassas, VA) were maintained in a tissue culture room solely
reserved for their culturing and transfected using a CaPO4 protocol to express the diheteromeric
NMDARs as previously described (Hansen et al., 2013). Cell lines were obtained directly from the
supplier, were expanded, frozen and kept in liquid nitrogen in a dewar. Mycoplasma testing was not
performed. cDNA ratios for transfection were 1:1:1 (GluN2D) or 1:1:5 (GluN2A) for GluN1:GluN2:
GFP. Cells were maintained in 5% CO2 at 37 C for 12–36 hr post-transfection. Prior to and during
˚
recording, cells were bathed in a solution containing (in mM) 150 NaCl, 10 HEPES, 3 KCl, 0.5 CaCl2,
0.01 EDTA (pH 7.4). Cells were patched with borosilicate glass micropipettes (3–4 MW) that were
filled with internal recording solution that contained (in mM) 95 CsGluconate, 5 CsCl, 40 HEPES, 8
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NaCl, 5 MgCl2, 10 BAPTA, 0.6 EGTA, 2 Na2ATP, and 0.3 NaGTP (pH 7.35). The whole cell recording
conformation was achieved, and the cell lifted into the flow of solution from a two barreled theta
glass or a triple barrel square glass perfusion system to perform rapid solution exchange experi-
ments. The theta/triple barreled glass was translated using a piezoelectric manipulator to exchange
the solution around the cell. Calibration of the perfusion manifold was performed each day to ensure
the 10–90% rise time of the solution exchange (switching between 0/100% and 50/50% H2O/external
solution) around an open tip was less than 1.5 ms for a theta tube and 4 ms for a transition into an
outside lane and 2 ms for a transition between lanes for a triple barreled manifold. For some record-
ings, 1 mM 2-(hydroxypropyl)-b-cyclodextrin was added to wash solutions to ensure modulators did
not adhere to the tubing, valves, or recording chamber.
Statistical analysis
Concentration-response curves for both positive and negative modulators were fitted by the Hill
equation:
!
h
I
½MŠ
½MŠhþEC5h0
¼ 1 þ Extent
I
½MŠ¼0
where I is the measured response of receptor activation, Extent is the maximal predicted modula-
tion of the glutamate/glycine response, [M] is the concentration of the modulator being used, h is
the Hill slope, and EC50 is the half-maximal effective concentration of the modulator. Modulator con-
centration response data were plotted as a percentage of unmodulated response and displayed fit-
ted curves were obtained by fitting all data simultaneously.
Glutamate and glycine concentration-response curves were fit with the Hill equation as follows:
!
h
I
½AŠ
½AŠhþEC5h0
¼
IMax
where I is the measured response of receptor activation, [A] is the concentration of the agonist, h
is the Hill slope, and EC50 is the half-maximally effective concentration of the agonist. Agonist con-
centration-response data, unless otherwise stated, were plotted as a percentage of maximal
response and displayed fitted curves were obtained by fitting all data simultaneously with the appro-
priate Hill equation.
The time course for the onset and offset of positive and negative modulation was fitted using a
single exponential function:
ꢀ
ꢁ
Àt
It ¼ A Ã 1 À e t þ c
where t is time, It is the difference in current at a given time point as compared to the response
at t = 0, A is the amplitude of the exponential, t is the time constant for the exponential and c is a
constant. Additionally, some of the complex modulator time-courses were fitted with the sum of a
single exponential equation and a linear component
ꢀ
ꢁ
Àt
It ¼ A Ã 1 À e t þ mt þ c
where m is the slope.
Measurements are given as mean SEM unless otherwise indicated. The number of replicate
experiments used was chosen to ensure a power level of at least 0.80 when a = 0.05 and when
detecting appropriate effect sizes. EC50 and IC50 values and confidence intervals reported were cal-
culated by averaging the log(EC50) or log(IC50) value, determining confidence intervals for mean log
(EC50) or log(IC50), and converting back to units of molarity. Statistical significance evaluations (a set
to 0.05) were performed using a one-way ANOVA with Dunnett’s multiple comparison test, a paired
t–test, or other appropriate tests as described.
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Modelling receptor function
Matlab (Mathworks, Natick, MA) scripts were written based on previously described approaches to
modelling channel function (Colquhoun and Hawkes, 1977; Colquhoun and Hawkes, 1995).
Briefly, a Q matrix was constructed for a model that accounts for macroscopic responses
(Lester and Jahr, 1992). The glutamate association and dissociation rates were from (Erreger et al.,
2005), and unidirectional rate constants for gating and modulator binding were chosen to allow the
model to be used for modulator competition. The differential equations derived by the Q matrix
were solved using an ordinary differential equation solver (ode23s, Matlab) and occupancy of each
state was determined at equilibrium given the concentrations of the theoretical ligands.
Supplemental methods
Synthetic chemistry experimental details
All reagents were obtained from commercial suppliers and used without further purification unless
specified otherwise, and were > 90% pure as determined by the manufacturer. Reaction progress
was monitored by thin layer chromatography (TLC) on pre-coated glass plates (silica gel 60 F254,
0.25 mm) or liquid chromatography-mass spectrometry (LC-MS) using an analytical column (ZORBAX
Eclipse XDB-C18, 4.6 x 50 mm, 3.5 mm, Agilent). When noted, flash chromatography was performed
on a Teledyne (Lincoln, NE) ISCO Combiflash Companion with prepackaged Teledyne Redisep or
Silicycle disposable normal phase silica columns. Proton and carbon NMR spectra were recorded on
an INOVA-400 (400 MHz), VNMRS 400 (400 MHz), INOVA-600 (600 MHz), or Unity-600 (600 MHz).
All chemical shifts are reported in parts per million and referenced to the residual solvent peak.
Mass spectra were performed by the Emory University Mass Spectroscopy Center on either a VG
70-S Nier Johnson or JEOL instrument. The IR spectra were acquired with a Nicolet Avatar 370
DTGS. Final compound purity was established either through high-performance liquid chromatogra-
phy (HPLC) under the conditions listed or by elemental analysis performed by Atlanta Microlab Inc.
where C, H, N agreed with proposed structures within 0.4% of theoretical values unless indicated
otherwise.
Separation of EU1794-4 enantiomers
Semi-preparative separation of EU1794-4 enantiomers from racemic EU1794-4 (0.020 g) was
accomplished using a ChiralPak OD-RH (21 mm  250 mm, 5 mm) with the following conditions: 10.4
mL/min flow rate, 10 mL injection volume (2 mg/1 mL), 30% ACN/70% water with 0.1% formic acid
over 120 min to afford (À)-EU1794-4, tR 62 min; (+)-EU1794-4, tR 65 min. The enantiomeric excess
(ee) was determined using a ChiralPak OD-RH column (4.6 mm  150 mm, 5 mm) with the following
conditions: 1 mL/min flow rate, 5 mL injection volume, 30% ACN/70% water with 0.1% formic acid.
(À)-EU1794-4: tR 29 min; 88% ee; [a]D20 = À185 (c 1.0, dry CHCl3). (+)-EU1794-4: tR 34 min, 73%
ee; [a]D20 = +191 (c 1.0, dry CHCl3).
Separation of EU1794-27 enantiomers
Semi-preparative separation of EU1794-27 enantiomers from racemic EU1794-27 (0.020 g) was
accomplished using a ChiralPak OD-RH (21 mm  250 mm, 5 mm) with the following conditions: 10.4
mL/min flow rate, 10 mL injection volume (2 mg/1 mL), 60% ACN/40% water with 0.1% formic acid
over 120 min to afford (À)-EU1794-27, tR 55 min; (+)-EU1794-27, tR 59 min. The enantiomeric
excess (ee) was determined using a ChiralPak OD-RH column (4.6 mm  150 mm, 5 mm) with the fol-
lowing conditions: 1 mL/min flow rate, 5 mL injection volume, 40% ACN/60% water with 0.1% formic
acid. (À)-EU1794-27: tR 13 min; 88% ee; [a]D20 = À172 (c 1.0, dry CHCl3). (+)-EU1794-27: tR 15
min, 46% ee; [a]D20 = +178 (c 1.0, dry CHCl3).
General Procedure A: Preparation of aminothiophene compounds in
alcohol solvent
To a solution of aldehyde/ketone (1.0 eq.) in alcohol (0.2-0.4 M, corresponding to the ester moiety
for each reaction, see below) was added cyanoacetate (1.1 eq.), elemental sulfur (1.1 eq.), and mor-
pholine (1.4 eq.). The reaction mixture was heated to 50 C in an oil bath. When TLC or LC-MS indi-
˚
cated complete conversion (12-24 h.), the reaction mixture was concentrated in vacuo. The resulting
residue was taken up in EtOAc and washed with water (2x) and brine (2x). The organic layer was
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dried over MgSO4 and filtered. The solvent was removed in vacuo. The resulting product was sub-
jected to flash column chromatography.
General Procedure B: Preparation of aminothiophene compounds in DMF
To a solution of aldehyde/ketone (1.0 eq.) in DMF (1.0 M) was added cyanoacetate (1.1 eq.), ele-
mental sulfur (1.1 eq.), and morpholine (1.4 eq.). The reaction mixture stirred at room temperature
for 24 hours and then concentrated in vacuo. The resulting residue was taken up in EtOAc and
washed with water (3x) and brine (3x). The organic layer was dried over MgSO4 and filtered. The sol-
vent was removed in vacuo. The resulting product was subjected to flash column chromatography.
General Procedure C: Preparation of (Z)-4-oxo-4-(thiophen-2-ylamino)but-2-
enoic acid compounds
A solution of the aminothiophene (1.0 eq.) in anhydrous ether was added dropwise to a solution of
maleic anhydride (1.0 eq.) in anhydrous ether for a total concentration of 0.1-0.2 M. Reaction was
allowed to stir at room temperature or refluxed until complete consumption of starting material was
observed by TLC or LC-MS (16 h.-6 days). The resulting precipitate was filtered, washed with ether,
and carried on without further purification.
General Procedure D: Preparation of 2-(2-imino-4-oxothiazolidin-5-yl)-N-
(thiophen-2-yl)acetamide compounds from (Z)-4-oxo-4-(thiophen-2-ylamino)
but-2-enoic acid using EDCI
To a solution of carboxylic acid (1 eq.) in dry DMF (0.15 M) was added HOBt (1.1 eq.) and EDCI (1.5
eq.). The reaction mixture was stirred at room temperature for 1 hour or until all materials were in
solution. To the solution was added thiourea (1.1 eq.). The reaction mixture was allowed to stir at
room temperature for an additional hour and then heated to 60 C for 24 hours. The reaction mix-
˚
ture was poured into EtOAc and washed with water (2x) and brine (2x). The organic extracts were
combined, dried over anhydrous MgSO4 and concentrated in vacuo. Crude material was dissolved in
hot methanol or ethanol and then slowly cooled to room temperature. The resulting precipitate was
filtered and washed with cold solvent.
General Procedure E: Preparation of aryl maleimide compounds
A mixture of carboxylic acid (1.0 eq.), acetic anhydride (3.3-7.0 eq.), and sodium acetate (0.2 eq.)
was heated to 75 C in an oil bath. After consumption of starting material, reaction mixture was
˚
cooled to room temperature where it stirred for 12-18 hours. The reaction was diluted with water
and extracted with DCM (3x). The organic extracts were dried over MgSO4, filtered, and concen-
trated. The resulting product was subjected to flash column chromatography.
General Procedure F: Preparation of 2-(2-imino-4-oxothiazolidin-5-yl)-N-
(thiophen-2-yl)acetamide compounds from aryl maleimides
A mixture of maleimide (1.0 eq.) and thiourea (1.0 eq.) in absolute EtOH (0.1 M) stirred at room tem-
perature for 24-48 hours. Reaction mixture was filtered, and solid material was washed with EtOH
and DCM.
Chemical structure 1. ethyl 2-amino-6-methyl-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (11b).
DOI: https://doi.org/10.7554/eLife.34711.028
Compound 11b was prepared according to procedure A using 4-methylcyclohexanone (1.6 mL,
13.4 mmol) and ethyl 2-cyanoacetate (1.6 mL, 14.7 mmol) in absolute ethanol (0.2-0.4 M). The crude
material was purified by flash column chromatography (ISCO, 120 g silica gel column, 0-10% EtOAc-
1
Hexanes over 25 minutes) to afford the title compound as a white crystalline solid (3.14 g, 98%). H
NMR [400 MHz, CDCl3] d 5.92 (br s, 2H), 4.26 (q, J = 7.0 Hz, 2H), 2.85-2.90 (m, 1H), 2.53-2.63 (m,
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2H), 2.11-2.17 (m, 1H), 1.81-1.88 (m, 2H), 1.29-1.37 (m, 4H), 1.05 (d, J = 6.4 Hz, 3H); 13C NMR [100
MHz, CDCl3] d 166.3, 162.0, 132.3, 117.4, 105.8, 59.5, 32.8, 31.3, 29.7, 26.9, 21.7, 14.7; HRMS (ESI)
[M+H]+, calc’d for C12H18NO2S 240.10528, found 240.10521.
Chemical structure 2. ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (11c)
DOI: https://doi.org/10.7554/eLife.34711.029
Compound 11c was prepared according to procedure A using cyclohexanone (5.3 mL, 50.9
mmol) and ethyl 2-cyanoacetate (6.0 mL, 56.0 mmol) in absolute ethanol (0.2-0.4 M). The crude
material was recrystallized from absolute ethanol to afford the title compound as light yellow crystals
(10.0 g, 87%). 1H NMR [400 MHz, CDCl3] d 5.96 (br s, 2H), 4.26 (q, J = 7.1 Hz, 2H), 2.73-2.69 (m,
2H), 2.52- 2.48 (m, 2H), 1.81-1.71 (m, 4H), 1.34 (t, J = 7.2 Hz, 3H); 13C NMR [100 MHz, CDCl3] d
166.3, 161.9, 132.6, 117.8, 105.9, 59.5, 27.1, 24.7, 23.4, 23.0, 14.7; HRMS (ESI) [M+H]+, calc’d for
C11H16NO2S 226.08963, found 226.08966; IR (solid ATR): 3400, 3294, 2982, 2939, 2895, 1644, 1593,
1488, 1271 cm-1.
Chemical structure 3. isopropyl 2-amino-6-methyl-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (11f)
DOI: https://doi.org/10.7554/eLife.34711.030
Compound 11f was prepared according to procedure A using 4-methylcyclohexanone (2.2 mL,
17.8 mmol) and isopropyl 2-cyanoacetate (2.5 mL, 19.6 mmol) in isopropanol (0.2-0.4 M). The crude
material was purified by flash column chromatography (ISCO, 80 g silica gel column, 0-20% EtOAc-
1
Hexanes over 25 minutes) to afford the title compound as an off-white solid (4.42 g, 98%). H NMR
[400 MHz, CDCl3] d 5.96 (br s, 2H), 5.16 (septet, J = 6.3 Hz, 1H), 2.84-2.91 (m, 1H), 2.52-2.65 (m,
2H), 2.10-2.18 (m, 1H), 1.80-1.90 (m, 2H), 1.25-1.35 (m, 1H), 1.32 (d, J = 6.4 Hz, 3H), 1.31 (d, J = 6.4
Hz, 3H), 1.04 (d, J = 6.7 Hz, 3H); 13C NMR [100 MHz, CDCl3] d 165.9, 161.9, 132.3, 117.4, 106.1,
66.8, 32.9, 31.3, 29.6, 27.0, 22.4, 21.7; HRMS (ESI) [M+H]+, calc’d for C13H20NO2S 254.12093, found
254.12052.
Chemical structure 4. isopropyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (11h)
DOI: https://doi.org/10.7554/eLife.34711.031
Compound 11h was prepared according to procedure A using cyclohexanone (2.1 mL, 20.4
mmol) and isopropyl 2-cyanoacetate (2.8 mL, 22.4 mmol) in isopropanol (0.2-0.4 M). The crude mate-
rial was purified by flash column chromatography (ISCO, 120 g silica gel column, 0-10% EtOAc-Hex-
anes over 25 minutes) to afford the title compound as a white solid (4.12 g, 84%). 1H NMR [400
MHz, CDCl3] d 6.12 (br s, 2H), 5.16 (septet, J = 6.2 Hz, 1H), 2.69-2.71 (m, 2H), 2.46-2.48 (m, 2H),
1.72-1.77 (m, 4H), 1.31 (d, J = 6.2 Hz, 6H); 13C NMR [100 MHz, CDCl3] d 165.7, 162.0, 132.3, 117.4,
105.6, 66.6, 27.0, 24.5, 23.2, 22.9, 22.1; HRMS (ESI) [M+H]+, calc’d for C12H18NO2S 240.10528,
found 240.10549; Elem. Anal. calc’d for C12H17NO2S, C, 60.22; H, 7.16; N, 5.85, found C, 60.49; H,
7.26; N, 5.79.
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Chemical structure 5. tert-butyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (11k)
DOI: https://doi.org/10.7554/eLife.34711.032
Compound 11k was prepared according to procedure A using cyclohexanone (1.6 mL, 15.3
mmol) and tert-butyl 2-cyanoacetate (2.4 mL, 16.8 mmol) in absolute ethanol (40.0 mL). The crude
material was purified by flash column chromatography (ISCO, 80 g silica gel, 0-20% EtOAc-Hexanes
over 25 minutes) to afford the title compound as a yellow oil (2.45 g, 63%). 1H NMR [400 MHz,
CDCl3] d 5.83 (br s, 2H), 2.67-2.64 (m, 2H), 2.49-2.46 (m, 2H), 1.76-1.69 (m, 4H), 1.52 (s, 9H); 13C
NMR [100 MHz, CDCl3] d 165.8, 161.2, 132.7, 117.7, 107.4, 80.2, 28.8, 27.4, 24.8, 23.5, 23.1; HRMS
(ESI) [M+H]+, calc’d for C13H20NO2S 254.12093, found 254.12087; IR (solid ATR): 3434, 3324, 2932,
1732, 1660, 1576, 1484, 1141 cmÀ1
.
Chemical structure 6. butyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (11l)
DOI: https://doi.org/10.7554/eLife.34711.033
Compound 11l was prepared according to procedure B using cyclohexanone (1.2 mL, 11.6 mmol)
and butyl 2-cyanoacetate (1.8 mL, 12.8 mmol). The crude material was purified by flash column chro-
matography (ISCO, 80 g silica gel, 0-10% EtOAc-Hexanes over 20 minutes) to afford the title com-
1
pound as a yellow oil (2.35 g, 80%). H NMR [400 MHz, CDCl3] d 5.96 (br s, 2H), 4.21 (t, J = 6.6 Hz,
2H), 2.72-2.69 (m, 2H), 2.52-2.49 (m, 2H), 1.82-1.66 (m, 6H), 1.45 (sextet, J = 7.4 Hz, 2H), 0.96 (t, J =
7.3 Hz, 3H); 13C NMR [100 MHz, CDCl3] d 166.5, 161.9, 132.6, 117.8, 106.0, 63.5, 31.1, 27.2, 24.7,
23.4, 23.1, 19.6, 114.0; HRMS (ESI) [M+H]+, calc’d for C13H20NO2S 254.12093, found 254.12061; IR
(solid ATR): 3433, 3325, 2932, 2857, 1664, 1576, 1485, 1265 cm-1.
Chemical structure 7. benzyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (11m)
DOI: https://doi.org/10.7554/eLife.34711.034
Compound 11m was prepared according to procedure B using cyclohexanone (0.7 mL, 6.5
mmol) and benzyl 2-cyanoacetate (1.1 mL, 7.1 mmol). The crude material was purified by flash col-
umn chromatography (ISCO, 40 g silica gel, 0-20% EtOAc-Hexanes over 15 minutes) to afford the
1
title compound as a yellow tinted oil (1.38 g, 74%). H NMR [400 MHz, CDCl3] d 7.43-7.33 (m, 5H),
6.00 (br s, 2H), 5.29 (s, 2H), 2.74-2.71 (m, 2H), 2.52-2.49 (m, 2H), 1.81-1.68 (m, 4H); 13C NMR [100
MHz, CDCl3] d 166.0, 162.4, 136.9, 132.5, 128.7, 128.1, 128.1, 117.8, 105.5, 65.4, 27.2, 24.7, 23.4,
23.0; HRMS (ESI) [M+H]+, calc’d for C16H18NO2S 288.10528, found 288.10519; IR (solid ATR): 3442,
3325, 2980, 2934, 1659, 1575, 1483, 1261 cm-1.
Chemical structure 8. (Z)-4-((3-(ethoxycarbonyl)-6-methyl-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)amino)-4-
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oxobut-2-enoic acid (13b)
DOI: https://doi.org/10.7554/eLife.34711.035
Compound 13b was prepared according to procedure C using compound 11b (2.6 g, 10.7
mmol). The crude yellow solid (3.54 g, 98%) was carried on without further purification. 1H NMR [400
MHz, CDCl3] d 12.22 (s, 1H), 6.51 (d, J = 12.8 Hz, 1H), 6.45 (d, J = 12.8 Hz, 1H), 4.38 (q, J = 7.0 Hz,
2H), 2.95-2.99 (m, 1H), 2.78 (dd, J = 4.9, 15.9 Hz, 1H), 2.66-2.75 (m, 1H), 2.29-2.35 (m, 1H), 1.89-
1.93 (m, 2H), 1.42 (t, J = 7.3 Hz, 4H), 1.09 (d, J = 6.7 Hz, 3H); 13C NMR [100 MHz, CDCl3] d 166.9,
164.3, 162.1, 144.4, 137.8, 132.1, 130.8, 130.0, 115.0, 61.6, 32.8, 31.0, 29.3, 26.3, 21.5, 14.4; HRMS
(ESI) [M+H]+, calc’d for C16H20NO5S, 338.10567, found 338.10550.
Chemical structure 9. (Z)-4-((3-(ethoxycarbonyl)-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)amino)-4-oxobut-2-enoic
acid (13c)
DOI: https://doi.org/10.7554/eLife.34711.036
Compound 13c was prepared according to procedure C using compound 11c (3.0 g, 13.3 mmol).
1
The crude yellow solid (4.02 g, 93%) was carried on without further purification. H NMR [400 MHz,
CDCl3] d 12.18 (br s, 1H), 6.49 (d, J = 12.8 Hz, 1H), 6.45 (d, J = 12.8 Hz, 1H,), 4.36 (q, J = 7.1 Hz,
2H), 2.78 (app. t, J = 5.2 Hz, 2H), 2.69 (app. t, J = 4.9 Hz, 2H), 1.78-1.83 (m, 4H), 1.40 (t, J = 7.3 Hz,
3H); 13C NMR [100 MHz, CDCl3] d 166.8, 164.3, 162.0, 144.2, 137.7, 132.3, 131.0, 130.0, 115.1,
61.5, 26.4, 24.7, 22.9, 22.7, 14.4; HRMS (ESI) [M+H]+, calc’d for C15H18NO5S 324.09002, found
324.08965.
Chemical structure 10. (Z)-4-((3-(isopropoxycarbonyl)-6-methyl-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)amino)-4-
oxobut-2-enoic acid (13f)
DOI: https://doi.org/10.7554/eLife.34711.037
Compound 13f was prepared according to procedure C using compound 11f (2.0 g, 7.9 mmol).
1
The crude yellow solid (2.69 g, 97%) was carried on without further purification. H NMR [400 MHz,
CDCl3] d 12.25 (br s, 1H), 6.49 (d, J = 12.8 Hz, 1H), 6.45 (d, J = 12.8 Hz, 1H), 5.22 (septet, J = 6.2
Hz, 1H), 2.93-2.97 (m, 1H), 2.64-2.79 (m, 2H), 2.26-2.33 (m, 1H), 1.84-1.92 (m, 2H), 1.29-1.43 (m, 7H),
1.08 (d, J = 6.1 Hz, 3H); 13C NMR [100 MHz, CDCl3] d 166.4, 164.3, 162.0, 144.2, 137.8, 132.0,
130.7, 130.1, 115.3, 69.5, 32.7, 31.0, 29.3, 26.3, 22.1, 21.5; HRMS (ESI) [M–H]-, calc’d for
C
17H22NO5S 350.10677, found 350.10666.
Chemical structure 11. (Z)-4-((3-(isopropoxycarbonyl)-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)amino)-4-oxobut-2-
enoic acid (13h)
DOI: https://doi.org/10.7554/eLife.34711.038
Compound 13h was prepared according to procedure C using compound 11h (1.5 g, 6.3 mmol).
1
The crude yellow solid (2.01 g, 95%) was carried on without further purification. H NMR [600 MHz,
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CDCl3] d 12.27 (br s, 1H), 6.50 (d, J = 12.8 Hz, 1H), 6.46 (d, J = 12.8 Hz, 1H), 5.23 (septet, J = 6.3
Hz, 1H), 2.79-2.80 (m, 2H), 2.69-2.70 (m, 2H), 1.81-1.82 (m, 4H), 1.39 (d, J = 6.0 Hz, 6H); 13C NMR
[150 MHz, CDCl3] d 166.5, 164.3, 162.1, 144.1, 137.8, 132.4, 131.1, 130.1, 115.5, 69.5, 26.6, 24.7,
22.9, 22.8, 22.2; HRMS (ESI) [M+H]+, calc’d for C16H20NO5S 338.10567, found 338.10578.
Chemical structure 12. (Z)-4-((3-(tert-butoxycarbonyl)-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)amino)-4-oxobut-2-
enoic acid (13k)
DOI: https://doi.org/10.7554/eLife.34711.039
Compound 13k was prepared according to procedure C using compound 11k (2.4 g, 9.6 mmol).
The crude yellow solid (2.88 g, 86%) was carried on without further purification. HRMS (ESI) [M+H]+,
calc’d for C17H22NO5S 352.12132, found 352.12107.
Chemical structure 13. ethyl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-6-methyl-4,5,6,7- tetrahydrobenzo[b]
thiophene-3-carboxylate (18b)
DOI: https://doi.org/10.7554/eLife.34711.040
Compound 18b was prepared according to procedure E using compound 13b (1.0 g, 3.0 mmol).
The crude material was purified by flash column chromatography (ISCO, 24 g silica gel, 0-18%
EtOAc-Hexanes over 15 minutes) to afford the title compound as a yellow oil that solidified upon
standing (0.769 g, 81%). HRMS (APCI) [M+H]+, calc’d for C16H18NO4S 320.09511, found 320.09520.
Chemical structure 14. isopropyl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-
carboxylate (18h)
DOI: https://doi.org/10.7554/eLife.34711.041
Compound 18h was prepared according to procedure E using compound 13h (0.8 g, 2.4 mmol).
The crude material was purified by flash column chromatography (ISCO, 24 g silica gel, 0-25%
EtOAc-Hexanes over 20 minutes) to afford the title compound as a golden brown oil (0.712 g, 94%).
1H NMR [400 MHz, CDCl3] d 6.91 (s, 2H), 5.09 (septet, J = 5.3 Hz, 1H), 2.84 (app. t, J = 5.8 Hz, 2H),
2.75 (app. t, J = 5.5 Hz, 2H), 1.80-1.86 (m, 4H), 1.20 (d, J = 6.1Hz, 6H); HRMS (APCI) [M+H]+, calc’d
for C16H18NO4S 320.09511, found 320.09527.
Chemical structure 15. tert-butyl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-
carboxylate (18k)
DOI: https://doi.org/10.7554/eLife.34711.042
Compound 18k was prepared according to procedure E using compound 13k (2.0 g, 5.7 mmol).
The crude material was purified by flash column chromatography (ISCO, 40 g silica gel, 0-18%
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EtOAc-Hexanes over 15 minutes) to afford the title compound as a light yellow solid (1.44 g, 76%).
1H NMR [400 MHz, CDCl3] d 6.90 (s, 2H), 2.83-2.79 (m, 2H), 2.75-2.72 (m, 2H), 1.86-1.76 (m, 4H),
1.44 (s, 9H); 13C NMR [100 MHz, CDCl3] d 169.1 (2C), 161.4, 136.6, 135.7, 135.1 (2C), 131.6, 130.4,
81.6, 28.4 (3C), 26.6, 25.4, 22.9, 22.6; HRMS (APCI) [M–Ot-Bu]+, calc’d for C13H10NO3S 260.03759,
found 260.03737; IR (solid ATR): 2918, 1709, 1565, 1410, 1393, 1367, 1136, 691 cmÀ1
.
Chemical structure 16. butyl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-
carboxylate (18l)
DOI: https://doi.org/10.7554/eLife.34711.043
Compound 18l was prepared according to procedure E using compound 13l (1.2 g, 3.4 mmol).
The crude material was purified by flash column chromatography (ISCO, 40 g silica gel, 0-18%
EtOAc-Hexanes over 15 minutes) to afford the title compound as a light yellow solid (1.04 g, 91%).
1H NMR [400 MHz, CDCl3] d 6.91 (s, 2H), 4.13 (t, J = 6.7 Hz, 2H), 2.84 (app. t, J = 5.8 Hz, 2H), 2.75
(app. t, J = 5.8 Hz, 2H), 1.87-1.78 (m, 4H), 1.57 (p, J = 7.2 Hz, 2H), 1.34 (sextet, J = 7.5 Hz, 2H), 0.91
(t, J = 7.5 Hz, 3H); 13C NMR [100 MHz, CDCl3] d 169.2 (2C), 162.21, 136.8, 135.9, 135.1 (2C), 133.0,
128.7, 64.7, 30.8, 26.6, 25.4, 22.8, 22.6, 19.3, 13.9; 62 HRMS (APCI) [M+H]+, calc’d for C17H20NO4S
334.11076, found 334.11128; IR (solid ATR): 3097, 2944, 1710, 1414, 1364, 1175, 836, 693 cmÀ1
.
Chemical structure 17. benzyl 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-
carboxylate (18m)
DOI: https://doi.org/10.7554/eLife.34711.044
Compound 18m was prepared according to procedure E using compound 13m (0.4 g, 1.0
mmol). The crude material was purified by flash column chromatography (ISCO, 24 g silica gel, 0-
25% EtOAc-Hexanes over 15 minutes) to afford the title compound as a light yellow solid (0.299 g,
78%). 1H NMR [400 MHz, CDCl3] d 7.40-7.21 (m, 5H), 6.44 (s, 2H), 5.07 (s, 2H), 2.91-2.78 (m, 2H),
2.75-2.64 (m, 2H), 1.88-1.71 (m, 4H); 13C NMR [100 MHz, CDCl3] d 169.2 (2C), 161.6, 137.0, 136.3,
135.4, 134.2 (2C), 133.3, 129.4 (2C), 128.8 (2C), 128.7, 128.3, 67.0, 26.4, 25.4, 22.8, 22.5; HRMS
(APCI) [M+H]+, calc’d for C20H18NO4S 368.09511, found 368.09512; IR (solid ATR): 2942, 2850,
1763, 1672, 1638, 1537, 1176, 1137, 691 cmÀ1
.
Chemical structure 18. (Z)-4-((3-((benzyloxy)carbonyl)-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)amino)-4-oxobut-2-
enoic acid (13m)
DOI: https://doi.org/10.7554/eLife.34711.045
Compound 13m was prepared according to procedure C using compound 11m (0.6 g, 2.1
mmol). The crude yellow solid (0.573 g, 70%) was carried on without further purification. HRMS (ESI)
[M+H]+, calc’d for C20H20NO5S 386.10567, found 386.10587.
Perszyk et al. eLife 2018;7:e34711. DOI: https://doi.org/10.7554/eLife.34711
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