Stereospecific Inhibition of Ethanol Potentiation on Glycine Receptor by M554 Stereoisomers

Article abstract of DOI:10.1021/acs.jcim.0c00943

Blocking the interaction between the Gβγprotein and the glycine receptor (GlyR) has emerged as a promising pharmacological strategy to treat acute alcohol intoxication by inhibiting ethanol potentiation on GlyR. M554 is a recently discovered small molecule capable of binding to Gβγwith potent in vitro and in vivo inhibitory activity. This compound has been tested as a mixture of diastereomers, and no information is available concerning the stereospecific activity of each species, which is critical to pursue efforts on lead optimization and drug development. In this work, we explored the differential activity of four M554 stereoisomers by in silico molecular dynamics simulations and electrophysiological experiments. Our results revealed that the (R,R)-M554 stereoisomer is a promising lead compound that inhibits ethanol potentiation of GlyR.

Full text of DOI:10.1021/acs.jcim.0c00943

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Article
Stereospecific Inhibition of Ethanol Potentiation on Glycine
Receptor by M554 Stereoisomers
́
Pilar A. Vasquez, Loreto San Martín, Yenifer Argel, Camila Riquelme, Josefa Torres, Felipe Vidal,
Francisca Cayuman, Patricio Castro, Jorge Fuentealba, Gustavo Moraga-Cid, Gonzalo Yevenes,
́
́
́
Chunyang Jin, Veronica A. Jimenez,* and Leonardo Guzman*
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ABSTRACT: Blocking the interaction between the Gβγ protein and the glycine receptor (GlyR) has emerged as a promising
pharmacological strategy to treat acute alcohol intoxication by inhibiting ethanol potentiation on GlyR. M554 is a recently
discovered small molecule capable of binding to Gβγ with potent in vitro and in vivo inhibitory activity. This compound has been
tested as a mixture of diastereomers, and no information is available concerning the stereospecific activity of each species, which is
critical to pursue efforts on lead optimization and drug development. In this work, we explored the differential activity of four M554
stereoisomers by in silico molecular dynamics simulations and electrophysiological experiments. Our results revealed that the (R,R)-
M554 stereoisomer is a promising lead compound that inhibits ethanol potentiation of GlyR.
perspective, ethanol potentiation of GlyR has been attributed
to the regulation of the intracellular signal transduction protein
Gβγ on the GlyR alpha subunit,7 as blocking Gβγ with
peptides or antibodies inhibits ethanol potentiation of GlyR.8,9
The protein residues relevant for the induction of ethanol
effects at the Gβγ−GlyR interface were identified from in vitro
and in vivo experiments using genetically modified knock-in
mice for the GlyR α1 subunit that has an impaired interaction
with Gβγ.8,10,11 Our findings revealed that amino acid clusters
of the cytoplasmatic domain of GlyR are critical for Gβγ
binding and can be targeted by peptides or small molecules
that disrupt the normal process of GlyR potentiation by
ethanol.9,12 Electrophysiological experiments in vitro and
behavioral assays in vivo confirmed the validity of the proposed
inhibitory mechanism using rationally designed peptides and
1. INTRODUCTION
Glycine receptor (GlyR) is a ligand gated ion channel member
of the cys-loop family. This receptor is expressed mainly in the
spinal cord, brain stem, and certain other areas in the brain and
participates in the control of voluntary movements, pain
transmission, and respiratory coordination among other related
functions. GlyR is formed by five subunits, each of them
composed of an extracellular domain, a transmembrane
domain, and a cytoplasmic domain. Five different subunits
are expressed, alpha 1, 2, 3, 4, and beta subunits, depending on
the developmental state and the neuronal type.1 The activation
of GlyR by the neurotransmitter glycine allows for the influx of
chloride ions, inducing the hyperpolarization of plasma
membranes. In the context of a synapse, this signaling inhibits
the formation of action potentials in the postsynaptic
neuron.1,2
The potentiation of GlyR by ethanol has been recognized as
a critical factor for behavioral effects associated with recrea-
tional ethanol consumption such as mild to severe motor
incoordination.3,4 In this regard, ethanol increases the
hyperpolarization of postsynaptic potential in synaptic net-
works that regulate voluntary muscle contraction leading to the
loss of extremities’ coordination.5,6 From a molecular
Received: August 12, 2020
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Figure 1. Structure and stereochemistry descriptors of the four stereoisomers of M554.
small-molecule inhibitors targeting the Gβγ interacting site.12
This Gβγ interaction site was further analyzed using in silico
modeling, showing that residues Asp186, Asp228, and Asp246
are essential for the interaction with the mentioned inhibitors
and thereby relevant for the rational discovery of novel
pharmacologically active compounds capable of inhibiting the
ethanol effects arising from acute alcohol intoxication.12 In a
recent attempt, massive virtual screening was carried out to
identify a set of novel compounds capable of inhibiting ethanol
potentiation of GlyR. Our results led to the discovery of the
small-molecule M554 (N-(butan-2-yl)-2-[3-(1H-indol-3-yl)-
propanamido]propenamide), which exhibited significant in-
hibition of ethanol potentiation in vitro and in vivo when
assayed as a mixture of stereoisomers. M554 is the first
reported molecule with this kind of activity under pharmaco-
logical doses of ethanol. Nevertheless, additional research is
required for further pharmacological development of this
molecule as the stereospecific inhibitory activity of M554
remains unknown.12
M554 has two chiral centers and four diastereomers, namely,
(R,R)-M554, (S,S)-M554, (R,S)-M554, and (S,R)-M554
(Figure 1). After successful synthesis and stereoisomeric
purification and characterization, in this work, we aimed at
examining the stereospecific ethanol potentiation inhibition
properties of M554 diastereomers using a combination of in
silico modeling and in vitro electrophysiological experiments.
The comprehensive information herein provided will be of
high relevance to develop novel active molecules with
improved efficacy to treat acute ethanol intoxication.
GA. Unless otherwise stated, reagent-grade chemicals were
obtained from commercial sources and were used without
further purification. All moisture- and air-sensitive reactions
and reagent transfers were carried out under dry nitrogen.
2.1.1. 2-Amino-N-(butan-2-yl)propanamide (4a). To a
stirred solution of Boc-DL-Ala-OH (1a) (473 mg, 2.5 mmol)
in CH3CN (10 mL) under nitrogen at room temperature was
added sec-butylamine (2a) (183 mg, 2.5 mmol), followed by
HBTU (1.14 g, 3.0 mmol) and triethylamine (693 μL, 5.0
mmol). After stirring for 3 h, the reaction mixture was diluted
with EtOAc (30 mL) and washed with ice-cold 10% citric acid
(2 × 10 mL), saturated NaHCO3 (2 × 10 mL), and brine (10
mL). The organic layer was dried (Na2SO4) and concentrated
1
to give crude 3a (650 mg) as a white sticky solid. H NMR
(300 MHz, CDCl3) δ 5.89 (br s, 1H), 4.98 (br s, 1H), 4.15−
4.02 (m, 1H), 3.95−3.82 (m, 1H), 1.50−1.42 (m, 2H), 1.45
(s, 9H), 1.35 (dd, J = 3.0, 6.0 Hz, 3H), 1.12 (dd, J = 3.0, 6.0
Hz, 3H), 0.90 (dt, J = 3.0, 7.5 Hz, 3H). To a stirred solution of
crude 3a (650 mg) in MeOH (10 mL) under nitrogen at room
temperature was added 4 M HCl in dioxane (3 mL). After
stirring overnight, the reaction mixture was concentrated. The
residue was titrated with Et2O to give crude 4a hydrochloride
(450 mg) as an off-white solid. 1H NMR (300 MHz, CD3OD)
δ 3.90−3.78 (m, 2H), 1.56−1.40 (m, 5H), 1.14 (d, J = 6.0 Hz,
3H), 0.96−0.86 (m, 3H); ESI MS [M + H]+ m/z 145.4.
2.1.2. (2R)-2-Amino-N-[(2R)-butan-2-yl]propanamide
(4b). The procedure for 4a was followed using 378 mg (2.0
mmol) of Boc-D-Ala-OH (1b) and 146 mg (2.0 mmol) of (R)-
sec-butylamine (2b) to give 360 mg of crude 4b hydrochloride
1
as a white solid. H NMR (300 MHz, CD3OD) δ 3.90−3.80
2. MATERIALS AND METHODS
(m, 2H), 1.60−1.46 (m, 5H), 1.14 (d, J = 6.0 Hz, 3H), 0.93 (t,
J = 7.5 Hz, 3H); ESI MS [M + H]+ m/z 145.4.
2.1. Synthesis of M554 Diastereomers. Nuclear
magnetic resonance (1H NMR and 13C NMR) spectra were
obtained on a Bruker Avance DPX-300 MHz NMR
spectrometer. Chemical shifts are reported in parts per million
(ppm) with reference to the internal solvent. Mass spectra
(MS) were run on a Perkin-Elmer Sciex API 150 EX mass
spectrometer equipped with APCI (atmospheric pressure
chemical ionization) or ESI (turbospray) sources or on a
Hewlett Packard 5989A instrument by electron impact.
Analytical thin-layer chromatography (TLC) was carried out
using EMD silica gel 60 F254 TLC plates. TLC visualization
was achieved with a UV lamp or in an iodine chamber. Flash
column chromatography was carried out on a CombiFlash
Companion system using Isco prepacked silica gel columns or
using EM Science silica gel 60 Å (230−400 mesh). Optical
rotations were obtained using an Autopol IV automatic
polarimeter (Rudolph Research Analytical Corporation) and
a glass cell (1.5 mL volume; 100 mm pathlength). Elemental
analyses were carried out by Atlantic Microlab Inc., Norcross,
2.1.3. (2R)-2-Amino-N-[(2S)-butan-2-yl]propanamide (4c).
The procedure for 4a was followed using 1.42 g (7.5 mmol) of
Boc-D-Ala-OH (1b) and 549 mg (7.5 mmol) of (S)-sec-
butylamine (2c) to give 1.25 g of crude 4c hydrochloride as a
1
white solid. H NMR (300 MHz, CD3OD) δ 3.90−3.75 (m,
2H), 1.58−1.46 (m, 5H), 1.13 (d, J = 6.0 Hz, 3H), 0.93 (t, J =
6.0 Hz, 3H); ESI MS [M + H]+ m/z 145.4.
2.1.4. (2S)-2-Amino-N-[(2R)-butan-2-yl]propanamide
(4d). The procedure for 4a was followed using 378 mg (2.0
mmol) of Boc-L-Ala-OH (1c) and 146 mg (2.0 mmol) of (R)-
sec-butylamine (2b) to give 362 mg of crude 4d hydrochloride
1
as an off-white solid. H NMR (300 MHz, CD3OD) δ 3.90−
3.78 (m, 2H), 1.60−1.46 (m, 5H), 1.13 (d, J = 6.0 Hz, 3H),
0.93 (t, J = 7.5 Hz, 3H); ESI MS [M + H]+ m/z 145.4.
2.1.5. (2S)-2-Amino-N-[(2S)-butan-2-yl]propanamide (4e).
The procedure for 4a was followed using 378 mg (2.0 mmol)
of Boc-L-Ala-OH (1c) and 146 mg (2.0 mmol) of (S)-sec-
butylamine (2c) to give 363 mg of crude 4e hydrochloride as a
B
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1
white solid. H NMR (300 MHz, CD3OD) δ 3.90−3.80 (m,
followed using 362 mg of 4d and 378 mg (2 mmol) of 3-
indolepropionic acid to give 350 mg of 5d (55% yield over
three steps) as a white solid: [α]D20 −45.0° (c 0.5, MeOH); 1H
NMR (300 MHz, CDCl3) δ 8.20 (s, 1H), 7.57 (d, J = 9.0 Hz,
1H), 7.33 (d, J = 9.0 Hz, 1H), 7.21−7.07 (m, 2H), 6.95 (d, J =
3.0 Hz, 1H), 6.46−6.32 (m, 2H), 4.55−4.40 (m, 1H), 3.90−
3.76 (m, 1H), 3.08 (t, J = 7.5 Hz, 2H), 2.57 (t, J = 7.5 Hz,
2H), 1.50−1.35 (m, 2H), 1.25 (d, J = 6.0 Hz, 3H), 1.12 (d, J =
6.0 Hz, 3H), 0.83 (t, J = 7.5 Hz, 3H); 13C NMR (75 MHz,
CDCl3) δ 172.65, 171.64, 136.37, 127.13, 122.07, 121.54,
119.33, 118.65, 114.80, 111.22, 48.88, 46.78, 37.08, 29.44,
21.23, 20.23, 18.32, 10.29; ESI MS [M + H]+ m/z 316.3. Anal.
calcd for C18H25N3O2·0.25H2O: C, 67.58; H, 8.03; N, 13.13.
Found: C, 67.47; H, 7.88; N, 13.06.
2.1.10. (2S)-N-[(2S)-Butan-2-yl]-2-[3-(1H-indol-3-yl)-
propanamido]propanamide (5e). The procedure for 5a was
followed using 363 mg of 4e and 378 mg (2 mmol) of 3-
indolepropionic acid to give 355 mg of 5e (56% yield over
three steps) as a white solid: [α]D20 −26.5° (c 0.5, MeOH); 1H
NMR (300 MHz, CDCl3) δ 8.20 (s, 1H), 7.57 (d, J = 9.0 Hz,
1H), 7.33 (d, J = 9.0 Hz, 1H), 7.21−7.07 (m, 2H), 6.95 (d, J =
3.0 Hz, 1H), 6.46−6.30 (m, 2H), 4.58−4.42 (m, 1H), 3.92−
3.76 (m, 1H), 3.08 (t, J = 7.5 Hz, 2H), 2.57 (t, J = 7.5 Hz,
2H), 1.50−1.35 (m, 2H), 1.26 (d, J = 6.0 Hz, 3H), 1.05 (d, J =
6.0 Hz, 3H), 0.88 (t, J = 7.5 Hz, 3H); 13C NMR (75 MHz,
CDCl3) δ 172.58, 171.70, 136.36, 127.14, 122.06, 121.52,
119.33, 118.66, 114.83, 111.21, 48.91, 46.73, 37.10, 29.54,
21.22, 20.27, 18.72, 10.31; ESI MS [M + H]+ m/z 316.3. Anal.
calcd for C18H25N3O2·0.25H2O: C, 67.58; H, 8.03; N, 13.13.
Found: C, 67.48; H, 7.93; N, 13.21.
2.2. Cell Culture and Transfection. HEK293 cells were
transfected with the human GlyR α1 subunit inserted in a
specialized plasmid for the expression of proteins in
mammalian cells, using the Xfect Transfection Reagent kit
(Clontech). At the same time, co-transfection was carried out
with a plasmid that allows for the expression of GFP, which
was used as a marker of positively transfected cells. Recordings
were made after 18−36 h after transfection.
2.3. Electrophysiology. Whole-cell recordings were
obtained under a voltage clamp, using a holding potential of
−60 mV. Patch electrodes contained 140 mM KCl, 10 mM
BAPTA, 10 mM HEPES, 4 mM MgCl2, 2 mM ATP, and 0.5
mM GTP at pH 7.4 and 290−310 mOsm, with or without the
indicated small molecule at 200 μM. In this way, the molecules
were able to diffuse into the cytoplasm. The external solution
contained 150 mM NaCl, 5.4 mM KCl, 2.0 mM CaCl2, 1.0
mM MgCl2, 10 mM HEPES, and 10 mM glucose at pH 7.4
and 315−320 mOsm. For the recording of ethanol-mediated
potentiation on GlyR, a previously described methodology was
used.12 Briefly, ethanol (100 mM) was co-applied with glycine
(15 μM) externally perfused. The evoked currents, in the
presence and absence of ethanol, were recorded just after the
clamping and after 15 min. The results were expressed as the
percentage of ethanol potentiation after 15 min of intracellular
diffusion.
2H), 1.60−1.46 (m, 5H), 1.14 (d, J = 6.0 Hz, 3H), 0.93 (t, J =
7.5 Hz, 3H); ESI MS [M + H]+ m/z 145.4.
2.1.6. N-(Butan-2-yl)-2-[3-(1H-indol-3-yl)propanamido]-
propanamide (5a). To a stirred solution of crude 4a (1.82
g, 10 mmol) in CH3CN (40 mL) under nitrogen at room
temperature was added 3-indolepropionic acid (1.89 g, 10
mmol), followed by HBTU (4.55 g, 12 mmol) and Et3N (2.77
mL, 20 mmol). After stirring for 4 h, the reaction mixture was
diluted with EtOAc (100 mL) and washed with ice-cold 10%
citric acid (2 × 20 mL), saturated NaHCO3 (2 × 20 mL), and
brine (20 mL). The organic layer was dried (Na2SO4) and
concentrated. Flash column chromatography of the crude
product on silica gel using 0−100% EtOAc in hexanes
provided a partially purified product, which was then
recrystallized from EtOAc/hexanes to give 5a (2.04 g, 65%
1
yield over 3 steps) as a white solid: H NMR (300 MHz,
CDCl3) δ 8.03 (s, 1H), 7.61 (d, J = 9.0 Hz, 1H), 7.35 (d, J =
9.0 Hz, 1H), 7.25−7.10 (m, 2H), 6.99 (d, J = 3.0 Hz, 1H),
6.25−6.05 (m, 2H), 4.48−4.36 (m, 1H), 3.90−3.78 (m, 1H),
3.11 (t, J = 7.5 Hz, 2H), 2.60 (t, J = 7.5 Hz, 2H), 1.52−1.36
(m, 2H), 1.24 (dd, J = 3.0, 6.0 Hz, 3H), 1.09 (dd, J = 6.0, 9.0
Hz, 3H), 0.87 (dt, J = 9.0, 6.0 Hz, 3H); 13C NMR (75 MHz,
CDCl3) δ 172.74, 172.66, 171.84, 171.77, 136.39, 127.16,
122.00, 121.57, 119.27, 118.65, 114.76, 111.25, 48.86, 48.82,
46.79, 46.74, 37.02, 29.51, 29.43, 21.25, 20.26, 20.23, 18.74,
18.30, 10.33; ESI MS [M + H]+ m/z 316.3. Anal. calcd for
C18H25N3O2: C, 68.54; H, 7.99; N, 13.32. Found: C, 68.65; H,
7.98; N, 13.22.
2.1.7. (2R)-N-[(2R)-Butan-2-yl]-2-[3-(1H-indol-3-yl)-
propanamido]propanamide (5b). The procedure for 5a was
followed using 360 mg of 4b and 378 mg (2 mmol) of 3-
indolepropionic acid to give 340 mg of 5b (54% yield over
three steps) as a white solid: [α]D20 +26.4° (c 0.5, MeOH); 1H
NMR (300 MHz, CDCl3) δ 8.16 (s, 1H), 7.57 (d, J = 9.0 Hz,
1H), 7.33 (d, J = 9.0 Hz, 1H), 7.20−7.10 (m, 2H), 6.95 (d, J =
3.0 Hz, 1H), 6.50−6.34 (m, 2H), 4.55−4.45 (m, 1H), 3.90−
3.76 (m, 1H), 3.08 (t, J = 7.5 Hz, 2H), 2.57 (t, J = 7.5 Hz,
2H), 1.52−1.40 (m, 2H), 1.27 (d, J = 6.0 Hz, 3H), 1.06 (d, J =
6.0 Hz, 3H), 0.90 (t, J = 7.5 Hz, 3H); 13C NMR (75 MHz,
CDCl3) δ 172.59, 171.71, 136.36, 127.14, 122.06, 121.53,
119.33, 118.66, 114.82, 111.21, 48.92, 46.74, 37.09, 29.53,
21.22, 20.27, 18.73, 10.32; ESI MS [M + H]+ m/z 316.3. Anal.
calcd for C18H25N3O2·0.5H2O: C, 66.64; H, 8.08; N, 12.95.
Found: C, 66.30; H, 7.78; N, 12.79.
2.1.8. (2R)-N-[(2S)-Butan-2-yl]-2-[3-(1H-indol-3-yl)-
propanamido]propanamide (5c). The procedure for 5a was
followed using 430 mg of 4c and 378 mg (2 mmol) of 3-
indolepropionic acid to give 400 mg of 5c (63% yield over
three steps) as a white solid: [α]D20 +45.2° (c 0.5, MeOH); 1H
NMR (300 MHz, CDCl3) δ 8.21 (s, 1H), 7.57 (d, J = 9.0 Hz,
1H), 7.33 (d, J = 9.0 Hz, 1H), 7.20−7.06 (m, 2H), 6.95 (d, J =
3.0 Hz, 1H), 6.50−6.35 (m, 2H), 4.55−4.42 (m, 1H), 3.90−
3.76 (m, 1H), 3.11 (t, J = 7.5 Hz, 2H), 2.60 (t, J = 7.5 Hz,
2H), 1.52−1.40 (m, 2H), 1.22 (d, J = 6.0 Hz, 3H), 1.09 (d, J =
6.0 Hz, 3H), 0.86 (t, J = 7.5 Hz, 3H); 13C NMR (75 MHz,
CDCl3) δ 172.65, 171.65, 136.38, 127.14, 122.05, 121.54,
119.32, 118.65, 114.80, 111.22, 48.87, 46.78, 37.08, 29.45,
21.24, 20.24, 18.33, 10.30; ESI MS [M + H]+ m/z 316.3. Anal.
calcd for C18H25N3O2: C, 68.54; H, 7.99; N, 13.32. Found: C,
68.25; H, 7.91; N, 13.11.
2.4. Data Analysis. In vitro obtained data were expressed
as the mean S.E.M., and statistical analyses were performed
using analysis of variance (one-way or two-way ANOVA
followed by Bonferroni or Tukey post hoc tests). Values of p <
0.05 were considered statistically significant. Origin 6.0
(MicroCal) software was used for these statistical analyses.
2.5. Computational Modeling. The coordinates of the
high-resolution structure of the Gβγ dimer of the hetero-
2.1.9. (2S)-N-[(2R)-Butan-2-yl]-2-[3-(1H-indol-3-yl)-
propanamido]propanamide (5d). The procedure for 5a was
C
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trimeric G protein transducing were retrieved from the Protein
Data Bank server under PDB code 1TBG.13 Initial structures of
the four stereoisomers of M554 were obtained using the
Molefacture plugin of the VMD software, based on the
absolute configuration information provided by RTI Interna-
tional (Figure 1). Topology and force field parameter files
consistent with the GAFF force field were obtained for the
series of M554 stereoisomers using the Antechamber
implementations in Amber 16 software, with atomic charges
calculated at the AM1-BCC semiempirical method.14 The set
of M554 isomers were minimized using the pmemd.CUDA
software under the GPU-enhanced implementations of Amber
16.15 Minimized structures were used to conduct molecular
docking calculations against the Gβγ dimer using the
AutoDock Vina software.16 Autodock Tools was used to add
polar hydrogens, calculate Gasteiger charges, and select the set
of active bonds to enable ligand flexible docking. The searching
space comprised the aspartic residues Asp186, Asp228, and
Asp246 in a cubic grid of 25 Å. The best-ranked docked
conformations were employed as starting structures to conduct
fully atomistic molecular dynamics (MD) simulations in
ligand−Gβγ dimer complexes with the series of M554
stereoisomers under study. Systems were solvated with
TIP3P explicit waters in a cubic box with a solvation shell of
10 Å length measured from the protein surface. An appropriate
number of Na+ counterions were added to maintain charge
neutrality. Long-range electrostatics was treated using the
particle-mesh Ewald approach. The cutoff for nonbonded
terms was 10 Å. The SHAKE algorithm was employed to
constrain all bonds involving hydrogen. Proteins were
simulated using the ff14SB force field. All MD simulations
were performed using the pmemd.CUDA program under
GPU-enhanced implementations of Amber 16. Simulation
protocols consisted of (a) 1500 steepest descent minimization
steps, followed by 3500 conjugate gradient minimization steps
for water molecule relaxation, (b) 1500 steepest descent
minimization steps, followed by 6500 conjugate gradient
minimization steps for the entire system, (c) 500 ps of
progressive NVT heating from 0 to 300 K, (d) 500 ps of NVT
equilibrium at 300 K with restrains applied to the protein
backbone, (e) 20 ns of NVT equilibrium at 300 K, and finally
(f) 150 ns of unrestrained NPT production dynamics at 300 K
and 1 bar. Trajectory analysis was carried out from 1000
snapshots retrieved from the last 50 ns of MD simulations.
Trajectory alignment, ligand root-mean-square deviation
(RMSD), and root-mean-square fluctuation (RMSF) calcu-
lations for Cα atoms were carried out using MDLovoFit
implementations.17 Ligand cluster analysis was carried out
using the Clustering VMD plugin. MM/GBSA binding free
energy calculations were used to estimate the strength of
ligand−Gβγ interactions using 1000 snapshots retrieved from
the last 50 ns of each MD run using the MMPBSA.py module
in Amber 16 under a single-trajectory approach.18 GB
calculations were carried out using the modified GB model
(igb=5) with mbondi2 and α, β, and γ values of 1.0, 0.8, and
4.85, respectively.
with the signaling dimer Gβγ from the heterotrimeric G
protein. According to this model, small molecules capable of
inhibiting the GlyR−Gβγ interaction can exhibit pharmaco-
logical activity to treat the symptoms of acute ethanol
intoxication by acting as inhibitors of the ethanol potentiation
on GlyR. Major progress in this field was attained by the recent
structure-based discovery of the small-molecule M554, which
exhibited in vitro and in vivo activity when tested as a mixture
of stereoisomers.12 Building on these findings, herein we
present the synthesis and in silico and in vitro evaluation of
M554 diastereomers as potential inhibitors of ethanol
potentiation on GlyR.
3.1. Synthesis of M544 Diastereomers. Compound
M554 (a mixture of stereoisomers) and the isolated
diastereomers (R,R)-M554, (R,S)-M554, (S,R)-M554, and
(S,S)-M554 were synthesized in accordance with the reaction
in Scheme 1. Coupling of Boc-alanine 1a−1c with sec-
Scheme 1. Synthesis of Racemic M554 and Pure
Diastereomers
butylamine 2a−2c using N,N,N′,N′-tetramethyl-O-(1H-benzo-
triazol-1-yl)uronium hexafluorophosphate (HBTU) in acetoni-
trile afforded crude amides 3a−3e. Boc group deprotection
with hydrochloric acid in dioxane gave crude amines 4a−4e.
Finally, coupling of 4a−4e with 3-indolepropionic acid using
HBTU provided target compounds 5a−5e in 54−65% yield
over three steps. All target compounds were characterized by
1H NMR, 13C NMR, MS, and elemental analysis and were
>95% pure.
3.2. Molecular Dynamics Simulations. Fully atomistic
MD simulations were carried out to provide exploratory
molecular-level information concerning the differential binding
of M554 diastereomers to the Gβγ dimer. Protein-aligned
trajectories were analyzed in the search for the preferential
binding poses for the series of M554 diastereomers under
study (Figure 2). Ligand RMSD calculations and cluster
analysis in protein-aligned trajectories revealed that the (R,R)-
M554 and (S,S)-M554 enantiomers adopt more rigid and less
mobile binding modes compared with (R,S)-M554 and (S,R)-
M554 stereoisomers. Both the (R,R)-M554 and the (S,S)-
M554 compounds exhibit one highly populated cluster that
determines their association with the Gβγ protein. On the
other hand, the (R,S)-M554 and (S,R)-M554 stereoisomers
show more flexible conformations and more mobile binding
poses that span a wider protein binding region. Representative
structures of the Gβγ complexes indicate that (R,R)-M554 and
(R,S)-M554 bind to a protein region close to the Asp186,
3. RESULTS AND DISCUSSION
This work is aimed at evaluating the stereospecific activity of
M554 diastereomers in the search for inhibitors of ethanol
potentiation on GlyR. Our working model establishes that the
effects of ethanol on motor coordination involve the
intracellular interaction of the cytoplasmic domain of GlyR
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Figure 2. Ligand RMSD, ligand cluster analysis, and representative structures of M554−Gβγ complexes with the series of M554 diastereomers
obtained from protein-aligned MD trajectories.
Asp228, and Asp246 residues, which have been recognized as
critical residues for the binding of active inhibitors. On the
other hand, (S,S)-M554 and (S,R)-M554 bind preferentially to
Gβγ residues located far from the aspartic triad (Figure 2).
Besides ligand−Gβγ interactions, a relevant factor to
examine is the conformational change exhibited by the protein
as a consequence of ligand association. Figure 3 shows
representations of the most-populated protein conformations
in the series of ligand−Gβγ complexes, in which the most
mobile protein regions have been highlighted. Our results
indicate that M554 ligands restrain the mobility of the N-
terminal domains of the Gβ and Gγ subunits compared with
the ligand-free protein. Three stereoisomers, namely, the
(R,R), (S,S), and (S,R), led to equilibrated structures in which
the N-terminal domains adopt conformations similar to that
exhibited by the ligand-free protein. On the other hand, the
association of the (R,S) stereoisomer induces a strong
restructuring of the N-terminal domain of the Gγ subunit,
which might be related to a loss of protein functionality.
Exploratory energetic information concerning the relative
strength of M554−Gβγ complexes for the series of M554
diastereomers was estimated from MM/GBSA calculations
under a single-trajectory approach (Table 1). Binding
enthalpies were obtained from the analysis of 1000 trajectory
frames retrieved from the last 50 ns out of 150 ns simulations.
Calculated binding enthalpies account for similar affinities to
the Gβγ dimer for (R,R)-M554, (S,S)-M554, and (R,S)-M554,
whereas (S,R)-M554 exhibits a weaker association with the
protein. Due to computational costs, binding entropies were
estimated from normal mode analysis on a reduced set of 50
trajectory frames. Calculated binding entropies have large
negative terms that are in line with the loss of ligands’
translational, rotational, and vibrational degrees of freedom
after binding. Nevertheless, these results should be interpreted
with caution as they do not consider the expected favorable
entropic contribution of the solvent arising from the
desolvation of polar groups of the ligand and polar/charged
binding site residues. Thereby, they are reported separately
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Figure 3. Conformation of the Gβγ protein in the ligand-free state and the complexed state with the series of M554 stereoisomers. Mobile regions
corresponding to the N-terminal domains of the Gβ and Gγ subunits are highlighted in red.
Table 1. MM/GBSA Calculations for M554−Gβγ
consistent with their more restrained binding poses in the
Gβγ complexes. Nevertheless, a large overlap between the
standard deviations of the binding entropies was found, which
suggests that the differences between the calculated values are
not statistically significant. Based on these results, it is expected
that the binding profiles for the set of M554 stereoisomers are
governed mostly by enthalpic factors.
3.3. Electrophysiological Assays. Patch-clamp assays in
whole-cell configuration were performed in HEK293 cells that
overexpress the GlyR α1 subunit. Solutions of small molecules
(200 μM) were included inside the recording pipette to allow
diffusion into the cytoplasm. Glycine (15 μM, equivalent to
EC10) was extracellularly perfused to the cell, and the evoked
current was registered with or without co-perfusion of ethanol
(100 mM). Recordings of the glycine-evoked current were
registered as soon as the whole-cell configuration was
established and after 15 min (Figure 4). None of the
compounds used in this report had any basal effect on GlyR
activity. The change in glycine-evoked current with or without
a
Complexes with M554 Stereoisomers
diastereomer
ΔHbind (kcal mol−1
)
ΤΔSbind (kcal mol−1
)
(R,R)-M554
(S,S)-M554
(R,S)-M554
(S,R)-M554
−22.1 2.9
−21.3 2.2
−24.0 2.9
−14.1 4.9
−20.6 2.5
−21.9 3.7
−17.7 2.9
−17.9 3.5
a
ΔHbind values were obtained from MM/GBSA calculations without
including the entropic contribution and are reported in kcal mol−1
standard deviation. ΤΔSbind terms were estimated at T = 300 K using
normal mode analysis and are reported in kcal mol−1
deviation.
standard
from binding enthalpy results. According to this, binding
entropy estimates suggest more favorable contributions to
binding for (R,S)-M554 and (S,R)-M554, in agreement with
the high degree of mobility exhibited by these ligands in their
complexed states. On the other hand, (R,R)-M554 and (S,S)-
M554 show larger negative binding entropies that are
Figure 4. Inhibition of the ethanol potentiation of GlyR by stereoisomers of M554 in patch-clamp electrophysiological assays. (A) Representative
current traces activated with 15 μM glycine in the presence or absence of 100 mM ethanol using 200 μM (S,S)-M554 or (R,R)-M554 intracellularly
applied in HEK293 cells transfected with α1 GlyR. (B) Percentage of ethanol-induced potentiation of the glycine-activated current after 15 min of
intracellular application of racemic M554 (M554 (rac)) and its stereoisomers: (S,S)-M554, (R,R)-M554, (R,S)-M554, and (S,R)-M554. The values
represent the mean SEM from 4 to 6 cells. One-way ANOVA. *p < 0.05, **p < 0.01, and ***p < 0.001.
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ethanol was calculated and expressed as the percentage of
current evoked by glycine alone. In the control condition,
without any M554 diastereomer, ethanol potentiates the GlyR
activity in a 47.7 3.0% in agreement with previous reports.
At the same time, the inclusion of the molecule M554 as a
mixture of stereoisomers significantly attenuated the ethanol
Loreto San Martín − Departamento de Fisiología, Edificio
Arco de Ciencias Biológicas, Universidad de Concepción,
Concepción, Región del Bio Bio 4030000, Chile
Yenifer Argel − Departamento de Fisiología, Edificio Arco de
Ciencias Biológicas, Universidad de Concepción, Concepción,
Región del Bio Bio 4030000, Chile
Camila Riquelme − Departamento de Fisiología, Edificio Arco
de Ciencias Biológicas, Universidad de Concepción,
Concepción, Región del Bio Bio 4030000, Chile
potentiation on GlyR to 30.1
2.0%. Interestingly, the
different diastereomers induce different changes in ethanol
potentiation: (R,R)-M554, 29.3 1.9%; (S,S)-M554, 53.3
3.8%; (R,S)-M554, 52.5
3.7%; and (S,R)-M554, 33.2
Josefa Torres − Departamento de Fisiología, Edificio Arco de
Ciencias Biológicas, Universidad de Concepción, Concepción,
Región del Bio Bio 4030000, Chile
Felipe Vidal − Departamento de Fisiología, Edificio Arco de
Ciencias Biológicas, Universidad de Concepción, Concepción,
Región del Bio Bio 4030000, Chile
4.8%. According to this, (R,R)-M554 exhibits the largest
reduction in ethanol potentiation followed by (S,R)-M554.
Both systems lead to levels equivalent to those reached by the
mixture of stereoisomers. The other two, (R,S)-M554 and
(S,S)-M554, did not exhibit any detectable activity.
Francisca Cayuman − Departamento de Fisiología, Edificio
Arco de Ciencias Biológicas, Universidad de Concepción,
Concepción, Región del Bio Bio 4030000, Chile
Patricio Castro − Departamento de Fisiología, Edificio Arco de
Ciencias Biológicas, Universidad de Concepción, Concepción,
Región del Bio Bio 4030000, Chile
Jorge Fuentealba − Departamento de Fisiología, Edificio Arco
de Ciencias Biológicas, Universidad de Concepción,
Concepción, Región del Bio Bio 4030000, Chile
Gustavo Moraga-Cid − Departamento de Fisiología, Edificio
Arco de Ciencias Biológicas, Universidad de Concepción,
Concepción, Región del Bio Bio 4030000, Chile;
orcid.org/0000-0002-0260-8744
Gonzalo Yevenes − Departamento de Fisiología, Edificio Arco
de Ciencias Biológicas, Universidad de Concepción,
Concepción, Región del Bio Bio 4030000, Chile
Chunyang Jin − Center for Drug Discovery, Research Triangle
Institute, Research Triangle Park, North Carolina 27709,
United States; orcid.org/0000-0001-6733-3094
4. CONCLUSIONS
The stereospecific activity of M554 stereoisomers as inhibitors
of ethanol potentiation on GlyR was evaluated in silico and in
vitro. MD simulations indicate that all stereoisomers are
capable of binding to the Gβγ protein in regions close to the
putative active site for inhibitors of ethanol potentiation on
GlyR. Nevertheless, the (R,R)-M554 isomer showed a higher
preference for binding to a protein region close to the Asp186,
Asp228, and Asp246 residues. On the other hand, the
remaining isomers either bind to a distant site, exhibiting
large mobility in the complexed states, or induce a marked
conformational change in the protein after binding. These
factors could be related to the highest inhibitory activity of
(R,R)-M554 as revealed from the analysis of evoked currents
by extracellular glycine perfusion in the presence and absence
of ethanol. In vitro experiments revealed that (R,R)-M554 is
the most active diastereomer, followed by (S,R)-M554. The
reduced activity of the latter compound can be a consequence
of its large mobility in the complexed state, which reduces the
inhibitory efficacy that arises from effective interactions with
the Asp186, Asp228, and Asp246 residues. In the same
direction, the absence of inhibitory effects for (S,S)-M554 can
be related to its stable binding to a protein region far from the
putative active site, whereas in the case of (R,S)-M554, its
negligible activity can be a consequence of the conformational
changes induced on the protein after binding. Importantly,
future modifications of the M554 molecule should consider
(R,R) isomer as the lead structure to introduce or modify
groups that increase the efficacy for ethanol inhibition.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jcim.0c00943
Author Contributions
The manuscript was written through the contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We would like to thank Laurie Aguayo and Carolina Benitez
for valuable discussion and technical assistance. This work was
supported by FONDECYT under Grant 1170853 (L.G.).
Computational modeling resources were provided by FON-
DECYT under Grant 1160060 (V.A.J.).
AUTHOR INFORMATION
Corresponding Authors
■
́
́
Veronica A. Jimenez − Departamento de Ciencias Químicas,
Facultad de Ciencias Exactas, Universidad Andrés Bello,
Talcahuano, Región del Bio Bio 4300866, Chile;
orcid.org/0000-0002-6783-5657;
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■
Email: veronica.jimenez@unab.cl
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