Novel N-phenyl-substituted thiazolidinediones protect neural cells against glutamate- and tBid-induced toxicity

Article abstract of DOI:10.1124/jpet.114.213777

Mitochondrial demise is a key feature of progressive neuronal death contributing to acute and chronic neurological disorders. Recent studies identified a pivotal role for the BH3-only protein B-cell lymphoma-2 interacting domain death antagonist (Bid) for such mitochondrial damage and delayed neuronal death after oxygen-glucose deprivation, glutamate-induced excitotoxicity, or oxidative stress in vitro and after cerebral ischemia in vivo. Therefore, we developed new N-phenyl-substituted thiazolidine-2,4-dione derivatives as potent inhibitors of Bid-dependent neurotoxicity. The new compounds 6, 7, and 16 were identified as highly protective by extensive screening in a model of glutamate toxicity in immortalized mouse hippocampal neurons (HT-22 cells). These compounds significantly prevent truncated Bid-induced toxicity in the neuronal cell line, providing strong evidence that inhibition of Bid was the underlying mechanism of the observed protective effects. Furthermore, Bid-dependent hallmarks of mitochondrial dysfunction, such as loss of mitochondrial membrane potential, ATP depletion, as well as impairments in mitochondrial respiration, are significantly prevented by compounds 6, 7, and 16. Therefore, the present study identifies a class of N-phenyl thiazolidinediones as novel Bid-inhibiting neuroprotective agents that provide promising therapeutic perspectives for neurodegenerative diseases, in which Bid-mediated mitochondrial damage and associated intrinsic death pathways contribute to the underlying progressive loss of neurons. Copyright

Full text of DOI:10.1124/jpet.114.213777

Supplemental Material can be found at:
http://jpet.aspetjournals.org/content/suppl/2014/05/20/jpet.114.213777.DC1.html
1521-0103/350/2/273–289$25.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
http://dx.doi.org/10.1124/jpet.114.213777
J Pharmacol Exp Ther 350:273–289, August 2014
Copyright ª 2014 by The American Society for Pharmacology and Experimental Therapeutics
Novel N-Phenyl–Substituted Thiazolidinediones Protect Neural
Cells against Glutamate- and tBid-Induced Toxicitys
Sina Oppermann, Florian C. Schrader, Katharina Elsässer, Amalia M. Dolga,
Anna Lena Kraus, Nunzianna Doti, Christof Wegscheid-Gerlach, Martin Schlitzer,
and Carsten Culmsee
Institute for Pharmacology and Clinical Pharmacy, Faculty of Pharmacy (S.O., K.E., A.M.D., C.C.) and Institute for Pharmaceutical
Chemistry, Faculty of Pharmacy, Philipps University of Marburg, Marburg, Germany (F.C.S., A.L.K., C.W.-G., M.S.); and Institute
of Biostructures and Bioimaging, Naples, Italy (N.D.)
Received February 5, 2014; accepted May 16, 2014
ABSTRACT
Mitochondrial demise is a key feature of progressive neuronal Bid–induced toxicity in the neuronal cell line, providing strong
death contributing to acute and chronic neurological disorders. evidence that inhibition of Bid was the underlying mechanism of
Recent studies identified a pivotal role for the BH3-only protein the observed protective effects. Furthermore, Bid-dependent
B-cell lymphoma-2 interacting domain death antagonist (Bid) for hallmarks of mitochondrial dysfunction, such as loss of mito-
such mitochondrial damage and delayed neuronal death after chondrial membrane potential, ATP depletion, as well as impair-
oxygen-glucose deprivation, glutamate-induced excitotoxicity, ments in mitochondrial respiration, are significantly prevented by
or oxidative stress in vitro and after cerebral ischemia in vivo. compounds 6, 7, and 16. Therefore, the present study identifies
Therefore, we developed new N-phenyl–substituted thiazolidine- a class of N-phenyl thiazolidinediones as novel Bid-inhibiting
2,4-dione derivatives as potent inhibitors of Bid-dependent neuroprotective agents that provide promising therapeutic
neurotoxicity. The new compounds 6, 7, and 16 were identi- perspectives for neurodegenerative diseases, in which Bid-
fied as highly protective by extensive screening in a model of mediated mitochondrial damage and associated intrinsic death
glutamate toxicity in immortalized mouse hippocampal neurons pathways contribute to the underlying progressive loss of
(HT-22 cells). These compounds significantly prevent truncated neurons.
studies demonstrated a pivotal role for the proapoptotic Bcl-2
Introduction
family protein Bcl-2 interacting domain death antagonist (Bid)
Neuronal cell death is a consequence of both acute and
in neuronal cell death (Culmsee et al., 2005; Becattini et al.,
chronic neurologic insults and is associated with oxidative
2006; Landshamer et al., 2008; Grohm et al., 2010) where Bid
stress, breakdown of the mitochondrial membrane potential,
mediated detrimental effects on mitochondrial integrity and
and release of mitochondrial death–promoting factors that
mediate detrimental DNA damage (Culmsee and Landshamer,
fragmentation, thereby accelerating glutamate toxicity and
oxidative stress (Landshamer et al., 2008; Grohm et al., 2010,
2006). In particular, emerging evidence suggests that disturbed
2012).
mitochondrial dynamics and integrity are key decision points
Bid is a widely expressed cytosolic BH3-only protein compris-
in the sequence of intrinsic neuronal death signaling (Grohm
ing 195 amino acid residues (22 kDa) in its full-length form;
et al., 2010). Therefore, the regulators of intrinsic pathways of
it can be cleaved to a truncated proapoptotic protein of 15 kDa,
programmed cell death, such as the proapoptotic members of
termed truncated Bid (tBid) (Li et al., 1998). In apoptotic cells,
the B-cell lymphoma-2 (Bcl-2) family, may serve as promising
detrimental Bid cleavage is mediated by caspase-8 or calpains,
therapeutic targets for neuroprotection. In fact, our recent
resulting in translocation of tBid to the mitochondrial outer
membrane, where it mediates membrane permeabilization and
the subsequent release of death-promoting proteins, such as
cytochrome c, Smac/DIABLO, or apoptosis-inducing factor
(AIF) into the cytosol (Gross et al., 1999; McDonnell et al.,
This work was supported by the research funds of the University of
Marburg.
dx.doi.org/10.1124/jpet.114.213777.
s
This article has supplemental material available at jpet.aspetjournals.org.
ABBREVIATIONS: AIF, apoptosis-inducing factor; Bcl-2, B-cell lymphoma-2; Bid, Bcl-2 interacting domain death antagonist; BI-6C9, N-[4-[(4-
aminophenyl)thio]phenyl]-4-[[(4-methoxyphenyl)sulfonyl]amino]-butanamide; CI, cell index; DMSO, dimethylsulfoxide; EI-HRMS, electron ionization
high-resolution mass spectrometry; GFP, green fluorescent protein; HEK, human embryonic kidney (cells); HT-22, immortalized mouse
hippocampal neurons; MRC, mitochondrial respiratory capacity; MS, mass spectrometry; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide; NA, numerical aperture; NCI, normalized cell index; OCR, oxygen consumption rate; PBS, phosphate-buffered saline; PI, propidium iodide;
pIRES-tBid, tBid expressing plasmid; PPAR, peroxisome proliferator activator receptor; RTCA, real-time cell analyzer; siRNA, small interfering
RNA; Smac/DIABLO, second mitochondria-derived activator of caspase/direct IAP binding protein with low pI; tBid, truncated Bid; TMRE,
tetramethylrhodaminethylester.
273

274
Oppermann et al.
1999; Brustovetsky et al., 2003; Culmsee and Plesnila, 2006). General Procedure A: Preparation of N-Substituted
Our previous studies showed that inhibition of Bid by using Thiazolidinediones Derivatives
small interfering RNA provides neuroprotective effects in vitro
by inhibiting tBid-induced mitochondrial fragmentation, loss
of mitochondrial membrane integrity, mitochondrial AIF re-
lease, and cell death (Landshamer, et al., 2008). In vivo, a
pivotal role for Bid in mechanisms of delayed neuronal death
has been confirmed in models of cerebral ischemia and brain
trauma, where Bid knockout mice revealed significantly re-
duced brain damage compared with wild-type controls (Plesnila,
et al., 2001; Yin, et al., 2002; Bermpohl et al., 2006). These
results exposed Bid as a promising target for the development
of novel therapeutic strategies of neuroprotection with high
relevance for the treatment of age-related diseases of the
nervous system, where intrinsic death pathways cause im-
paired mitochondrial integrity and neuronal dysfunction.
The respective amine (1.1 equivalent) and methyl thioglycolate
(1.0 equivalent) were added to an ice-cooled solution of 1,19-
carbonyldiimidazole (1.1 equivalent) in 30 ml of absolute methylene
chloride. The reaction mixture was stirred at room temperature for
3 days and then washed three times with 3 M HCl. The solvent was
removed, and the crude solid was purified by recrystallization from
toluene or by flash column chromatography.
3-(4-Phenoxyphenyl)Thiazolidine-2,4-Dione (Compound 5). Ac-
cording to procedure A from 4-phenoxyaniline (1.14 g, 6.17 mmol),
1,19-carbonyldiimidazole (1.00 g, 6.17 mmol) and thioglycolic acid
methyl ester (0.50 ml, 5.61 mmol), purification was as follows:
recrystallization from toluene to yield a white solid (1.54 g, 5.38
mmol, 96%); 1H-NMR [dimethylsulfoxide (DMSO)-d6] d 7.41–7.47
(m, 2H), 7.29–7.33 (m, 2H), 7.18–7.22 (m, 1H), 7.07–7.10 (m, 4H),
4.30 (s, 2H); 13C-NMR (DMSO-d6) d 171.8, 171.3, 157.1, 124.1, 128.0,
So far, only a few small molecules targeting Bid were 129.5, 130.1, 118.2, 34.1. Mass spectrometry (MS) electron ionization
high-resolution mass spectrometry (EI-HRMS) m/z calculated for
C
3-Phenylthiazolidine-2,4-Dione (Compound 6). According to
designed based on multidisciplinary NMR approaches
(structure-activity relationships by interligand Nuclear Over-
hauser Effects) (Becattini et al., 2006). These first Bid
inhibitors inhibited tBid-induced second mitochondria-derived
activator of caspase/direct IAP binding protein with low pI
(Smac/DIABLO) release from isolated mitochondria and at-
tenuated mitochondrial damage, AIF release, and neuronal cell
death in models of glutamate toxicity and oxygen-glucose dep-
rivation in neuronal cell lines and primary neurons in vitro
(Becattini et al., 2004, 2006; Landshamer et al., 2008; Tobaben
et al., 2011).
The aim of the present study was to develop a novel class of
small-molecule compounds that target Bid and provide
protection against neurotoxic insults. Based on the structures
of available Bid inhibitors, a series of N-phenyl–substituted
thiazolidine-2,4-dione derivatives were designed and screened
for neuroprotection in a model of glutamate toxicity in im-
mortalized mouse hippocampal HT-22 neurons. In these cells,
which lack ionotropic glutamate receptors, glutamate-induced
cell death is mediated by inhibition of the cellular cystine
import, subsequent glutathione depletion, and enhanced
formation of reactive oxygen species through increased
lipid peroxidation and Bid-dependent mitochondrial damage
(Murphy et al., 1989; Burdo et al., 2006). The protective
effects of the synthesized compounds were further investi-
gated in a model of glutamate-induced excitotoxicity in
cultured cerebrocortical neurons. To investigate the inter-
ference with the proapoptotic activities of Bid, we examined
the effects of the newly synthesized compounds after tBid
expression in HT-22 cells and further analyzed the com-
pounds’ ability to prevent glutamate-induced Bid-mediated
impairments in mitochondrial integrity and function.
15H11NO3S: 285.0460; found: 285.0452 [M]1.
procedure A from aniline (0.56 ml, 6.17 mmol), 1,19-carbonyldiimida-
zole (1.00 g, 6.17 mmol) and thioglycolic acid methyl ester (0.50 ml,
5.61 mmol), purification was as follows: recrystallization from toluene
to yield a white solid (856 mg, 4.43 mmol, 79%); 1H-NMR (DMSO-d6) d
7.46–7.50 (m, 3H), 7.29–7.32 (m, 2H), 4.31 (s, 2H); 13C-NMR (DMSO-d6) d
171.7, 171.2, 133.3, 129.0, 128.6, 127.7, 34.1. MS (EI-HRMS) m/z cal-
culated for C9H7NO2S: 193.0198; found: 193.0206 [M]1.
3-o-Tolylthiazolidine-2,4-Dione (Compound 7). According to
procedure A from o-toluidine (661 mg, 6.17 mmol), 1,19-carbonyl-
diimidazole (1.00 g, 6.17 mmol) and thioglycolic acid methyl ester
(0.50 ml, 5.61 mmol), purification was as follows: flash column
chromatography (EtOAc, isohexane; 4:1) to yield a white solid (360 mg,
1,74 mmol, 31%); 1H-NMR (DMSO-d6) d 7.37–7.39 (m, 2H), 7.31–7.34
(m, 1H), 7.22–7.24 (m, 1H), 4.33 and 4.47 (AB, JAB 5 17.4 Hz, 2H), 2,09
(s, 3H); 13C-NMR (DMSO-d6) d 171.4, 171.1, 135.8, 132.4, 130.6, 129.4,
128.7, 126.8, 34.3, 16.8. MS (EI-HRMS) m/z calculated for C10H9NO2S:
207.0345; found: 207.0354 [M]1.
3-p-Tolylthiazolidine-2,4-Dione (Compound 8). According to
procedure A from p-toluidine (661 mg, 6.17 mmol), 1,19-carbonyl-
diimidazole (1.00 g, 6.17 mmol) and thioglycolic acid methyl ester
(0.50 ml, 5.61 mmol). Purification was as follows: recrystallization
from toluene to yield a white solid (801 mg, 3.87 mmol, 69%); 1H-NMR
(DMSO-d6) d 7.29–7.31 (m, 2H), 7.16-7.18 (m, 2H), 4.29 (s, 2H), 2.35
(s, 3H); 13C-NMR (DMSO-d6) d 171.8, 171.3, 138.4, 130.7, 129.5, 127.5,
34.1, 20.6. MS (EI-HRMS) m/z calculated for C10H9NO2S: 207.0345;
found: 207.0349 [M]1.
N-(3-(2,4-Dioxothiazolidin-3-yl)Phenyl)Acetamide (Compound
9). According to procedure A from N-(3-aminophenyl)acetamide (927 mg,
6.17 mmol), 1,19-carbonyldiimidazole (1.00 g, 6.17 mmol), and
thioglycolic acid methyl ester (0.50 ml, 5.61 mmol). Purificationwas as
follows: recrystallization from toluene to yield a white solid (428 mg,
1.61 mmol, 29%); 1H-NMR (DMSO-d6) d 10.17 (s, 1H), 7.65–7.56
(m, 1H), 7.55–7.57 (m, 1H), 7.38–7.44 (m, 1H), 6.95–6.97 (m, 1H), 4.31
(s, 2H), 2.06 (s, 3H). 13C-NMR (DMSO-d6) d 171.9, 171.3, 169.0, 140.0,
133.5, 129.2, 122.3, 119.2, 118.2, 34.3, 24.0. MS (EI-HRMS) m/z
calculated for C11H10N2O3S: 250.0412; found: 250.0439 [M]1.
Materials and Methods
1H-NMR and 13C-NMR spectra were recorded on Jeol ECA-500 and
Jeol EX-400 spectrometers (Tokyo, Japan). Mass spectra were
recorded on a Varian MAT CH7a and a S.I.S. VG 7070. Reagents
and solvents were purchased from abcr (Karlsruhe, Germany), Alfa
Aesar (Karlsruhe, Germany), Fluorochem (Hatfield, UK), Matrix
Scientific (Columbia, SC), Merck (Darmstadt, Germany), Sigma-
Aldrich (Steinheim, Germany), and Thermo Fisher Scientific
(Schwerte, Germany) and were purified by distillation or recry-
stallization, if necessary. Chromatography refers to column flash
5-(2,4-Dioxothiazolidin-3-yl)-2-Methylbenzonitrile (Compound
10). According to procedure A from 5-amino-2-methylbenzonitrile
(815 mg, 6.17 mmol), 1,19-carbonyldiimidazole (1.00 g, 6.17 mmol),
and thioglycolic acid methyl ester (0.50 ml, 5.61 mmol). Purification
was as follows: recrystallization from toluene to yield a white solid
(588 mg, 2.37 mmol, 42%); 1H-NMR (DMSO-d6) d 7.78–7.79 (m, 1H),
7.56–7.62 (m, 2H), 4.31 (s, 2H), 2.53 (s, 3H); 13C-NMR (DMSO-d6) d
171.7, 171.0, 142.5, 132.7, 131.6, 131.5, 131.3, 117.0, 112.3, 34.4, 19.7.
chromatography using the indicated eluent and Macherey-Nagel MS (EI-HRMS) m/z calculated for C11H8N2O2S: 232.0306; found:
(Düren, Germany) silica gel (0.040–0.063 mm).
232.0322 [M]1.

Thiazolidinediones as Novel Neuroprotectants
275
3-(4-(Methylsulfonyl)Phenyl)Thiazolidine-2,4-Dione (Com-
GmbH (Sankt Augustin, Germany). Pictures for two-dimensional
pound 11). According to procedure A from 4-(methylsulfonyl)aniline representation of the docking solution were generated with pose
(1.06 g, 6.17 mmol), 1,19-carbonyldiimidazole (1.00 g, 6.17 mmol), and view (Stierand and Rarey, 2007), as implemented in LeadIT, and
thioglycolic acid methyl ester (0.50 ml, 5.61 mmol). Purification was
exported from the program in the same way as the three-dimensional
as follows: recrystallization from toluene to yield a white solid (673 mg, representation.
2.48 mmol, 44%); 1H-NMR (DMSO-d6) d 8.07–8.10 (m, 2H), 7.62–7.64
(m, 2H), 4.33 (s, 2H), 3.29 (s, 3H); 13C-NMR (DMSO-d6) d 171.7, 171.0,
Expression and Purification of Recombinant Bid
140.9, 137.6, 128.8, 128.0, 43.3, 34.5; MS (EI-HRMS) m/z calculated
for C10H9NO4S: 270.9973; found: 270.9973 [M]1.
Full-length Bid with a hexa-histidine tag at the N terminus
was expressed in the pET15b vector (Addgene, Cambridge, MA) in
competent Escherichia coli Rosetta 2 (DE3) cells (Novagene, Merck
KGaA, Darmstadt, Germany). The protein was recovered in the
soluble bacteria fraction and purified by ÄKTAprime plus chro-
matography on a Ni Sepharose HisTrap FF column, followed by an
ion exchange HiTrap Q HP column and a HiLoad 16/600 Superdex
75-pg column (GE Healthcare Bio-Science AB, Uppsala, Sweden).
Highly pure protein was eluted as shown by SDS-PAGE and
Coomassie staining and stored in 20 mM Tris pH 7.4, 50 mM NaCl
at 4°C until use.
3-(3,4-Dimethylphenyl)Thiazolidine-2,4-Dione (Compound
12). According to procedure A from 3,4-dimethylaniline (748 mg,
6.17 mmol), 1,19-carbonyldiimidazole (1.00 g, 6.17 mmol), and
thioglycolic acid methyl ester (0.50 ml, 5.61 mmol), purification
was as follows: recrystallization from toluene to yield a white solid
(533 mg, 2.41 mmol, 43%); 1H-NMR (DMSO-d6) d 7.25–7.27 (m, 1H),
6.99–7.06 (m, 2H), 4.29 (s, 2H), 2.26 (s, 3H), 2.24 (s, 3H); 13C-NMR
(DMSO-d6) d 171.9, 171.4, 137.3, 137.2, 130.9, 129.9, 128.4, 125.0,
34.1, 19.2, 19.0. MS (EI-HRMS) m/z calculated for C11H11NO2S:
221.0496; found: 221.0515 [M]1.
3-(5,6,7,8-Tetrahydronaphthalen-2-yl)Tiazolidine-2,4-
Dione (Compound 13). According to procedure A from 5,6,7,
8-tetrahydronaphthalen-2-amine (908 mg, 6.17 mmol), 1,19-carbonyl-
diimidazole (1.00 g, 6.17 mmol), and thioglycolic acid methyl ester (0.50
ml, 5.61 mmol), purification was as follows: Flash column chromatog-
raphy (EtOAc, isohexane; 4:1) to yield a white solid (592 mg, 4.43
mmol, 43%). 1H-NMR (DMSO-d6) d 7.16-7.18 (m, 1H), 6.97–
6.99 (m, 2H), 4.28 (s, 2H), 2.73–2.75 (m, 4H), 1.70–1.78 (m, 4H).
13C-NMR (DMSO-d6) d 172.2, 171.4, 137.6, 137.5, 130.6, 129.5,
127.9, 124.7, 34.1, 28.6, 28.4, 22.4, 22.3. MS (EI-HRMS) m/z
calculated for C13H13NO2S: 247.0667; found: 247.0670 [M]1.
3-(4-Methoxyphenyl)Thiazolidine-2,4-Dione (Compound 14). Ac-
cording to procedure A from 4-methoxyaniline (0.76 g, 6.17 mmol), 1,19-
carbonyldiimidazole (1.00 g, 6.17 mmol), and thioglycolic acid methyl
ester (0.50 ml, 5.61 mmol), purification was as follows: recrystallization
from toluene to yield a white solid (1.02 mg, 4.76 mmol, 74%); 1H-NMR
(DMSO-d6) d 7.20–7.22 (m, 2H), 7.03–7.05 (m, 2H), 4.28 (s, 2H), 3.79
(s, 3H); 13C-NMR (DMSO-d6) d 172.0, 171.5, 159.3, 129.0 125.8, 114.3,
55.3, 34.1. MS (EI-HRMS) m/z calculated for C16H13NO2S: 223.0303;
found: 223.0296 [M]1.
Label-Free Biochemical Assay
Biochemical assays were performed using the Corning Epic
label-free technology on the EnSpire Multimode Plate Reader
(PerkinElmer, Rodgau, Germany) (O’Malley et al., 2007). Bid im-
mobilization on the optical biosensors was accomplished by adding
200 mg/ml protein in 20 mM sodium acetate, pH 4.0, using a
12-channel Thermo Scientific matrix multichannel equalizer pi-
pette followed by overnight incubation at 4°C. The microplate was
subsequently washed three times with phosphate-buffered saline
(PBS; pH 7.4) buffer with 0.005% Tween by a robotic wash station.
After washing, the plate was equilibrated in the assay buffer (PBS,
1% DMSO, pH 7.4) for 2 hours (30 ml). After the incubation, a
baseline reading was recorded. After another washing step, 15 ml of
the assay buffer, together with 15 ml of various thiazolidinediones,
prepared as described previously, was dispensed in the plate. The final
readings were taken over a period of 1 hour. The label-free re-
sponses were measured as shifts in reflected wavelength and were
expressed in picometers. Results were analyzed using the EnSpire
label-free user interface software. The difference between the last
baseline measurements and the maximum signal was used to de-
termine the KD values. Graphs were generated using GraphPad
PrismR V-5.01 (GraphPad Software, La Jolla, CA). Compounds used
for the Bid assay were diluted with the assay buffer (PBS, 1%
DMSO, pH 7.4) at a working concentration of 200 mM (100 mM final
concentration in the plate) and then further diluted in the assay buffer
directly in a 384-well polypropylene plate for a total of four different
concentrations.
3-(4-Benzylphenyl)Thiazolidine-2,4-Dione (Compound 15). Accord-
ing to procedure A from 4-benzylaniline (1.13 g, 6.17 mmol), 1,19-
carbonyldiimidazole (1.00 g, 6.17 mmol), and thioglycolic acid methyl
ester (0.50 ml, 5.61 mmol), purification was as follows: recrystallization
from toluene to yield a white solid (1.35 g, 4.76 mmol, 85%), 1H-NMR
(DMSO-d6) d 7.26–7.37 (m, 6H), 7.18–7.22 (m, 3H), 4.29 (s, 2H), 3.99
(s, 2H); 13C-NMR (DMSO-d6) d 171.9, 171.4, 142.2, 140.7, 131.2, 129.2,
128.7, 128.5, 127.8, 126.1, 40.6, 34.2. MS (EI-HRMS) m/z calculated for
C
16H13NO2S: 283.0667; found: 283.0654 [M]1.
3-(3,4-Dimethoxyphenyl)Thiazolidine-2,4-Dione (Compound
16). According to procedure A from 3,4-dimethoxyaniline (945 mg,
6.17 mmol), 1,19-carbonyldiimidazole (1.00 g, 6.17 mmol) and
thioglycolic acid methyl ester (0.50 ml, 5.61 mmol), purification was as
follows: recrystallization from toluene to yield a white solid (865 mg,
3.42 mmol, 63%); 1H-NMR (DMSO-d6) d 7.04–7.06 (m, 1H,), 6.92–
6.93 (m, 1H), 6.81–6.83 (m, 1H), 4.28 (s, 2H), 3.79 (s, 3H), 3.73
(s, 3H) 13C-NMR (DMSO-d6) d 166.2, 165.0, 151.0, 149.0, 134.3,
121.9, 115.9, 115.8, 55.7, 34.7. MS (EI-HRMS) m/z calculated for
Cell Culture of HT-22 Cells
HT-22 cells, derived from immortalized hippocampal neurons were
cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, Karls-
ruhe, Germany) supplemented with 10% heat-inactivated fetal calf
serum, 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mM
glutamine (PAA Laboratories GmbH, Cölbe, Germany). Cells were
cultured at densities of 10,000 to 12,000 cells/well either in standard
96-well plates (Greiner, Frickenhausen, Germany) for MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide] assays or in
E-plates 96 (Roche Diagnostics, Penzberg, Germany) for impedance
measurement. For plasmids and gene transfer and measuring of tBid-
induced toxicity, cells were cultured at densities of 40,000 cells/wells
in standard 24-well plates (Greiner).
C11H11NO4S: 253.0409; found: 253.023 [M]1
.
Molecular Docking
According to Becattini et al. (2006), docking studies were conducted
using the NMR solution structure of mouse BID from the Protein Data
Bank entry PDB ID 1DDB (McDonnell et al., 1999). All compounds
were converted from SMILES notation into Sybyl mol2 format using
CORINA (Molecular Networks GmbH, Erlangen, Germany). Protein
preparation and docking were performed using FlexX (Rarey et al.,
1996), available within the LeadIT version 2.1.3 from BioSolveIT
Primary Cerebrocortical Neuron Culture
Primary cortical neurons were prepared from embryonic brains
(E16-18) of rats. Meninges were removed and the cortical neurons

276
Oppermann et al.
separated by mechanical dissociation and mild trypsinization. Cells
were plated at a density of 16,000 cells/well (96-well plates) on
polyethyleneimine precoated plates. Neurobasal medium (Invitrogen)
supplemented with 5 mM HEPES, 1.2 mM glutamine, 2% (v/v) B27
supplement (Invitrogen) was used as a culture medium. After 7 or 8
days of in vitro culture, cortical neurons were used for further ex-
monitoring of changes in cell proliferation and cell viability recorded
as the differences in cell impedance, which is displayed as the cell
index (CI) as function of time. HT-22 cells were cultured at a density of
10,000 cells/well in 96-well E-plates and treated with glutamate
(3 mM) and/or novel compounds (0.1–50 mM) 24 hours after seeding.
Recording of CI values and normalization were performed using the
periments. Compounds were prepared as a stock solution in DMSO. RTCA Software 1.2 (Roche Diagnostics). Background impedance
The final concentration of DMSO in all experiments was less than caused by the media was recorded before seeding of the cells and
0.5% v/v, revealing no effect on glutamate-induced cell death. Com- subtracted automatically by the RTCA software.
pounds were applied at a concentration of 25 mM 1 hour before the
glutamate challenge and additionally as cotreatment with glutamate.
For induction of excitotoxicity, cerebrocortical neurons were exposed
to 150 mM glutamate for 24 hours. Experiments were repeated at
least three times, and analyses were performed without knowledge
of the treatment history.
ATP-Luminescence Measurements
For analysis of total ATP levels, HT-22 neurons were seeded
in white 96-well plates (Greiner) for luminescence measurements.
Twenty-four hours after seeding, cells were treated with 5 mM
glutamate and when indicated additionally with the Bid inhibitor
BI-6C9 (10 mM) and novel compounds (20 mM), respectively. Cellular
ATP levels were detected 20 hours after glutamate exposure using the
ViaLightTM Plus-Kit (Lonza, Verviers, Belgium). After treatment of
cells with the nucleotide-releasing reagent and injection of the ATP
monitoring reagent into each well, luminescence was detected im-
mediately by the FluoStar plate reader (BMG Labtech). The values
were given as relative values in percent of control cells. The ex-
periments were repeated at least three times with an n 5 8 per
treatment condition.
Induction of Neuronal Cell Death in HT-22 Cells
Neuronal cell death was induced 24 hours after seeding of the cells.
Induction of apoptosis was performed by either glutamate-induced
toxicity or tBid overexpression.
Glutamate-Induced Toxicity
For measuring glutamate toxicity, cell growth medium was re-
moved and replaced by standard cell culture medium containing
glutamate (3 mM) (Sigma-Aldrich, Munich, Germany) and/or novel
inhibitors at final concentrations of 0.1 to 100 mM. BI-6C9 (N-[4-
[(4-aminophenyl)thio]phenyl]-4-[[(4-methoxyphenyl)sulfonyl]amino]-
butanamide; Sigma-Aldrich) was added to the media at a final
concentration of 10 mM and used as a positive control for neuropro-
tection. Between 14 and 16 hours later, cell viability was analyzed.
Measurements of Oxygen Consumption Rate
Oxygen consumption rate (OCR) measurements were assessed using
an XF96 Extracellular Flux Analyzer (Seahorse Bioscience, North
Billerica, MA), which directly records the OCR in cells that remain
attached to the culture plate by using calibrated optical sensors. The
OCR recordings were carried out as previously described with minor
modifications (Gohil et al., 2010). Briefly, HT-22 cells were seeded in XF
96-well cell culture microplates (Seahorse Bioscience) at a density of
10,000 cells/well in 4.5 g/liter of standard culture medium and incubated
at 37°C and 5% CO2 for ∼24 hours and afterward treated with glutamate
(5 mM) and/or novel compounds (20 mM) for 20 hours. Before starting
the measurements, the growth medium was washed and replaced with
∼180 ml of assay medium (with 4.5 g/liter of glucose as the sugar source,
2 mM glutamine, 1 mM pyruvate, pH 7.35), and cells were incubated at
37°C for at least 60 minutes. Three baseline measurements were
recorded before the addition of compounds. To assess mitochondrial
dysfunction, respiratory chain poisons were used. The ATP synthase
inhibitor oligomycin was injected in port A at a final concentration of
3 mM to measure OCR in the absence of oxidative phosphorylation. The
protonophore carbonylcyanide-4-(trifluormethoxy)-phenylhydrazone
was subsequently injected in port B at a concentration of 0.4 mM to
dissipate the proton gradient across the inner mitochondrial membrane
and thereby to assess mitochondrial respiratory capacity (MRC).
Coinjection of the complex I/III inhibitors rotenone/antimycin A in port
C at concentrations of 1 mM respectively was used to inhibit O2
consumption by the mitochondrial electron transport chain and thus to
address nonmitochondrial respiration. Three measurements were
performed after the addition of each compound by a 4-minute mix cycle
used to oxygenate the medium and a 3-minute measurement cycle to
assess respiration. For comparing OCR after compound exposure to
OCR in nontreated cells, the absolute cell number is insignificant as
a result of the same population of cells being compared. Therefore,
results are indicated as normalized OCR (% baseline rate) for each
individual cell population to minimize variability resulting from slight
differences in plating and viability during culture and treatment time
(∼48 hours).
tBid-Induced Toxicity
One hour before transfection with a tBid-expressing plasmid
(pIRES-tBid), HT-22 cells were pretreated with BI-6C9 (Sigma-
Aldrich) at a final concentration of 10 mM or with novel compounds at
final concentrations of 1 to 100 mM. Sixteen to 20 hours after tBid-
induced toxicity, cell morphology and cell viability were analyzed.
Cell Morphology
For analysis of cellular morphology transmission light microscopy
of living HT-22 cells was performed using an Axiovert 200 microscope
(Carl Zeiss, Jena, Germany) equipped with a Lumenera Infinity 2
digital camera (Lumenera Corporation, Ottawa, ON, Canada). Light
was collected through a 10 ꢀ 0.25 numerical aperture (NA) objective
(Carl Zeiss), and images were captured using phase contrast. The
INFINITY ANALYZE software (Lumenera Corporation) was used for
digital image recording and image analysis.
Cell Viability Assay and Impedance Measurements
Quantification of cell viability in HT-22 cells was performed either
in standard 96-well plates or in standard 24-well plates by MTT
(Sigma-Aldrich) reduction at 0.25 mg/ml for 1 hour. After terminating
the reaction by removing the media and freezing the plate at 280°C
for at least 1 hour, the MTT dye was dissolved in DMSO and
absorbance was determined at 590 nm versus 630 nm (FluoStar
OPTIMA; BMG Labtech, Offenburg, Germany). Cell viability levels
were demonstrated as the percentage of absorption levels in un-
treated control cells (100% cell viability). DMSO control was used as
solvent control. For statistical analysis, experiments were repeated at
least three times.
In addition, real-time detection of cellular viability was conducted
using the xCELLigence real-time cell analyzer (RTCA)-MP (Roche
Diagnostics) by measuring the electrical impedance between micro-
Detection of Mitochondrial Membrane Potential (Dcm)
For the detection of mitochondrial membrane potential (Dcm), cells
electrodes integrated into the bottom of custom-made tissue culture were treated with 5 mM glutamate and/or the Bid inhibitor BI-6C9
plates (E-plates 96). High electrical resistance of cells enables (10 mM) or the novel compounds (20 mM) 24 hours after cell seeding in

Thiazolidinediones as Novel Neuroprotectants
277
24-well plates. Twenty-hours after glutamate exposure, Dcm in whole
cells was determined using the MitoPT tetramethylrhodaminethy-
leste (TMRE) kit (Immunochemistry Technologies, Hamburg, Germany)
followed by flow cytometric measurements using the Guava Easy Cyte
6-2 L system (Merck Millipore, Schwalbach, Germany). As a positive
control for a complete loss of Dcm, the protonophore carbonyl cyanide
m-chlorophenylhydrazone (50 mM) was applied to intact cells. After
respective treatment, cells were collected and washed with PBS. After
incubation with 0.2 mM TMRE for 30 minutes at 37°C, cells were
washed and resuspended in assay buffer. Flow cytometry was per-
formed using fluorescence excitation at 488 nm and TMRE emission
at 680 nm. Data were collected from 10,000 cells per condition. High
red TMRE fluorescence indicates intact mitochondria, whereas mito-
chondrial membrane depolarization is evidenced by the drop in red
fluorescence. For quantitative analysis, the GuavaSoft Software pack-
age was used. Measurements are representative of at least three to
five independent experiments, each with n 5 3.
Immunostaining and Confocal Laser Scanning Microscopy
For analysis of Bid localization and translocalization, HT-22 cells
were seeded in ibidi m-slide eight-well plates at a density of 20,000
cells/well and transfected with the pDSRed2-Bid vector. Mitochondria
were visualized by transfection of cells with mito-green fluorescent
protein (GFP) as previously described (Landshamer et al., 2008).
Plasmid transfection was performed analogous to the tBid trans-
fection as described already herein. Cells were cotreated with
glutamate (3 mM) and thiazolidinediones (25 mM) 48 hours after
transfection. Immunoanalysis of pDSRed2-Bid and mGFP was
performed 18 hours after treatment. After fixation of cells with 4%
paraformaldehyde, images were acquired using a confocal laser
scanning microscope (Leica SP5; Leica, Wetzlar, Germany). Light
was collected through a 63 ꢀ 1.4 NA, oil immersion objective. mGFP
fluorescence was excited at 488 nm, and emissions were detected
between 500 and 535 nm bandwidth. DSred2-Bid fluorescence was
detected by excitation at 633 nm and emission between 640 nm and
750 nm. For digital imaging, the software LSM Image Browser 4.2.0
(Carl Zeiss) was used.
Plasmids and Gene Transfer
Plasmid pCDNA 3.11 was obtained from Invitrogen. The pIRES-
tBid vector was generated as previously described (Kazhdan et al.,
2006). The ApoAlert pDSRed2-Bid vector, which encodes a biologically
active fluorescent fusion protein of Bid and DsRed monomer, was
derived from Clontech (Palo Alto, CA).
Concentration-Response Curves and EC50 Values
To compare the potency of different synthesized compounds, EC50
values were determined based on the concentration-response curves
for each of the substances. To determine the concentration required to
achieve maximal neuroprotective effects, initially all compounds were
screened by their ability to prevent glutamate-induced cell death in
concentrations of 10, 20, 30, 40, and 50 mM. Since quantification of cell
viability (MTT assay) revealed maximal protective effects of the
compounds in a concentration of 20 mM and greater, we could not
generate concentration ranges for this (Supplemental Fig. 1). There-
fore, compounds with an EC50 , 10 mM were screened in lower
concentrations of 0.1 mM up to 50 mM for maximum protection. Cell
viability data were normalized between untreated control conditions
(0%) and maximum amplitude at 50 mM (100%). Using OriginPro8.5
software (OriginLab Corporation, Northampton, MA), the resulting
data were fitted with a sigmoid function following the equation:
tBid Overexpression in HT-22 Cells
For plasmid transfections of HT-22 cells, 4 ꢀ 104 cells/well were
seeded in 24-well plates 24 hours before transfection and incubated
under normal growth conditions (37°C, 5% CO2). On the day of
transfection, standard growth medium was replaced with 500 ml of
fresh standard growth medium. Pretreatment with different sub-
stances was performed 1 hour before the following plasmid trans-
fection. pIRES-tBid or empty vector pcDNA 3.11 was dissolved
separately in antibiotic- and serum-free medium, and Attractene
transfection reagent (Qiagen, Hilden, Germany) was added to the
DNA solution. To allow complex formation, samples were incubated
for 10 to 15 minutes at room temperature. Afterward, 60 ml of the
transfection mixture was added to the cell culture medium at a final
concentration of 2 mg of DNA/well and 4.5 ml of Attractene/well.
Control cells were treated with 60 ml antibiotic- and serum-free cell
culture medium only, and vehicle controls were additionally treated
with 4.5 ml of Attractene/well. Cells were incubated under normal
growth conditions until cell morphology and cell viability were
analyzed 16 to 20 hours after tBid overexpression.
.
ꢀ
ꢀ
ꢁ
y 5 A1 1 ðA2 2 A1Þ 1 1 10
^ðLOGx0 2 xÞppÞ ;
and EC50 values for all substances were calculated.
Statistical Analysis
All data are given as means 6 standard deviation. Statistical
multiple comparisons were performed by analysis of variance followed
by Scheffé’s post hoc test. Calculations were performed with the
Winstat standard statistical software package (R. Fitch Software, Bad
Krozingen, Germany).
tBid Overexpression in Human Embryonic Kidney 293 Cells
Human embryonic kidney (HEK293) cells were cultured in Dulbecco’s
modified Eagle medium (Invitrogen) supplemented with 10% heat-
inactivated fetal calf serum, 100 U/ml penicillin, 100 mg/ml strepto-
mycin, and 2 mM glutamine (PAA Laboratories GmbH). For tBid
overexpression, cells were seeded in 12-well plates at a density of 10,000
cells/well. Twenty-four hours after seeding, cells were transfected with
0.5 mg of pIRES-tBid using effectene (Qiagen) and allowed to express the
recombinant proteins for 24 hours. Compound 16 (25 mM) and BI-6C9
Results
In an effort to identify structurally novel small-molecule
compounds that provide protection against intrinsic mito-
chondrial death pathways, we focused on the development of
(10 mM) were applied on HEK293 cells 1 hour after pIRES-tBid trans- potent inhibitors of Bid-mediated neurotoxicity. The pre-
fection. To analyze the toxicity mediated by tBid overexpression, we
performed PI flow cytometric measurements using the Guava Easy Cyte
6-2L system (Merck Millipore). Data were collected from 10,000 cells per
treatment condition. For quantification of the data, the GuavaSoft
Software package was used. PI staining was used to determine the
apoptotic and necrotic cells. Cells were incubated with 2.5 ml PI
(PromoKine, Heidelberg, Germany) for 10 minutes at room tem-
perature, and fluorescence of PI was detected with the red filter
at 690/50 nm. tBid was detected by the green fluorescent IRES
with the green filter at 525/530 nm, and all green cells were gated and
analyzed for PI-positive cells.
viously described Bid inhibitors were obtained by estimating
the binding affinity of different fragments through Nuclear
Overhauser Effects by Becattini et al. (2006). The binding
fragments (i.e., BI-2A7 and BI-2A1) showed binding affinity,
but only BI-2A1 reduced the Bid-mediated release of the
proapoptotic factor Smac/DIABLO. Combination of two of the
fragments yielded BI-6C9, the most active inhibitor described
in the series of Becattini et al. (2004) (Fig. 1). We initially
started the development of alternative Bid-inhibitors by
substituting the p-phenoxyaniline for fragment BI-2A7 (1),

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Oppermann et al.
hand) and 7 (right hand), are shown in Fig. 2. The reduced
flexibility of compound 7 might improve its binding to Bid and
thereby increase its neuroprotective property compared with
more flexible structures (e.g., compound 8) (Fig. 2).
N-Phenyl–Substituted Thiazolidinediones Provide
Neuroprotection against Glutamate-Induced Toxicity.
In a first approach, all synthesized structures were screened
for their potential to attenuate glutamate-induced and Bid-
dependent neuronal cell death in HT-22 cells. Therefore,
HT-22 cells were treated with the synthesized compounds in
concentration ranges of 0.1 mM up to 50 mM and exposed to
toxic glutamate concentrations of 3 mM. To investigate the
protective and toxic properties of all compounds, cell viability
was determined by the MTT assay 14 hours after cotreatment.
In the first screening setup, examining substance concen-
trations between 10 and 50 mM, most of the compounds
revealed maximal neuroprotection at a concentration of
20 mM, whereas concentrations higher than 30 mM and up
to 50 mM could not further increase the protective properties
(Supplemental Fig. 1). To examine the doses required to
achieve maximal protection, compounds were further tested
in concentration ranges between 0.1 mM and up to 10 mM, and
the concentration of 50 mM was set as steady-state dose
providing 100% protection. Figure 3 shows the protective
effects of three representative 3-aryl substituted thiazolidi-
nedione derivatives, which significantly preserved cell viability
of glutamate-treated HT-22 neurons in a concentration-
dependent manner between 5 and 50 mM (Fig. 3, A, C, and E).
Notably, compounds 6, 7, and 16 achieved full protection
against glutamate-induced toxicity at concentrations greater
than 20 mM (Fig. 3, A, C, and E; Supplemental Fig. 1, A–E).
This pronounced protective effect was comparable to the
protective effect of the available Bid inhibitor BI-6C9 that was
applied in the screening experiments as a reference for maxi-
mal protection in this cell death model. Importantly, treatment
of cells with the newly synthesized substances alone re-
vealed no toxic effects in concentrations up to 50 mM (Fig. 3,
A, C, and E; Supplemental Fig. 1, A–E). Similar results
were obtained for all synthesized compounds mentioned in
Fig. 1. Development of thiazolidine-2,4-dione structure 5 from BI-6C9 (3).
BI-6C9 was developed by Becattini et al. (2004) using the fragments
BI-2A1 (2) and BI-2A7 (1).
thereby omitting the metabolically labile primary aro- Table 1.
matic amine and the thioether partial structure. Since the
To further compare the neuroprotective potency of all tested
p-phenoxyaniline (4) and its propanoyl derivatives pro- compounds, EC50 values were calculated from the cell viabil-
vided slightly neuroprotective activities in a study conducted ity data at the applied concentration ranges. Since our data
in parallel, structure 4 was used to further explore structure- revealed full protective effects of the compounds at concen-
activity relationships of this type of compound. Therefore, we trations of 50 mM (positive control for highest protection), cell
incorporated the acylamide partial structure of 4 into a viability data were normalized between 0% for glutamate-only
thiazolidine-2,4-dione, concurrently reducing conformational treated cells (vehicle) to 100% protection at 50 mM and
flexibility (Fig. 1).
concentration-response curves were fitted with a sigmoid
To establish structure-activity relationships, several function (dose-response fit). Concentration-response curves
thiazolidine-2,4-dione derivatives were prepared by varying for compounds 6, 7, and 16, which represent the highest
the 3-aryl substituent. The desired compounds were obtained therapeutic potency of the tested compounds, are shown in
following a procedure described by Geffken (1987). Briefly, the Fig. 2, B, D, and F, and calculated EC50 values of all struc-
appropriately substituted aniline, thioglycolic acid methyl tures are provided in Table 1.
ester, and N,N-carbodiimidazole gave the target thiazolidine-
Real-Time Monitoring of Cell Impedance Confirms
2,4-diones in a one-pot reaction. These compounds were Neuroprotection by the N-Phenyl–Substituted Thi-
further analyzed for their ability to target Bid and their azolidinediones. The neuroprotective properties of the
neuroprotective efficiency (Table 1).
N-phenyl–substituted thiazolidinediones detected by end-
Of note, for some molecules (e.g., compound 7), a rotational point MTT assays were substantiated by real-time analysis of
barrier is observed as demonstrated by the AB system for the cell impedance, expressed as normalized cell index (NCI) (Fig.
protons at C-5 in the 1H-NMR spectrum. The spatial structures, 4). High electrical resistance of cells allows the investigation
a two-dimensional illustration concerning the barrier and the of cell proliferation and viability, as well as cell morphology
corresponding C-5 proton NMR-signals of compound 8 (left and adhesion, since alterations in these cell properties are

Thiazolidinediones as Novel Neuroprotectants
279
TABLE 1
Neuroprotective properties of N-phenyl–substituted thiazolidinediones
Chemical structures of newly synthesized compounds are shown. EC50 values are given as the mean drug concentration required to inhibit cell
death by 50% compared with controls. Toxicity values indicate toxic drug concentrations found to induce cell death without cotreatment with
glutamate (analysis of variance Scheffé’s test).
Compound ID
Structure
EC50 (mM)
Toxicity
mM
5
13.27
10#
50###
6
7
8
9
9.87
6.78
—
—
—
—
18.10
14.14
10
11
8.76
—
—
15.71
12
13
9.59
9.60
—
—
(continued)

280
Oppermann et al.
TABLE 1—Continued
Compound ID
Structure
EC50 (mM)
Toxicity
14
13.68
—
15
16
13.42
9.39
—
—
#P , 0.05; ###P , 0.001 compared with untreated control.
recorded as changes in cell impedance (Diemert et al., 2012). rat neurons. Glutamate challenge mediated significant neuro-
The RTCA revealed that cellular impedance significantly nal toxicity, which was partially rescued by pretreatment
decreased in cell populations exposed to glutamate (3 mM) 5 with our synthesized thiazolidinediones, particularly with
to 8 hours after treatment. In contrast, NCI was fully pre- compound 16 (Fig. 4D).
served to control levels by cotreating the cells with glu-
N-Phenyl–Substituted Thiazolidinediones Attenuate
tamate (3 mM) and compounds 6, 7, or 16 at a concentration tBid-Induced Neuronal Cell Death. Our previous studies
of 50 mM, respectively (Fig. 4, A–C). Notably, once initiated, demonstrated a pivotal role for the BH3-only protein Bid in
glutamate exposure triggered the complete loss of cellular neuronal apoptotic cell death in the present model of glutamate
impedance within a small time window of 2–4 hours, in- toxicity in HT-22 cells (Landshamer et al., 2008; Grohm et al.,
dicating that neuronal cell death occurred rapidly and in 2010). Pharmacological Bid inhibition by the available small-
a highly synchronized manner. Nevertheless, this decrease molecule inhibitor BI-6C9 prevented cell death by inhibiting
in CI was persistently blocked by our novel inhibitor com- tBid translocation to the mitochondrial membrane, the sub-
pounds. Further monitoring of cell impedance confirmed the sequent release of AIF, cytochrome c, and Smac/DIABLO into
concentration-dependent protective effects of the tested com- the cytosol (Becattini et al., 2004, 2006). To examine the role of
pounds that were shown by the MTT assay (Fig. 3, A–C). our newly synthesized compounds in Bid-mediated neuronal cell
Compounds 6 and 16 revealed transient neuroprotection death, the novel compounds were applied to HT-22 neurons
at concentrations of 5, 10, and 25 mM (6) but showed a transfected with a tBid-encoding plasmid, and cell morphology
sustained protection over time at concentrations of 50 mM and cell viability were analyzed (Fig. 5). After overexpression of
(Fig. 4, B and C). Similarly, compound 7 was only tran- tBid, HT-22 neurons showed excessive alterations in cell
siently protective at concentrations lower than 5 mM but morphology: cells clearly appeared shrunken, rounded up, and
attenuated the complete decline of NCI already at concen- detached from the bottom of the culture plate (Fig. 5A). In
trations of 10 mM, although not to levels of control cells. contrast, 1 hour of pretreatment of the cells with the novel
However, persistent neuroprotection against glutamate- compounds (1 mM) rescued HT-22 cells from tBid-induced
induced toxicity was achieved at a concentration of 50 mM toxicity and preserved spindle-shaped cell morphology (Fig.
(Fig. 3A). These results accentuate that real-time measure- 5A). Notably, treatment of the cells with the compounds 6, 7, and
ments of cell proliferation not only confirmed our findings 16 at low concentrations of 1 mM induced significant protective
detected by MTT assay but further allowed for distinction effects (Fig. 5). Quantification of cell viability determined by
between transient and persistent protective effects. Therefore, MTT assay 20 hours after tBid transfection confirmed the
this method provided a better estimation of the protective compound-mediated protection against tBid-induced cell death.
potential of the novel compounds.
Expression of tBid reduced cell viability of HT-22 cells to 20–30%
To transfer the compound’s neuroprotective effects estab- of control levels, whereas cell viability was significantly restored
lished in the neuronal HT-22 cell line to primary neuronal by pretreatment of cells with the compounds 6, 7, or 16 at
cells, the thiazolidinediones were applied to a model of concentrations of 1 mM, respectively (Fig. 5, B–D). Similar
glutamate-induced excitotoxicity in primary cerebrocortical results were observed with pretreatment of the cells with the

Thiazolidinediones as Novel Neuroprotectants
281
Fig. 2. (Top) Spatial structure of compound
7 and 8. (Center) Rotation characteristics
of the exemplary N-phenyl-thiazolidine-
2,4-diones. (Bottom) The H-NMR experi-
ment shows an AB-type spin system for
the C5 methylene protons resulting from
their diastereotopic nature.
indicated compounds at higher concentrations of 10 and 50 mM, compounds were able to rescue HT-22 cells after tBid-
whereas no toxic effects of the compounds were detected when overexpression, thereby providing strong evidence for our
applied under control conditions (data not shown). To estimate hypothesis that the newly synthesized compounds provided
the transfection efficiency of tBid-transfected cells, parallel protection by targeting the proapoptotic protein Bid.
transfection experiments in HT-22 cells were performed using
To examine whether the proposed protective potency of the
the current protocols for DNA vector transfection (Attractene; compounds against tBid-induced cell death is specific for
Qiagen). Cells transfected with a GFP of a comparable vector neuronal cells or whether they can be transferred to non-
size as the tBid-vector were analyzed by fluorescence micros- neuronal cells, we investigated the compound’s effect on
copy, revealing a transfection efficiency of approximately HEK293 cells (Supplemental Fig. 3A). To this end, HEK293
70% (Supplemental Fig. 2). These data correlate well to the cells were transfected with the tBid-encoding plasmid in the
percentage of tBid-induced cell death and are similar to previous presence and absence of the synthesized thiazolidinediones.
results on the transfection efficiency in HT-22 cells (Landshamer Notably, tBid overexpression promoted severe cell damage,
et al., 2008). Thus, it is suggested that treatment of cells with the which occurred rapidly within 15 hours after transfection,
thiazolidinediones protected all transfected cells. These findings indicating that this cell line is more sensitive to tBid-induced
showed that the new N-phenyl–substituted thiazolidinedione cell death than the current used HT-22 cell line. In HEK293

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Oppermann et al.
Fig. 3. N-Phenyl–substituted thiazolidinediones provide neuroprotection against glutamate-induced toxicity. HT-22 cells were cultured in 96-well plates with
10,000 cells/well. MTT assay was used to determine cell viability 14 hours after cotreatment with glutamate (3 mM) and respective small molecules in different
concentrations from 0.1 to 50 mM. BI-6C9 (10 mM) was used as a positive control for neuroprotection. Compounds 7 (A), 16 (C), and 6 (E) attenuated glutamate-
induced toxicity in a concentration-dependent manner. (A) DMSO treatment was used as solvent control. The presented data are normalized to the cell viability
of untreated controls (100% cell viability). For statistical analysis, experiments were independently repeated at least three times with an n = 8 per treatment
condition. Results are presented as mean 6 S.D. ***P , 0.001 compared with glutamate-treated control (vehicle), analysis of variance, Scheffé’s test. (B, D, and
F) Based on these data, concentration-response curves and EC50 values were calculated for each substance. Cell viability data were fitted from 0 for glutamate-
treated control (vehicle) to 100 for highest protection at 50 mM, and dose-response fits for compound 7 (B), 16 (D), and 6 (F) are shown.
cells, cotreatment with BI-6C9 (10 mM) provided only a slight
For a better understanding of the protective effects provided
protection against tBid toxicity. Interestingly, the protective by the novel compounds and to confirm their ability to inhibit
effect achieved by cotreatment of cells with compound 16 was Bid, the compounds were subjected to docking analysis into the
more pronounced, revealing a small but significant increase in three-dimensional structure of Bid. Thereby a possible binding
HEK293 cell survival compared with tBid-mediated damage mode of compound 7, which revealed the highest protective
(Supplemental Fig. 3A). These data indicate that the pro- potency, was derived by means of docking with the program
tective potential of the newly synthesized thiazolidinediones FlexX (Fig. 6). The thiazolidinedione moiety points into a deeply
is not specific for neurons, although their potency might vary buried pocket on the surface of Bid, like BI-6C9, and allows
between different cell lines.
interactions with the amino acids Val186 and Ile86 of Bid.

Thiazolidinediones as Novel Neuroprotectants
283
Fig. 4. Real-time monitoring of cell impedance confirms concentration-dependent neuroprotection mediated by the novel compounds. HT-22 cells were
seeded in 96-well E-plates at a density of 10,000 cells/well and exposed to 3 mM glutamate (Glut) 24 hours after seeding. The indicated cell groups were
cotreated with the novel compounds 7 (A), 16 (B), and 6 (C) in concentrations of 1 to 50 mM. Cellular impedance was continuously monitored by the
xCELLigence system (Roche), and cell index was normalized at 0 hour (treatment time point). Cotreatment with glutamate and BI-6C9 (10 mM) was
used as a positive control for persistent protection. Each compound revealed a dose-dependent transient protection at concentrations from 10 to 25 mM
yet a sustained protection over time at 50 mM. (D) Primary rat cortical neurons were cultured in 96-well plates at a density of 16,000 cells/well. MTT
assays revealed a reduction in cell viability 24 hours after glutamate-induced excitotoxicity (150 mM glutamate) up to 50% compared with control cells.
Pretreatment with the indicated concentrations of compound 16 attenuated glutamate-induced cell damage. Results are provided as mean 6 S.D. with
n = 6–8 per treatment group. All experiments were independently repeated three times.
Further, a hydrophobic interaction between the aromatic ring caspase-independent cell death (Konig et al., 2007; Landshamer
of compound 7 with the side chain of Tyr185 of Bid is suggested et al., 2008; Grohm et al., 2010). Since these intrinsic cell death
(Fig. 6, A and B) and substantiate the intended specificity of features are Bid-dependent and initiated by the translocation of
newly synthesized compounds to target Bid. Finally, we could Bid to mitochondria (Plesnila et al., 2001; Culmsee and Plesnila,
confirm the compound’s specificity in a cell-free system using 2006; Landshamer et al., 2008; Grohm et al., 2010), we further
purified recombinant Bid protein. The binding of the presented addressed the compound’s protective potential against Bid-
thiazolidinediones to recombinant Bid was measured using the dependent mitochondrial dysfunction.
EnSpire label-free platform (Fig. 6, C–E) (O’Malley et al.,
To examine the compound’s ability to prevent mitochondrial
2007). Using this biochemical assay, the target Bid was initially Bid translocation after the glutamate challenge, HT-22 cells
immobilized at a concentration of 200 mg/ml onto the amino- were transfected with a pDsRed2-Bid vector that results in the
coupling surface of the EnSpire label-free biochemical micro- expression of a red fluorescing Bid residing in the cytosol (Fig.
plate biosensor. After washout of the unbound target and 7A). To visualize mitochondria, cotransfection of cells with
further equilibration of the biosensor, several concentrations mGFP was performed. In line with our previous findings in
of the appropriate compounds were added to the immo- HT-22 cells, under physiologic conditions, Bid is distributed in
bilized Bid protein. The background-corrected responses the cytosol, whereas glutamate treatment causes the trans-
were well fitted by a one-site interaction model, yielding a location of Bid to mitochondria (Fig. 7A, yellow in merged)
KD value in the low mM range for each compound tested (Landshamer et al., 2008). Cotreatment of cells with the com-
(KD 17.7 6 5.2 mM (6), KD 2.2 6 0.9 mM (7), KD 0.76 6 0.32 mM pound 16, as a representative of the thiazolidinediones, prevented
(16); Fig. 6, C–E). These findings underscore that the identified the accumulation of Bid at the mitochondria and retrained the
thiazolidinediones are able to bind efficiently to Bid.
homogeneous distribution of Bid in the cytosol (Fig. 7A). To
Novel Compounds Prevent Glutamate-Induced Bid further investigate whether hallmarks of Bid-dependent cell
Translocation and Bid-Mediated Mitochondrial Impair- death downstream of mitochondrial Bid transactivation are
ments. Glutamate-induced toxicity involves mainly im- prevented by the presented compounds, we analyzed mito-
pairments in mitochondrial integrity and function. Loss of chondrial membrane potential (Dcm) in HT-22 cells. After the
mitochondrial membrane potential (Dcm) and mitochondrial translocation of Bid to mitochondria, a significant loss of Dcm
fragmentation trigger the release of death promoting factors was detected, indicated by a drop in red TMRE fluorescence
into the cytosol, thereby preceding the final execution of 20 hours after glutamate treatment (Fig. 7, B and C). The

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Fig. 5. N-Phenyl–substituted thiazolidinediones attenuate tBid-induced neuronal cell death. HT-22 cells were cultured in 24-well plates with a density
of 40,000 cells/well, and tBid-induced toxicity was determined 20 hours after transfection of cells with a tBid-encoding plasmid (pIRES-tBid). Cell
morphology was analyzed by microscopy (A), and cell viability was determined by MTT assay. (B–D) Pretreatment with the novel compounds 7, 16, and 6
(1 mM) was performed 1 hour before pIRES-tBid transfection. BI-6C9 (10 mM) was used as a positive control for inhibition of tBid-induced toxicity. (A)
Photomicrographs (10 ꢀ 0.25 NA objective) show typical alterations in cell morphology of HT-22 cells 20 hours after tBid overexpression (pIRES-tBid,
control). Pretreatment with novel compounds preserved cell morphology of HT-22 cells. (B–D) Quantification of cell viability confirms significant
protective effects against tBid-induced toxicity mediated by compound 7 (B), compound 16 (C), and compound 6 (D). The presented data are normalized
to untransfected control (100% cell viability). For statistical analysis, experiments were independently repeated at least three times with n = 3 per
treatment condition, and results are reported as mean 6 S.D. ***P , 0.001 compared with tBid treated control cells, analysis of variance, Scheffé’s-test.
uncoupler carbonyl cyanide m-chlorophenylhydrazone was levels of compound-treated HT-22 cells were examined in the
used as a positive control to induce depolarization of the presence and absence of glutamate (Fig. 7D). Indeed, the rapid
mitochondrial membrane (Fig. 7C). Of note, the application of depletion in cytoplasmic ATP caused by glutamate-induced cell
20 mM of the novel compounds 7, 16, or 6 did not affect Dcm injury was completely attenuated by compounds 7, 6, and 16,
under control conditions but almost completely attenuated the whereas ATP levels of control cells were not altered (Fig. 7D).
pronounced breakdown of Dcm after glutamate challenge Therefore, these data confirmed at a functional level that the
(Fig. 7, B and C). The protective effects mediated by the novel novel small molecules preserve mitochondrial integrity and
compounds were comparable to that of the available Bid energy production of glutamate-treated HT-22 cells.
inhibitor BI-6C9, suggesting that the newly synthesized
compounds preserve Bid translocation to mitochondria, there- by N-Phenyl–Substituted Thiazolidinediones. Broader
by maintaining Dcm. insights into the compounds’ ability to rescue metabolic
Mitochondrial Respiratory Activity Is Maintained
To address whether the N-phenyl thiazolidinediones are also functions were derived from further investigations of mito-
able to prevent the functional integrity of mitochondria, ATP chondrial bioenergetics (Fig. 8). Therefore, HT-22 cells were

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Fig. 6. Two- and three-dimensional representation of the binding mode of compound 7 for inhibition of Bid. (A) Three-dimensional docking of compound
7 in the hydrophobic groove of Bid. (B) Two-dimensional representation of the docking solution of (A) reveals interaction of compound 7 with Val186 and
Ile86 and Tyr185 of Bid. (A and B) For the inhibitor compound, the carbon atoms are shown in gray, oxygen in red, nitrogen blue, and sulfur yellow; the
solvent-accessible surface of Bid is represented with formal charges (red, negative; blue, positive). (C–E) EnSpire assay of molecule binding to
immobilized Bid. Binding curves of molecules 4, 5, and 6 to Bid proteins. Data points are the mean of three independent measurements (n = 3) 6 S.D. The
data were fitted with a simple one-site binding model, yielding a KD value of 17.7, 2.2, and 0.76 mM for compounds 6, 7, and 16, respectively.
challenged with glutamate and/or the novel compounds, and Whereas rotenone/antimycin A–sensitive OCR specifically
O2 consumption was analyzed using the Extracellular Flux indicates respiration in mitochondria, rotenone/antimycin
Analyzer (Seahorse Bioscience) designed for the exposure of A–nonresistant rate identifies nonmitochondrial respiration
mitochondria to defined substrates and inhibitors to modulate and allows for calculation of mitochondrial maximum respi-
mitochondrial respiration in adherent cells. In accordance ration. After onset of glutamate exposure, mitochondrial
with previous studies reporting that glutamate-induced maximum respiration was markedly reduced in control cells
mitochondrial dysfunction involves changes in mitochondrial but was preserved by the application of compounds 7, 6, and
acidification and OCR, glutamate exposure of HT-22 cells 16 at concentrations of 20 mM, respectively (Fig. 8, A–C). In
decreased the OCR, indicating a glutamate-induced reduc- summary, the prevention of glutamate-induced reduction in
tion in mitochondrial respiration (Fig. 8). After record- mitochondrial respiratory capacity and maximum respiration
ing the basal mitochondrial respiration, the ATP synthase provides additional evidence for rescued metabolic functions
inhibitor oligomycin was added to uncouple mitochondrial and maintained energy metabolism in mitochondria of
respiration from ATP production. While the OCR of control cells glutamate-treated HT-22 cells that are mediated by the
was decreased after oligomycin injection, glutamate-treated N-phenyl–substituted thiazolidinediones.
cells responded less to oligomycin (Fig. 8). The further applica-
tion of the protonophor carbonylcyanide-4-(trifluormethoxy)-
phenylhydrazone increased OCR to a maximum extent,
Discussion
allowing the examination of the maximum oxygen flux and
In the present study, we developed a series of novel
MRC of cells. After glutamate treatment, both maximum 3-phenyl–substituted thiazolidine-2,4-dione derivatives that
oxygen flux and MRC were significantly decreased but were display protective effects against glutamate-induced toxicity
notably restored by cotreatment of cells with the compounds and, in addition, rescued HT-22 cells against tBid-mediated
7, 6, or 16, respectively (Fig. 8, A–C). Finally, the mitochon- cell death. Moreover, key features of mitochondrial dysfunc-
drial respiratory chain was inhibited by the application of the tion, such as loss of mitochondrial membrane potential
complexes I and III inhibitors rotenone and antimycin A. described as “point of no return” in the cells commitment to

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Fig. 7. N-Phenyl–substituted thiazolidinediones preserve mitochondrial Bid translocation and mitochondrial function. (A) Fluorescence photomicro-
graphs of HT-22 cells expressing a Bid-DsRed fusion protein and a green fluorescent mGFP. Control cells show homogeneous cytosolic distribution of
Bid, whereas glutamate-treated cells (3 mM) reveal colocalization of mitochondrial staining and DsRed signal (yellow in merged panels), indicating Bid
accumulation at mitochondria. Cotreatment of cells with compound 16 prevented mitochondrial translocation of Bid. (B) Mitochondrial membrane
potential (Dcm) was determined by TMRE-flow cytometric recordings. HT-22 cells were seeded in 24-well plates with a density of 40,000 cells/well and
treated with glutamate (5 mM) alone or cotreated with glutamate (5 mM) and novel compounds (20 mM). Twenty hours after treatment, cells were
stained with MitoTM TMRE dye, and red fluorescence of 10,000 cells per treatment condition was analyzed. Glutamate-treated cells (vehicle) showed
significant reduction of red fluorescence (loss of Dcm) compared with control vehicle; novel compounds prevented the breakdown of Dcm as indicated by
the preservation of red TMRE fluorescence. Numbers are mean percentages 6 S.D. of TMRE fluorescence of the indicated cell groups with n = 3. High red
fluorescence levels indicate intact mitochondria (right); decrease in red fluorescence depicts loss of Dcm (left). (C) Quantification of TMRE fluorescence
(B) confirmed the breakdown of Dcm in glutamate exposed vehicle cells, which was significantly prevented by the novel compounds 7, 6, and 16. The
uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), which causes a fast breakdown of the mitochondrial membrane potential, was used as
a positive damage control. (B and C) The experiment was independently repeated four times with n = 3. Data are presented as mean 6 S.D.

Thiazolidinediones as Novel Neuroprotectants
287
Fig. 8. Novel small molecules preserve mitochondrial respiration. (A–C) Time course of OCR in HT-22 cells in the presence and absence of glutamate.
HT-22 cells were seeded in XF 96-well microplates (Seahorse Bioscience) at a density of 10,000 cells/well and treated with glutamate (5 mM) and/ or
novel substances 7 (A), 6 (B), or 16 (C), respectively. Twenty hours after treatment, mitochondrial respiration was analyzed using the XF Mito Stress kit
and recorded as the rate of OCR by the Extracellular Flux Analyzer (Seahorse Bioscience). As controls, Olygomycin (3 mM) was used to inhibit ATP-
linked OCR, carbonylcyanide-4-(trifluormethoxy)-phenylhydrazone (FCCP; 0.4 mM) to measure uncoupled respiration and rotenone/antimycin (1 mM) to
inhibit complex I/III–dependent respiration. Glutamate exposure of HT-22 cells decreased oxygen consumption rate and thereby mitochondrial
respiration, which was significantly restored by cotreatment with the novel compounds (20 mM). Data were depicted as normalized OCR (% baseline
rate) for each individual population of cells and representative of four independent experiments, each with n = 6 for all treatment groups, respectively.
die (Kroemer et al., 2007), as well as impairments in compounds’ ability to preserve the oxygen consumption rate of
mitochondrial energy metabolism, were effectively prevented HT-22 cells, and thereby mitochondrial bioenergetics, de-
by the novel compounds. The observed protective effect in the scribed to be defective in neurodegenerative disorders (Clerc
model of oxidative stress is already promising since the new and Polster, 2012). In addition, we revealed that the proposed
compounds may provide a protective benefit against path- protective effects of the presented compounds found in HT-22
ologic pathways occurring in neurodegenerative diseases cells can be transferred to other cells. Parallel experiments in
where oxidative stress triggers the onset of neuronal cell primary cultured neurons demonstrated the neuroprotective
death. Further, our finding that the indicated structures effects of the N-phenyl–substituted thiazolidinediones in
preserved cell viability in HT-22 cells after tBid overex- a model of glutamate-induced excitotoxicity. Furthermore,
pression strongly supports the intended specificity of the the compounds provided slight protection against tBid-
N-phenyl–substituted thiazolidinediones to provide protec- induced cell death in HEK293 cells, indicating the com-
tion by targeting the proapoptotic protein Bid, which was pound’s protective potency independent of the cell type.
substantiated by virtual docking analysis. The specific bind- Recent studies demonstrated a crucial role for the BH3-only
ing of the compounds to the target Bid was finally con- protein Bid in the model of glutamate-induced oxidative
firmed by applying the compounds to recombinant Bid stress in HT-22 cells where small interfering RNA mediated
protein in biochemical cell-free assay. Of note, we report that Bid gene silencing and pharmacological Bid inhibition pro-
glutamate-induced mitochondrial translocation of Bid and the vided neuroprotection in vitro (Landshamer et al., 2008).
subsequent hallmarks of Bid-dependent intrinsic cell death Further, these previous studies identified depolarization of
were significantly prevented by the N-phenyl–substituted the mitochondrial outer membrane, mitochondrial fission,
thiazolidinediones (Fig. 7) and further demonstrated the and ATP depletion as key events of glutamate toxicity, and
(D) ATP-levels of HT-22 cells, treated with 20 mM of the compounds 7, 6, or 16 as indicated, were analyzed in the presence or absence of glutamate (5 mM,
20 hours) by ATP-luminescence measurements. Compound treatment did not affect basal ATP levels but significantly prevented ATP depletion after
glutamate exposure. Experiment was repeated three times with n = 8 per treatment condition, and results are provided as mean 6 S.D. ***P , 0.001
compared with glutamate-treated vehicle (analysis of variance, Scheffé’s test) (C and D).

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Oppermann et al.
these effects were apparently also Bid dependent (Landshamer
In conclusion, we report here the design and synthesis
et al., 2008; Grohm et al., 2010, 2012). A pivotal role of Bid in of N-phenyl–substituted thiazolidinediones as novel inhib-
neuronal cell death was further substantiated by previous itors against neurotoxicity and indicated inhibition of the
findings that reduced Bid expression prevented cell death in BH3- only protein Bid as a key mechanism of the neuro-
a model of oxygen glucose deprivation in vitro and also protective properties of the novel compounds. In particular,
reduced brain damage in models of cerebral ischemia and the thiazolidinediones derivatives provided significant pro-
brain trauma in vivo (Plesnila et al., 2001; Culmsee et al., tection against glutamate toxicity and tBid overexpression in
2005). Moreover, recent reports showed that full-length Bid, hippocampal HT-22 cells and further preserved detrimental
as well as the truncated form tBid, is sufficient to induce impairments of mitochondrial integrity and function, thereby
mitochondrial dysfunction and cell death in neurons and that inhibiting the final execution of intrinsic cell death. Future
they differ only in kinetics and cell death pathways (Konig optimization of the structures should result in compounds
et al., 2007). Whereas tBid is suggested to induce rapid with favorable pharmaceutical properties that are suitable
mitochondrial damage in a caspase-dependent manner, full- for therapeutic strategies in the treatment of neuronal and
length Bid is thought to be involved in caspase-independent non-neuronal diseases where Bid has been implicated (Plesnila
apoptosis with slower kinetics (Ward et al., 2006). In the et al., 2001; Guegan et al., 2002; Friedlander, 2003; Waldmeier,
described model system of glutamate toxicity in HT-22 cells, 2003; Culmsee and Plesnila, 2006).
however, activated full-length Bid as well as tBid may con-
tribute to neuronal cell-death pathways.
Acknowledgments
Overall, these findings in model systems of neuronal and
non-neuronal death in vitro and in vivo strongly suggest that
targeting Bid might be a promising tool to develop pharma-
ceutical drugs for neuroprotection. Indeed, it was recently
demonstrated that pharmacological inhibition of Bid with the
available inhibitor BI-6C9, which was used as positive control
for neuroprotection in the present study, strongly reduced tBid-
induced release of Smac/DIABLO from isolated mitochondria
in concentrations as low as 20 mM in vitro and prevented ef-
fectively tBid-mediated cell death at concentrations of 10–50 mM
in cell-based assays of caspase-dependent (Becattini et al.,
2004) and caspase-independent cell death (Culmsee et al.,
2005; Landshamer et al., 2008; Grohm et al., 2010; Tobaben
et al., 2011). In particular, the available Bid-inhibitors reduced
caspase-3 activity in tBid-transfected HeLa cells at 50 mM and
persistently blocked caspase activity at 100 mM (Becattini
et al., 2004). In our model systems in HT-22 neurons, the Bid
inhibitor BI-6C9 provided protection against glutamate toxicity
as well as tBid-induced death at a concentration of 10 mM.
Notably, the newly synthesized compounds provided protective
effects against glutamate-induced cell death at concentrations
of 5 mM up to 50 mM and attenuated tBid-induced toxicity even
at concentrations as low as 1 mM.
The authors thank Emma Esser for careful editing of the
manuscript and Dr. Alexander Seiler (Roche Diagnostics GmbH) for
providing support with the xCELLigence system. The authors further
thank Dr. Cornelius Krasel for excellent expertise on HEK293 cell
transfection and Dr. Hans-Peter Steffens for providing support with
the Enspire Multimode Plate Reader (PerkinElmer).
Authorship Contributions
Participated in research design: Oppermann, Culmsee, Schlitzer.
Conducted experiments: Oppermann, Elsässer, Dolga, Doti.
Contributed new reagents or analytic tools: Schrader, Kraus.
Performed data analysis: Oppermann, Wegscheid-Gerlach, Culmsee.
Wrote or contributed to the writing of the manuscript: Oppermann,
Culmsee, Schlitzer.
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Address correspondence to: Prof. Dr. Carsten Culmsee, Institute for
Pharmacology and Clinical Pharmacy, Faculty of Pharmacy, Philipps-University
of Marburg, Karl-von-Frisch-Str. 1, 35032 Marburg, Germany. E-mail: culmsee@
staff.uni-marburg.de