Development and Application of a Chemical Probe Based on a Neuroprotective Flavonoid Hybrid for Target Identification Using Activity-Based Protein Profiling

Article abstract of DOI:10.1021/acschemneuro.0c00589

Alzheimer's disease (AD) is the most common form of dementia, and up to now, there are no disease-modifying drugs available. Natural product hybrids based on the flavonoid taxifolin and phenolic acids have shown a promising pleiotropic neuroprotective profile in cell culture assays and even disease-modifying effects in vivo. However, the detailed mechanisms of action remain unclear. To elucidate the distinct intracellular targets of 7-O-esters of taxifolin, we present in this work the development and application of a chemical probe, 7-O-cinnamoyltaxifolin-alkyne, for target identification using activity-based protein profiling. 7-O-Cinnamoyltaxifolin-alkyne remained neuroprotective in all cell culture assays. Western blot analysis showed a comparable influence on the same intracellular pathways as that of the lead compound 7-O-cinnamoyltaxifolin, thereby confirming its suitability as a probe for target identification experiments. Affinity pulldown and MS analysis revealed adenine nucleotide translocase 1 (ANT-1) and sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) as intracellular interaction partners of 7-O-cinnamoyltaxifolin-alkyne and thus of 7-O-esters of taxifolin.

Full text of DOI:10.1021/acschemneuro.0c00589

pubs.acs.org/chemneuro
Research Article
Development and Application of a Chemical Probe Based on a
Neuroprotective Flavonoid Hybrid for Target Identification Using
Activity-Based Protein Profiling
Sandra Gunesch, David Soriano-Castell, Stephanie Lamer, Andreas Schlosser, Pamela Maher,*
and Michael Decker*
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ABSTRACT: Alzheimer’s disease (AD) is the most common form of dementia, and up to now, there are no disease-modifying
drugs available. Natural product hybrids based on the flavonoid taxifolin and phenolic acids have shown a promising pleiotropic
neuroprotective profile in cell culture assays and even disease-modifying effects in vivo. However, the detailed mechanisms of action
remain unclear. To elucidate the distinct intracellular targets of 7-O-esters of taxifolin, we present in this work the development and
application of a chemical probe, 7-O-cinnamoyltaxifolin-alkyne, for target identification using activity-based protein profiling. 7-O-
Cinnamoyltaxifolin-alkyne remained neuroprotective in all cell culture assays. Western blot analysis showed a comparable influence
on the same intracellular pathways as that of the lead compound 7-O-cinnamoyltaxifolin, thereby confirming its suitability as a probe
for target identification experiments. Affinity pulldown and MS analysis revealed adenine nucleotide translocase 1 (ANT-1) and
sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) as intracellular interaction partners of 7-O-cinnamoyltaxifolin-alkyne and thus
of 7-O-esters of taxifolin.
KEYWORDS: Alzheimer’s disease, natural product hybrids, CuAAC, neuroinflammation
on compounds with a pleiotropic neuroprotective profile to
deal with the multifactorial nature of AD,6 particularly as the
one-target strategy has not been successful in drug discovery so
far.7
Natural products have been used in traditional medicine and
are gaining increased attention for their potential as neuro-
protectants interfering with disease progression.8−10 The well-
established antioxidant features of natural products, i.e., their
radical scavenging abilities, are important for neuroprotection
INTRODUCTION
■
Alzheimer’s disease (AD) is the most common form of
dementia and due to aging societies, the number of affected
people is increasing dramatically.1 AD is characterized by
abnormal protein aggregation of hyperphosphorylated tau
protein forming neurofibrillary tangles and amyloid β
accumulation as extracellular plaques.2 Neuroinflammation
and oxidative stress are strong contributors to neurodegenera-
tion and AD pathology.3,4 Enhancing the pathophysiology of
protein aggregation, oxidative stress and neuroinflammation
may even represent key factors in AD development.5
Currently, there are no disease-modifying drugs on the market.
The only available treatments are symptomatic and cannot halt
or cure the disease.1 The complexity of AD and the lack of
understanding of the details of its cause and progression are
hampering drug development. It is important to broaden the
knowledge about the molecular causes of the disease and focus
Received: September 8, 2020
Accepted: October 13, 2020
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Figure 1. Workflow of the CuAAC approach for target identification. During incubation, the chemical probe binds to its native targets inside the
cells. The cells are lysed and submitted to the CuAAC reaction to tag the chemical probe, which is covalently bound to its intracellular targets. After
pulldown purification, target proteins are analyzed.
Figure 2. Chemical structures of cinnamic acid, the flavonoid taxifolin, and 7-O-cinnamoyltaxifolin (7CT).
induced by oxidative stress.11 However, polyphenols as plant
secondary metabolites have also shown to be neuroprotective
by activating several intracellular pathways in addition to their
antioxidative features.12 The flavonoid fisetin, for example, was
shown to activate the Ras-extracellular signal-regulated kinase
(ERK) cascade and induced cAMP response element-binding
protein (CREB) phosphorylation in rat hippocampal slices and
therefore enhanced memory in mice.13 Recently, it has been
shown that the flavonoid taxifolin prevented neuroinflamma-
tion in a cerebral amyloid angiopathy mouse model by
suppressing the ApoE-ERK1/2-amyloid-β precursor protein
axis.14 Phenolic acid esters of the flavonolignan silibinin are
potent neuroprotectants and activate mouse microglia,15 and
esters of taxifolin upregulated the antioxidant response element
(ARE) via nuclear factor (erythroid-derived 2)-like 2 (Nrf2)
activation.16,17 Even though the antioxidant and anti-
inflammatory effects of these natural products are well-
established and have been shown to translate into in vivo AD
models where they ameliorated memory deficits,13,17 the
molecular mode of action and the exact intracellular targets of
these compounds remain largely elusive.
To identify cellular targets of compounds, a bioorthogonal
Cu(I)-catalyzed [3 + 2] azido-alkyne cycloaddition (CuAAC)
approach can be used.18−20 An alkyne tagged chemical probe
of the compound of interest undergoes covalent interactions
with its target proteins when incubated with cells (Figure 1).
The alkyne probe binds to its native targets and can then be
tagged with any azide in a CuAAC reaction. Depending on the
application, tags can be fluorescent dyes or other markers for
visualization and identification of target proteins. A widely
used tag is biotin, as the high affinity for streptavidin is
convenient for the enrichment of bound proteins and enables
characterization by MS analysis.19
In our previous work, we showed that the 7-O-ester of the
flavonoid taxifolin and cinnamic acid, namely, 7-O-cinnamoyl-
taxifolin (7CT), represented a highly neuroprotective com-
pound acting in an overadditive manner (Figure 2).17
Specifically, at the concentrations tested, the individual
components taxifolin and cinnamic acid or the equimolar
mixture of both were not protective, whereas the ester hybrid
7-O-cinnamoyltaxifolin displayed pronounced neuroprotective
activity. We observed the overadditive effect of 7-O-
cinnamoyltaxifolin in a set of assays inducing protection
against oxidative stress in neuronal HT22 cells but also
investigating inhibition of inflammatory processes in BV-2
microglial cells.17 Furthermore, the overadditive effect of 7-O-
cinnamoyltaxifolin was translated to animals using an Aβ25−35
-
induced memory-impaired AD mouse model where the
compound was able to ameliorate short-term memory
deficits.17 The significant and overadditive effect in vivo of 7-
O-cinnamoyltaxifolin showed that these natural product
hybrids can be considered as a class of neuroprotective
compounds with a distinct pharmacological profile. This is
supported by structurally closely related 7-O-silibinin esters,
which are sensitive to minor chemical modifications, indirectly
ruling out unspecific effects underlying these properties.15
However, to further develop the compound, it is indispensable
to understand its mechanism of action and determine its
intracellular targets.
The goal of this study was to design and synthesize a
chemical probe of 7-O-cinnamoyltaxifolin suitable for target
identification with the CuAAC approach. Previously, targets
for xanthumol, a phytochemical of hops,21 have been identified
using this approach. Furthermore, a functional probe for
flavonoid catabolites was developed by Nakashima et al. and
used in the CuAAC reaction.22 Here, we present 7-O-
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Figure 3. Chemical structures. (A) Design of the chemical probe without altering electrophilic moieties (encircled) of 7-O-cinnamoyltaxifolin. (B)
The chemical structure of 7CT-alkyne (1).
a
Scheme 1. Synthesis of 7CT-Alkyne (1), a 7-O-Cinnamoyltaxifolin-Based Chemical Probe, and the Control Compound (2)
a
(i) NaI, dry acetone, reflux, overnight; (ii) H2SO4, ethanol, reflux, 6 h; (iii) 3, K2CO3, dry acetone, 0 °C to reflux, overnight; (iv) NaOH, water,
ethanol, room temperature; (v) (1) Oxalyl chloride, DMF, dry THF, rt, 30 min; (2) Taxifolin, triethylamine, rt, overnight.
cinnamoyltaxifolin-alkyne (7CT-alkyne, 1, Figure 3B), a
derivative of 7-O-cinnamoyltaxifolin (7CT), functioning as a
chemical probe for intracellular target identification. Equivalent
effects in relevant assays of neuroprotection and neuro-
inflammation of 7CT-alkyne 1 compared to those of 7CT
proved the probes’ interaction with the targets of the parent
compound. Among others, ANT-1 and SERCA were identified
as targets of 1 and 7CT.
probe for target identification purposes. For 7-O-cinnamoyltax-
ifolin, its simultaneous influence on several proteins and
signaling pathways could be mediated through the Michael
acceptor sites at the cinnamoyl moiety and the catechol
residue, allowing covalent conjugation to proteins (Figure 3A).
This is supported by comprehensive structure−activity
relationship studies (SARs) on the respective silibinin
ester.15 The attack of a nucleophilic side chain, for example,
of cysteinyl thiolates, to the electrophilic β-carbon could be
one potential mechanism to form covalent adducts inducing
cellular responses.23 To preserve potential reactive sites in 7-O-
cinnamoyltaxifolin, the aromatic moiety of the acid was chosen
to introduce the alkyne tag necessary for CuAAC. In a previous
SAR study with phenolic acid esters of silibinin15 and also with
7-O-feruloyltaxifolin as a potent neuroprotectant,17 we showed
that this entity tolerates modification without loss of activity.
Therefore, 7CT-alkyne (1) (Figure 3B) was synthesized
starting from 3-hydroxy cinnamic acid (4). The carboxylic
acid moiety was protected using ethanol to selectively react
with 5-iodopentyne (3), which was synthesized via a
Finkelstein reaction, at the aromatic hydroxyl position.
Deprotection under basic conditions gave compound 7.
Regioselective esterification represents a considerable synthetic
RESULTS AND DISCUSSION
■
Aiming to identify intracellular targets of flavonoid esters and
investigate their intracellular mode of neuroprotection, the
chemical probe of 7-O-cinnamoyltaxifolin (7CT), compound
1, was designed and synthesized to be used in a CuAAC
reaction for affinity-based protein profiling. The suitability of 1
as a probe for 7CT was investigated in cell-based phenotypic
screening assays and Western blots. Microscopic analysis of
Cy3-coupled 1 suggested mitochondria as one of the
subcellular locations of internalized 1, which was supported
by the identification of the mitochondrial ATP/ADP carrier
ANT-1 in MS analysis of the pulldown.
Chemistry. It is of utmost importance not to alter the
mode of action of the compound when designing a chemical
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Figure 4. Phenotypic screening assays in HT22 hippocampal nerve cells and neuroinflammation in BV-2 cells. (A) Toxicity of the compounds
7CT, 7CT-alkyne (1), and 7CT-alkane (2). (B) Neuroprotective effects of 7CT, 1, and 2 against oxytosis. Glutamate (5 mM) was used to induce
toxicity (red). (C) Neuroprotection of 7CT, 1, and 2 against ferroptosis induced by 0.3 μM RSL3 (red). (D) Neuroprotective effects of 7CT, 1,
and 2 against ATP depletion. ATP loss was induced with 20 μM iodoacetic acid (IAA, red). Data are presented as means SEM of three
independent experiments, and results refer to untreated control cells (black). Statistical analysis was performed using One-way ANOVA followed
by Dunnett’s multiple comparison post-tests using GraphPad Prism 5 referring to untreated controls in part A or cells treated with the respective
insult only in parts B, C, and D (red bars). Levels of significance: * p < 0.05; **p < 0.01; ***p < 0.001. (E) Effects of 7CT, 1, and 2 on NO
production in LPS-induced neuroinflammation in BV-2 microglial cells. Cells were treated overnight with 50 ng/mL LPS alone or in the presence
of 7CT, 1, or 2. Supernatants were cleared, and NO was quantified by the Griess assay. Data are given as means SEM and relative to BV-2 cells
treated with LPS only, which was set as 100%.
challenge due to the minor differences in reactivity of the
various hydroxyl groups in flavonoids. It can involve complex
sequences of full protection, regioselective deprotection,
esterification, and full deprotection. We previously described
direct regioselective esterification for both the flavonolignan
silibinin and taxifolin.15,17 Therefore, regioselective esterifica-
tion with taxifolin was achieved by forming the acid chloride of
compound 7 with oxalyl chloride. In situ esterification under
basic conditions with adjusted reaction times, as described
before,15 and extensive column purification gave the target
compound 7CT-alkyne (1). 7CT-alkane (2) was synthesized
as a control to compare the influence of an aliphatic
modification in the aromatic position on the neuroprotective
performance of the compound and served as a negative control
in the CuAAC reaction. The synthetic route for compound 2
was analogous to that of compound 1, replacing 5-iodopentyne
(3) with 1-iodopentane in step iii (Scheme 1).
Cell Culture Assays to Confirm a Comparable Mode
of Action. To validate the chemical probe, compounds 1 and
2 were subjected to phenotypic screening assays addressing
different age-related stresses, as age is the major risk factor of
neurodegenerative diseases.6 Activities of the chemical probe 1
and control compound 2 were investigated in the three
different assays oxytosis, ferroptosis, and ATP depletion in the
murine hippocampal cell line HT22. Oxytosis is a form of
programmed cell death due to oxidative stress induced by
intracellular glutathione (GSH) depletion due to glutamate
treatment.24 As GSH reduction is seen in the aging brain and is
accelerated in AD, this assay has a mechanistic association with
aging and AD.25 Closely related to oxytosis, if not identical, is
the cell death pathway ferroptosis.26 Here, oxidative stress is
induced by direct inhibition of glutathione peroxidase 4
(GPX4) with the compound RSL3. Distinct from oxytosis,
which is induced by glutamate inhibiting cysteine import by
blocking system xc−, ferroptosis is induced downstream in the
cascade. ATP depletion is of interest, as the breakdown in
neuronal energy production leads to decreased energy
metabolism and ATP levels in the aging brain, which is
associated with nerve cell damage and death in AD.27 To
induce ATP loss, HT22 cells were treated with iodoacetic acid
(IAA), an irreversible inhibitor of the glycolytic enzyme
glyceraldehyde 3-phosphate dehydrogenase.
Figure 4 shows the activity of the chemical probe 1 and the
control compound 2 compared to that of the lead compound
7-O-cinnamoyltaxifolin (7CT). Even though there is a slight
increase in toxicity by the introduction of the aliphatic moiety
(Figure 4A), compound 1 proved to be neuroprotective
throughout all assays, as was control compound 2. At a
concentration of 5 μM, compound 1 was as protective as 7CT
in the phenotypic screening assays for protection against
oxytosis (Figure 4B), ferroptosis (Figure 4C), and ATP
depletion (Figure 4D) proving that the compound is active
and therefore suitable for use as a chemical probe for target
identification.
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Furthermore, we were also interested in whether compound
1 could be effective against neuroinflammation. As shown in
Figure 4E, bacterial lipopolysaccharide (LPS)-induced inflam-
mation in BV-2 mouse microglial cells was reduced by
compounds 1 and 2. The production of nitric oxide (NO)
as a proinflammatory mediator was quantified to assess
inflammation. At 10 μM, the highest concentration tested,
NO levels are reduced to around 45% compared to those of
LPS treatment alone, confirming the anti-neuroinflammatory
effect of 1 and 2.
The results of the phenotypic screening assays and
microglial activation showed that compound 1 was protective
in all assays tested and was not altered in its activity compared
to that of 7CT, which was of utmost importance for using 1 as
a chemical probe. Compound 1 exhibited neuroprotection
addressing different characteristics of neurodegeneration and
aging in oxytosis, ferroptosis, ATP depletion, and neuro-
inflammation at low micromolar concentrations.
Intracellular Pathways Are Modified by Compound 1.
The nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is an
important transcription factor regulating many antioxidant and
detoxification enzyme genes. It is the main regulator of the
antioxidant response element (ARE) and is crucial for
maintaining the cellular redox balance by preventing the
accumulation of reactive oxygen species (ROS) and oxidative
stress, one of the hallmarks of AD.28 It has been shown that a
taxifolin ester with gallic acid upregulated Nrf2 pathway in
RAW264.7 cells,16 and we previously found 7CT to induce
Nrf2 activation in microglial BV-2 cells.17 Therefore, we asked
whether compound 1 also modifies Nrf2 levels. Nuclear
fractions of HT22 cells treated with compound 1 and 7CT for
comparison were submitted to Western blot analysis. Figure 5
shows that compound 1 significantly induced Nrf2 upregula-
tion. Even though 7CT seemed more potent at higher
concentrations and led to nearly a 6-fold induction of Nrf2
levels at 10 μM, increased Nrf2 levels were significant for both
compounds at the same concentration of 5 μM (Figure 5B).
Upregulation of Nrf2 is, therefore, one potential mechanism
explaining the neuroprotective activity of the compounds. The
classical mechanism for Nrf2 activation is by its interaction
with Keap1. When bound to Keap1, Nrf2 is directed for
proteasomal degradation. Modification on cysteine residues of
Keap1, by oxidative stress, for example, leads to dissociation of
the complex and translocation of Nrf2 to the nucleus where it
activates several cellular responses including the ARE.
However, phosphorylation of Nrf2 by various protein kinases
can be an alternative mechanism of Nrf2 regulation.29 It is
known that extracellular signal-regulated protein kinase (ERK)
can play a role in Nrf2 activation, and levels of ERK
phosphorylation are modified by natural products.30 Fur-
thermore, ERK signaling is involved in cell proliferation,
differentiation, and survival or promotion of cell death and
therefore can be involved in neurodegeneration.31 We were
interested in whether ERK is activated by compound 1 and
7CT in HT22 cells, as modifications of mitogen-activated
protein (MAP) kinases have not been investigated for 7-O-
esters of taxifolin before. Figure 6 shows a time-course of
HT22 cells incubated with 10 μM 1 or 7CT. Both compounds
lead to a significant increase in ERK phosphorylation and
thereby activation after 5 min (Figure 6B). The effect ceased
with increased incubation times thereby excluding chronic
ERK activation, which is correlated with neurodegeneration.32
Another MAP kinase important in the context of AD is p38.
Figure 5. Western blot analysis of Nrf2. Nrf2 induction by 7CT and 1
in nuclear fractions of HT22 cells. (A) Cells were treated for 4 h with
DMSO as control or with increasing concentrations of 1 or 7CT.
Nuclear fractions of HT22 cells were prepared and analyzed by
Western blot for Nrf2. Levels of Nrf2 were normalized to actin, and
representative blots are shown. (B) Quantification of the results from
three independent experiments as shown in part A. Statistical
significance refers to DMSO-treated controls with * p < 0.05.
Postmortem brains of early stage AD patients showed
increased phosphorylation of p38 which is connected to
mediating Aβ-induced inflammation and impaired autophagy
in neurons leading to neurodegeneration.33−35 At a concen-
tration of 10 μM, compound 1 and 7CT both reduce p38
phosphorylation (Figure 6C). Decreased activation was
observed after 5 min of incubation and was significantly
reduced for the whole time-course of 2 h. The results for
activation of ERK and deactivation of p38 underline that 7CT
and compound 1 modify MAP kinases. Thus, these results
strengthen the reliability of compound 1 as suitable bait for
target identification.
CuAAC with Cy3-Azide. The introduction of the alkyne
tag in compound 1 enables the reaction of the compound with
different azide carriers. As a proof of concept for the CuAAC
reaction, and to visualize cellular uptake and the intracellular
localization of compound 1, we coupled 1 to an azide
conjugated fluorophore Cy3. First, adducts of the chemical
probe 1 with proteins of HT22 lysates were visualized by
Western blot analysis. Cy3-azide was coupled to compound 1
in a CuAAC reaction, and bands were detected with an anti-
Cy3 antibody. HT22 cells were incubated with increasing
concentrations of 5−80 μM 1 for 4 h, lysed, and submitted to
the CuAAC reaction using 20 μM Cy3-azide. Incubation of
HT22 cells with 1 led to the conjugation of a wide range of
proteins with increasing intensity which correlated with
increasing concentrations of the compound (see Supporting
Information (SI), Figure S1). In cells incubated with DMSO,
no adducts were detected. This was also the case for
incubation with compound 2 lacking the alkyne tag (SI,
Figure S2). Therefore, Cy3-coupling and the detection of
conjugated intracellular proteins bound to the probe 1 was
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microscopy using an Airyscan detector.42 As shown in Figure
7, compound 1 is found at several locations after 30 min of
incubation, including in unknown structures in the perinuclear
region. Importantly, the fluorescence profiles of mito-GFP and
Cy3 indicate that compound 1 is also strongly enriched in
mitochondria after 30 min of incubation (see graphs on the
right of Figure 7). No significant signal for Cy3 was detected in
control cells treated with DMSO. These results indicate that
the interaction partners of compound 1 and 7CT might be
associated with mitochondria.
Affinity Pulldown and MS Analysis. After demonstrating
that compound 1 is suitable as a chemical probe for 7CT, due
to pronounced neuroprotection by the same mode of action,
the chemical probe 1 was applied for target identification in an
affinity pulldown with HT22 cells. HT22 cells were incubated
with 200 μM of compound 1 or DMSO as a control for 2 h
before lysis and the CuAAC reaction with biotin-azide.
Proteins bound by 1 and the DMSO-control lysates were
purified on streptavidin magnetic beads, separated by SDS−
PAGE, and analyzed by nanoLC−MS/MS after tryptic
digestion in a label-free quantification. A total of 708 proteins
were identified and quantified. Of these, 70 were significantly
enriched in both replicates and thus classified as potential
target candidates (summed significance 4; Figure 8). Two of
the 70 hits were pursued further to investigate the interaction
between compound 1 and 7CT and the respective identified
proteins. One was the sarco/endoplasmic reticulum Ca2+-
ATPase (SERCA, Atp2a2), which was the protein with the
highest significance in the log2 protein ratio sample/control
and therefore the top target identified according to MS analysis
(Figure 8). As the microscopic analysis showed localization of
compound 1 in mitochondria, we were interested in
mitochondrial proteins among the targets. Indeed, the
mitochondrial carrier adenine nucleotide translocase 1
(ANT-1, Slc25a4) was among the top 4 most significant hits
and was chosen as a second target for further examination.
ANT-1 and SERCA Are Identified in MS Analysis.
Adenine nucleotide translocase-1 (ANT-1) was identified as a
potential target in MS/MS analysis. This finding is supported
by the microscopic analysis conducted, where colocalization of
Cy3-labeled 1 with mitochondria was observed (Figure 7).
ANT-1 is an ADP/ATP carrier at the inner mitochondrial
membrane and the most abundant protein in mitochondria,
contributing to 1−10% of total mitochondrial protein.43 ANT-
1 is the muscle and brain-specific isoform of ANT and is
important for mitochondrial physiology as well as general cell
function, as it transports ADP into the mitochondrial matrix
and ATP into the cytoplasm, undergoing extensive conforma-
tional changes during transport.44−46 ANT-1 is relevant as a
potential pharmacological target in the context of neuro-
degenerative disorders, particularly for AD, as mitochondrial
dysfunction is proposed to be one of the major hallmarks of
the disease.47,48 ANT-1 has been studied in the context of
apoptosis as well. The carrier was characterized as a
proapoptotic protein, as increased ATP export, which is
important for apoptosis, is mediated by enhanced levels of
ANT-1 leading to mitochondrial breakdown and the apoptotic
cascade.45,49,50 Its role in nonapoptotic forms of cell death, like
ferroptosis and oxytosis covered in this work, has not been
described so far. Therefore, HT22 cells transfected with ANT-
1 siRNA were tested for effects on oxytosis and ferroptosis.
ANT-1 knockdown protected against these insults. Signifi-
cantly more cells survived treatment with glutamate as an
Figure 6. Time-dependent modification of MAP kinases by 7CT and
1 in HT22 cells. (A) Cells were treated for the indicated time points
with 10 μM 1 or 7CT. DMSO treatment served as a control. Lysates
of HT22 cells were prepared in sample buffer and analyzed by
Western blot for phosphorylated ERK (P-ERK), total ERK (ERK),
phosphorylated p38 (P-p38), or total p38 (p38). Levels of the
phosphorylated protein were normalized to those of the total protein,
and representative blots are shown. (B) Quantification of the results
from three independent experiments, as shown in part A. Statistical
significance refers to DMSO-treated controls with * p < 0.05, ** p <
0.01.
dependent on the presence of the alkyne moiety and not due
to unspecific binding.
Microscopic Analysis. Mitochondria play a crucial role in
apoptosis,36 and in AD, neuronal cell death is associated with
mitochondrial dysfunction.37 Although the exact role of
mitochondria in ferroptosis is under debate, increasing
evidence strongly suggests that these organelles can also play
a key role in this cell death pathway.38−41 To determine
whether compound 1 is targeting the mitochondria, we used
fluorescence microscopy to analyze its intracellular location.
Therefore, HT22 cells stably expressing the green fluorescent
protein in mitochondria (mito-GFP) were incubated with 5
μM compound 1 or DMSO for 30 min, and after fixation, the
cells were reacted with Cy3-azide in a CuAAC reaction. The
specimens were then imaged by high-resolution fluorescence
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Figure 7. Microscopic analysis of HT22-mitoGFP cells with compound 1. Representative microscopic images of HT22-mitoGFP cells incubated
with 5 μM 1 or DMSO for 30 min. Red signals derive from Cy3 and correspond to adducted proteins; green signals are mitochondria-targeted
GFP. Arrows indicate mitochondrial structures to note the colocalization of Cy3 staining with mitochondria in cells incubated with compound 1
compared to the absence of a signal in DMSO-treated cells. To visualize the colocalization, a line was plotted in the merged images (magnified
images). Charts on the right show quantification of the fluorescence profile along this line for each channel. The X-axis indicates distance, and the
Y-axis represents fluorescence intensity in arbitrary units. Bars = 10 μm.
inducer of oxytosis (Figure 9A), or erastin and RSL3 as
inducers of ferroptosis (Figure 9B,C, respectively). These
findings strongly support the implication of ANT-1 inhibition
in the protective effect of compound 1 and consequently 7CT.
The treatment of ANT-1 knockdown cells with 7CT in
oxytosis conditions induced by glutamate showed that the
compound provides additional protection (Figure 9E). In the
absence of 7CT, the knockdown of ANT-1 is significantly
protective. However, upon treatment of 7CT, the control cells
are as protected as the ANT-1 knockdown cells indicating that
ANT-1 is not the exclusive target of 7CT, and other
mechanisms are also involved in the protection (Figure 9E).
ROS and oxidative stress lead to increased mitochondrial
membrane potential (MMP).51 It was shown that FCCP, a
mitochondrial uncoupler dissipating the MMP, also protects
cells from oxytosis.52 Furthermore, a study with quercetin
showed that low concentrations of the flavonoid reduced the
MMP and inhibited adenine nucleotide exchange by ANT-1.53
As the concentrations at which neuroprotection is observed in
our study are in the low micromolar range, we hypothesize that
1 and 7CT could act as mild uncouplers and inhibitors of
ANT-1 and thereby protect cells. It has been reported that the
nucleotide exchange by ANT-1 can potentially play a role in
maintaining the MMP,54,55 but increased oxidative damage by
apoptosis inducers leads to harmfully increased expression
levels of ANT-1.56,57 However, whether this is also the case for
oxytosis and ferroptosis as well as the exact role of ANT-1 in
these nonapoptotic pathways remains a subject of future
research.
A second interaction partner identified by MS analysis was
the sarco/endoplasmic reticulum Ca2+-ATPase 2 (SERCA).
Located in the endoplasmic reticulum (ER), the pump
regulates calcium influx into the ER under ATP consumption.
ER-stress concomitant with calcium dysregulation causes
neuronal impairment and death and is involved in the
progressive neurological decline of AD.58 Krajnak et al. showed
that activation of SERCA is neuroprotective and improves
memory and cognition in APP/PS1 mice.59 In this work,
SERCA was validated as a target for 7-O-esters of taxifolin by
knockdown experiments. In contrast to the results with ANT-
1, HT22 cells treated with SERCA siRNA were not protected
against glutamate-induced oxytosis (Figure 9D). However,
G
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Figure 8. Mass spectrometric identification of target candidates. Log2 transformed protein ratios sample/control of two replicates are shown.
Summed significance values were used to identify the best target candidates (significance 2 in both replicates = significance 4, marked in red). The
size of the dots corresponds to the number of razors and unique peptides of a protein.
SERCA knockdown significantly impaired the protection by
compound 7CT at the lower concentrations of 2.5 and 5 μM
(Figure 9E) suggesting an important role of SERCA in the
protective mechanism of 7CT. At a concentration of 10 μM
7CT, the decrease in protection by the absence of SERCA was
no longer seen. Higher concentrations of 7CT likely activate
other protective pathways due to the pleiotropic action of the
compound and hence compensate for SERCA knockdown.
cells and localization in mitochondria. MS analysis after
CuAAC of 1 with biotin-azide and following streptavidin
purification, revealed 70 potential target candidates by MS
analysis. Due to the pleiotropic mechanism of action, the
identification of many interaction partners was assumed. As
compound 1 carries a Michael system, which is prone to react
with nucleophiles like thiols, we expected to identify proteins
that were previously described to interact with electrophiles.21
HSP90, prohibitin, and 14-3-3β are examples of such proteins.
Indeed, HSP90, prohibitin and 14-3-3β were identified among
others in the MS analysis of the pulldown with compound 1
(for the full list of targets, see Supporting Information).
However, the top target candidate of compound 1 in MS
analysis was SERCA. In addition, because microscopic analysis
pointed at mitochondria as a subcellular location for
compound 1, we also looked at the top mitochondrial target
candidate in the analysis which was the mitochondrial protein
ANT-1. These two proteins were chosen to investigate for
their relevance as targets of compound 1 and 7CT in
knockdown experiments. As both proteins are crucial for cell
homeostasis and survival and are for the first time shown to be
modified by 7-O-esters of flavonoids, further work in this area
should focus on the role of ANT-1 and SERCA in AD and
oxytotic/ferroptotic cell death pathways as well as the
CONCLUSION
■
In summary, a chemical probe of the neuroprotective natural
product hybrid 7-O-cinnamoyltaxifolin bearing an alkyne tag
for CuAAC reaction was designed and synthesized. The probe
1 proved its suitability as a neuroprotectant in cell-based
phenotypic screening assays addressing different aspects of
neurodegeneration and aging. On a molecular level, Western
blots were used to confirm that the parent compound and the
probe act on the same pathways and to ensure that the alkyne
tag was not altering the mechanisms of action. Both
compounds, 1 and 7CT, induced significant upregulation of
Nrf2 in HT22 nuclear fractions, increased ERK phosphor-
ylation, and reduced phosphorylation of p38 in HT22 lysates.
Microscopic analysis of 1 coupled to the fluorescent dye Cy3-
azide showed efficient uptake of the compound into HT22
H
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Figure 9. Transfection of HT22 cells with ANT-1 siRNA and SERCA siRNA and investigations on neuroprotection. ANT-1 knockdown leads to
protection against glutamate as an inducer of oxytosis (A), and erastin (B) and RSL3 (C) as inducers of ferroptosis. (D) The transfection of HT22
cells with SERCA siRNA is not protective against glutamate-induced oxytosis. (E) ANT-1 (dark gray) and SERCA (light gray) knockdown cells
were treated with increasing concentrations of 7CT in the presence of glutamate. Data are presented as means SEM of four independent
experiments. Statistical analysis was rendered using Two-way ANOVA followed by Bonferroni posttests using GraphPad Prism 5. Levels of
significance: * p < 0.05; ***p < 0.001.
commercial suppliers in reagent grade. For anhydrous reaction
conditions, THF was dried before use by refluxing over sodium slices
for at least 2 days under an argon atmosphere. Thin-layer
chromatography was performed on silica gel 60 (alumina foils with
fluorescent indicator 254 nm). UV light (254 and 366 nm) was used
for detection. For column chromatography, silica gel 60 (particle size
0.040−0.063 mm) was used. Nuclear magnetic resonance (NMR)
spectra were recorded with a Bruker AV-400 NMR instrument
(Bruker, Karlsruhe, Germany) in a deuterated solvent. Chemical shifts
are expressed in parts per million (ppm) relative to the solvent applied
(2.50 ppm for 1H and 39.5 ppm for 13C in DMSO-d6; 7.26 ppm for
1H and 77.2 ppm for 13C for CDCl3; 2.05 ppm for 1H and 206.3
ppm for 13C for acetone-d6; 4.87 ppm for 1H and 49.0 ppm for 13C
for methanol-d4). The purity of the synthesis products was
identification and functional characterization of the site(s)
modified by the compound. Our findings are a step toward
understanding the specific interaction partners of natural
product-derived compounds like 7-O-esters of flavonoids. The
activity-based protein profiling approach with compound 1 as a
chemical probe revealed several proteins as interaction partners
that might also apply to other flavonoids and lead to new
approaches for drug development against neurodegenerative
diseases like AD.
METHODS
■
Chemical Synthesis. General Information. All reagents were
used without further purification and bought from common
I
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determined by HPLC (Shimadzu Products), containing a DGU-
20A3R degassing unit, an LC20AB liquid chromatograph, and an
SPD-20A UV/vis detector. UV detection was measured at 254 nm.
Mass spectra were obtained by an LCMS 2020 (Shimadzu Products).
As a stationary phase, a Synergi 4U fusion-RP (150 mm × 4.6 mm)
column was used, and as a mobile phase, a gradient of methanol/
water with 0.1% formic acid was used. Parameters: A = water, B =
methanol, V(B)/(V(A) + V(B)) = from 5 to 90% over 10 min, V(B)/
(V(A) + V(B)) = 90% for 5 min, V(B)/(V(A) + V(B)) = from 90 to
5% over 3 min. The method was performed with a flow rate of 1.0
mL/min. Compounds were only used for biological evaluation if the
purity was ≥95%. Melting points were determined using an OptiMelt
automated melting point system (Scientific Instruments GmbH,
Gilching, Germany).
General Procedure A: Protection. Acid (1.0 equiv) was
dissolved in ethanol, and H2SO4 was added. The solution was
refluxed until TLC showed full conversion. The reaction mixture was
concentrated in vacuo and dissolved in ethyl acetate to be washed with
5% sodium hydrogen carbonate in water. The organic layers were
combined, and the solvent was evaporated.
General Procedure B: Substitution. K2CO3 (1.5 equiv) was
dissolved in dry acetone under argon and cooled to 0 °C in an ice
bath when the protected acid (1.0 equiv) was added. The mixture was
stirred at 0 °C for 15 min, and the respective iodine (2.0 equiv) was
added. The reaction was heated to reflux for 6 h, diluted with water
and 1 M HCl, extracted with ethyl acetate (3 × 30 mL), washed with
brine, and dried over Na2SO4. The solvent was removed, and the
compound was purified by silica column chromatography using 20%
ethyl acetate in petroleum ether to give the compound as an oil.
General Procedure C: Deprotection. The compound was
dissolved in ethanol, and 0.52 M NaOH was added. The mixture was
stirred at room temperature for 5 h. The reaction was diluted with 5
mL of water and extracted with ethyl acetate twice. The aqueous
layers were combined and acidified to pH 1 with 1 M HCl to be again
extracted with ethyl acetate. The organic layers were combined,
washed with brine, and dried over Na2SO4.
General Procedure D: Esterification. Acid (1.1 equiv) was
dissolved in 5 mL of dry THF, and oxalyl chloride (2.0 equiv)
together with catalytic amounts of DMF (4 μL) were added. The
reaction was stirred at room temperature until TLC showed complete
conversion of the acid to the acid chloride. The acid chloride was then
added dropwise to a solution of taxifolin (1.0 equiv) and triethylamine
(4.5 equiv) in dry THF and stirred at room temperature for 2 h. The
reaction was quenched with water and 1 M HCl and extracted with
ethyl acetate. The organic layer was combined, washed with brine, and
dried over Na2SO4. The mixture was purified by flash column
chromatography using a gradient of 4−15% acetone in dichloro-
methane to give the pure compound.15,17
13C NMR (101 MHz, acetone-d6): δ 167.0, 158.7, 145.2, 136.8,
130.9, 120.5, 119.10, 118.3, 115.4, 60.8, 14.6 ppm.
Ethyl (E)-3-(3-(Pent-4-yn-1-yloxy)phenyl)acrylate (6). Following
general procedure B, 5 (300 mg, 1.5 mmol, 1.0 equiv) was reacted
with 3 (605 mg, 3.12 mmol, 2.0 equiv) and K2CO3(311 mg, 2.25
mmol, 1.5 equiv). Compound 6 was obtained as a yellow oil (352 mg,
1.36 mmol, 91% yield).
1H NMR (400 MHz, CDCl3): δ 7.64 (d, J = 16.0 Hz, 1H, HC
CHCO), 7.27 (t, 1H, arom), 7.11 (m, 1H, arom), 7.05 (m, 1H,
arom), 6.93−6.91 (m, 1H, arom), 6.41 (d, J = 16.0 Hz, 1H, HC
CHCO), 4.26 (quart, 2H, OCH2CH3), 4.08 (t, 2H, H
CCH2CH2CH2O), 2.40 (dt, 2H, HCCH2CH2CH2O), 2.03−1.97
(m, 3H, HCCH2CH2CH2O), 1.33 (t, 3H, OCH2CH3) ppm.
13C NMR (101 MHz, CDCl3): δ 167.0, 159.3, 144.6, 135.9, 130.0,
120.9, 118.7, 116.8, 113.6, 83.4, 69.1, 66.3, 60.6, 28.2, 15.2, 14.4 ppm.
(E)-3-(3-(Pent-4-yn-1-yloxy)phenyl)acrylic Acid (7). Following
general procedure C, 290 mg 6 was dissolved in 3 mL of ethanol,
and 5 mL of 0.52 M NaOH was added. The compound was obtained
as a white solid (191 mg, 0.82 mmol, 73% yield).
1H NMR (400 MHz, CDCl3): δ 7.74 (d, J = 16.0 Hz, 1H, HC
CHCO), 7.31 (t, 1H, arom), 7.15 (d, J = 8.0 Hz 1H, arom), 7.08 (m,
1H, arom), 6.97 (dd, J = 8.0 and 2.3 Hz, 1H, arom), 6.46 (d, J = 16.0
Hz, 1H, HCCHCO), 4.11 (t, 2H, HCCH2CH2CH2O), 2.42 (td,
2H, HCCH2 CH2 CH2 O), 2.06−1.98 (m, 3H, H
CCH2CH2CH2O) ppm.
13C NMR (101 MHz, CDCl3): δ 172.0, 159.3, 147.0, 135.4, 130.0,
121.2, 117.5, 117.2, 113.8, 83.3, 69.0, 66.3, 28.1, 15.2 ppm.
7-O-Cinnamoyltaxifolin-pentyne (1). Following general procedure
D, 7 (120 mg, 0.52 mmol, 1.1 equiv) was dissolved in 5 mL of dry
THF, and 85 μL of oxalyl chloride (127 mg, 1.00 mmol, 2 equiv) and
catalytic amounts of DMF were added. Taxifolin (150 mg, 0.52 mmol,
1 equiv) and triethylamine (228 mg, 2.25 mmol, 4.5 equiv) were
dissolved in dry THF, and the acid chloride was added dropwise.
Target compound 1 was obtained as a white foam (137 mg, 0.26
mmol, 53% yield).
1H NMR (400 MHz, DMSO-d6): δ 11.73 (s, 1H, 5-OH), 9.03 (s,
2H, 3′ and 4′-OH), 7.80 (d, J = 16.0 Hz, 1H, ArCHCH−CO), 7.40
(s, 1H, arom CA), 7.35 (d, J = 5.1 Hz, 2H, arom CA), 7.05−7.02 (m,
1H, arom CA), 6.93 (m, 1H, arom B-ring), 6.88 (d, J = 16.0 Hz, 1H,
ArCHCH−CO), 6.79−6.76 (m, 2H, arom B-ring), 6.45 (d, J = 2.0
Hz, 1H, 8-H), 6.42 (d, J = 2.0 Hz, 1H, 6-H), 5.92 (d, J = 6.2 Hz, 1H,
3-OH), 5.15 (d, J = 11.6 Hz, 1H, 2-H), 4.70 (dd, J = 11.5, 6.3 Hz, 1H,
3-H), 4.09 (t, 2H, HCC−CH2−CH2−CH2), 2.80 (t, 1H, HCC−
CH2−CH2−CH2), 2.34 (td, J = 2.6 Hz, 2H, HCC−CH2−CH2−
CH2), 1.90 (tt, J = J′ = 6.5 Hz, 2H, HCC−CH2−CH2−CH2) ppm.
13C NMR (101 MHz, DMSO-d6): δ 199.6, 164.0, 162.0, 161.9,
158.9, 158.1, 147.2, 146.0, 145.0, 135.2, 130.1, 127.7, 121.5, 119.6,
117.7, 117.1, 115.5, 115.2, 113.8, 104.9, 102.8, 101.7, 83.7, 83.3, 71.9,
71.6, 66.2, 27.7, 14.5 ppm.
5-Iodo Pentyne (3). 5-Chloro pentyne (1.0 g, 9.76 mmol, 1.0
equiv) was dissolved in dry acetone, and sodium iodine (7.3 g, 48.8
mmol, 5.0 equiv) was added; the mixture was heated to reflux
overnight. The reaction was diluted with water and extracted with
dichloromethane (3 × 40 mL). The organic layer was combined,
washed with brine, and dried over Na2SO4, and the solvent was
evaporated. The compound was obtained as a yellow oil (1.8 g,
quantitative (quant)).
ESI: m/z calculated for C29H24O9 [M + H]+ 517.14, found 517.13;
HPLC purity = 99%.
Ethyl (E)-3-(3-(Pentyloxy)phenyl)acrylate (8). Following general
procedure B, 5 (500 mg, 2.6 mmol, 1.0 equiv) was reacted with 1-
iodopentane (1.03 g, 5.2 mmol, 2.0 equiv) and K2CO3(539 mg, 3.9
mmol, 1.5 equiv). Compound 8 was obtained as an off-white oil (330
mg, 1.26 mmol, 48% yield).
1H NMR (400 MHz, CDCl3): δ 3.28 (td, J = 6.7, 1.3 Hz, 2H),
2.34−2.26 (m, 2H), 2.01−1.92 (m, 3H) ppm.
1H NMR (400 MHz, CDCl3): δ 7.87 (d, J = 16.0 Hz, 1H), 7.49 (t,
1H), 7.30 (d, J = 7.9 Hz, 1H), 7.13 (dd, J = 7.9, 2.4 Hz, 1H), 6.64 (d,
J = 16.0 Hz, 1H), 4.48 (q, J = 7.1 Hz, 2H), 4.18 (t, 2H), 2.01 (quint,
2H), 1.72−1.59 (m, 4H), 1.56 (t, J = 7.1 Hz, 3H), 1.21−1.11 (m,
3H) ppm.
13C NMR (101 MHz, CDCl3): δ 82.4 (s), 69.6 (s), 32.0 (s), 19.6
(s), 5.2 (s) ppm.
Ethyl (E)-3-(3-Hydroxyphenyl)acrylate (5). Following general
procedure A, 500 mg of trans-3-hydroxycinnamic acid was dissolved
in 15 mL of ethanol, and 5 drops of sulfuric acid were added. After 6
h, TLC showed total consumption of the educt. Compound 5 was
obtained as an off-white solid (584 mg, quant).
13C NMR (101 MHz, CDCl3): δ 167.0, 159.5, 144.6, 135.8, 129.8,
120.6, 118.4, 116.7, 113.5, 68.1, 60.5, 28.9, 28.2, 22.5, 14.3, 14.0 ppm.
(E)-3-(3-(Pentyloxy)phenyl)acrylic Acid (9). Following general
procedure C, 250 mg of 8 was dissolved in 5 mL of ethanol, and 5
mL of 0.52 M NaOH was added. The compound was obtained as a
white solid (173 mg, 1.35 mmol, quant).
1H NMR (400 MHz, acetone-d6): δ 8.54 (s, 1H, −OH), 7.60 (d, J
= 16.0 Hz, 1H, HCCHCO), 7.26 (t, 1H, aromatic (arom)), 7.15−
7.11 (m, 2H, arom), 6.93−6.90 (m, 1H, arom), 6.43 (d, J = 16.0 Hz,
1H, HCCHCO), 4.20 (quart, 2H, OCH2CH3), 1.28 (t, 3H,
OCH2CH3) ppm.
1H NMR (400 MHz, CDCl3): δ 7.76 (d, J = 15.9 Hz, 1H), 7.31 (t,
J = 7.9 Hz, 1H), 7.13 (d, J = 7.7 Hz, 1H), 7.07 (s, 1H), 6.96 (dd, J =
8.2, 2.4 Hz, 1H), 6.44 (d, J = 15.9 Hz, 1H), 3.98 (t, J = 6.6 Hz, 2H),
J
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1.86−1.75 (m, 2H), 1.54−1.33 (m, 4H), 0.95 (t, J = 7.1 Hz, 3H)
ppm.
together with 1, 5, or 10 μM concentrations of the respective
compound for protection. After 24 h, cell viability was determined
using the MTT assay. Results are presented as percentages of
untreated control cells. Data are expressed as means SEM of three
independent experiments. The analysis was accomplished using
GraphPad Prism 5 Software applying One-way ANOVA followed
by Dunnett’s multiple comparison posttest. Levels of significance: * p
< 0.05; ** p < 0.01; *** p < 0.001.
13C NMR (101 MHz, CDCl3): δ 172.3, 159.7, 147.3, 135.5, 130.1,
121.1, 117.5, 117.4, 113.9, 68.3, 29.1, 28.3, 22.6, 14.2 ppm.
7-O-Cinnamoyltaxifolin-pentane (2). Following general proce-
dure D, 9 (134 mg, 0.57 mmol, 1.1 equiv) was dissolved in 5 mL of
dry THF, and 100 μL of oxalyl chloride (155 mg, 1.08 mmol, 2 equiv)
and catalytic amounts of DMF were added. Taxifolin (164 mg, 0.54
mmol, 1 equiv) and triethylamine (278 mg, 2.75 mmol, 4.5 equiv)
were dissolved in dry THF, and the acid chloride was added dropwise.
Compound 2 was obtained as a white foam (181 mg, 0.35 mmol, 64%
yield).
ATP Depletion in HT22 Cells. Cells (3 × 103 cells per well) were
seeded into sterile 96-well plates and incubated overnight. The next
day, medium was exchanged with fresh medium. Iodoacetic acid
(IAA, 20 μM) was added with vehicle (DMSO) as a negative control,
or together with 1, 5, or 10 μM concentrations of the respective
compound for protection. After 2 h of incubation at 37 °C in the
incubator, the medium was aspirated, and fresh medium was applied;
only the compounds at the same respective concentrations were
added without IAA. After 24 h, cell viability was determined using the
MTT assay. Results are presented as percentages of untreated control
1H NMR (400 MHz, DMSO-d6): δ 11.73 (s, 1H, 5-OH), 9.06 (s,
1H, 3′-OH), 9.00 (s, 1H, 4′-OH), 7.82 (d, J = 16.0 Hz, 1H, ArCH
CH−CO), 7.38 (s, 1H, arom H CA), 7.34−7.33 (m, 2H, arom H
CA), 7.03−7.00 (m, 1H, arom H CA), 6.93 (m, 1H, arom B-ring),
6.88 (d, J = 16.0 Hz, 1H, ArCH = CH−CO), 6.79−6.74 (m, 2H,
arom B-ring), 6.45 (d, J = 2.0 Hz, 1H, 8-H), 6.41 (d, J = 2.0 Hz, 1H,
6-H), 5.90 (d, J = 6.3 Hz, 1H, 3-OH), 5.13 (d, J = 11.6 Hz, 1H, 2-H),
cells. Data are expressed as means
SEM of three independent
4.69 (dd,
J
=
11.6, 6.3 Hz, 1H, 3-H), 4.01 (t, 2H,
experiments. The analysis was accomplished using GraphPad Prism 5
Software applying One-way ANOVA followed by Dunnett’s multiple
comparison posttest. Levels of significance: * p < 0.05; ** p < 0.01;
*** p < 0.001.
CH3CH2CH2CH2CH2O), 1.72 (tt, J = J’ = 6.8 Hz, 2H,
C H 3 C H 2 C H 2 C H 2 C H 2 O ) , 1 . 4 3 − 1 . 3 0 ( m , 4 H ,
CH3CH2CH2CH2CH2O), 0.89 (t, 3H, CH3CH2CH2CH2CH2O)
ppm.
Anti-Inflammatory Activity in BV-2 Cells. Cells (5 × 105 cells per
plate) were seeded in sterile 35 mm cell culture dishes. After
overnight incubation, medium was exchanged with fresh medium.
The cells were pretreated with the respective compounds at the
indicated concentrations for 30 min when 50 μg/mL bacterial
lipopolysaccharide (LPS) was added. After 24 h of incubation, the
medium was collected and spun briefly to remove floating cells, and
100 μL of the supernatant was assayed for nitrite using 100 μL of the
Griess Reagent in a 96-well plate. After incubation for 10 min at room
temperature, the absorbance at 550 nm was read on a microplate
reader. Results are normalized to cell number as assessed by the MTT
assay, as described above.
13C NMR (101 MHz, DMSO-d6): δ 199.6, 164.0, 162.0 161.9,
159.1, 158.1, 147.2, 146.0, 145.0, 135.1, 130.0, 127.7, 121.3, 119.6,
117.7, 117.0, 115.5, 115.2, 113.7, 104.8, 102.8, 101.7, 83.3, 71.9, 67.6,
28.4, 27.7, 21.9, 13.9 ppm.
ESI: m/z calculated for C29H28O9 [M + H]+ 521.17, found 521.14;
HPLC purity = 96%.
Cell Culture General Procedures. HT22 cells were grown in
Dulbecco’s Modified Eagle Medium (DMEM, Sigma-Aldrich,
Munich, Germany) supplemented with 10% (v/v) fetal calf serum
(FCS) and 1% (v/v) penicillin−streptomycin. BV-2 cells were grown
in low-glucose DMEM (Invitrogen, Carlsbad, CA, USA) supple-
mented with 10% FCS and 1% (v/v) penicillin−streptomycin. Cells
were subcultured every 2 days and incubated at 37 °C with 10% CO2
in a humidified incubator.
Western Blots. Sample Preparation. Cells (1.5 × 105HT22 cells
per 35 mm dish) were grown for 24 h before treatment with the
respective compounds at the indicated concentrations and incubation
times. For whole lysate analysis, cells were washed twice with PBS and
scraped into 100 μL of 2.5x SDS sample buffer; then, they were
sonicated and boiled for 5 min.
Compounds were dissolved in dimethyl sulfoxide (DMSO, Sigma-
Aldrich, Munich, Germany) as stock solutions and diluted further into
1x phosphate-buffered saline (PBS).
For determination of cell viability, a colorimetric 3-(4,5-
dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT,
Sigma-Aldrich, Munich, Germany) assay was used. MTT solution
(5 mg/mL in PBS) was diluted 1:10 with medium and added to the
wells after removal of the old medium. Cells were incubated for 3 h,
and then lysis buffer (10% SDS) was applied. The next day,
absorbance at 560 nm was determined with a multiwell plate
photometer (Tecan, SpectraMax 250).
For nuclear fractions, HT22 cells were rinsed twice in ice-cold Tris-
buffered saline (TBS), scraped into ice-cold nuclear fractionation
buffer (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM
EGTA, 1 mM DTT, 1 mM Na3VO4, 1x protease inhibitor cocktail,
and 1x phosphatase inhibitor cocktail), and incubated on ice for 15
min. NP40 at a final concentration of 0.6% was added, and cells were
vortexed; the nuclei were pelleted by centrifugation. Nuclear proteins
were extracted by sonication of the nuclear pellet in nuclear
fractionation buffer, and the extracts were cleared by additional
centrifugation. Protein concentrations were quantified by the
bicinchoninic acid assay (Pierce, Rockford, IL, USA) and adjusted
to equal concentrations. Western blot (5x) sample buffer (74 mM
Tris−HCl pH 8.0, 6.25% SDS, 10% β-mercaptoethanol, 20% glycerol)
was added to a final concentration of 2.5x, and samples were boiled
for 5 min.
Neurotoxicity and Oxytosis in HT22 Cells. Cells (5 × 103 cells per
well) were seeded into sterile 96-well plates and incubated overnight.
For the neurotoxicity assay, the medium was removed, and 1, 5, 10, or
25 μM of the compound diluted with medium from a 0.1 M stock
solution was added to the wells. DMSO (0.05%) in DMEM served as
a control. Cells were incubated for 24 h at which neurotoxicity was
determined using the colorimetric MTT assay.
For the oxytosis assay, 3 × 103 cells per well were treated with 5
mM glutamate (monosodium-L-glutamate, Sigma-Aldrich, Munich,
Germany) together with 1, 5, or 10 μM concentrations of the
respective compounds and incubated for 24 h. Cell viability was
determined using the MTT assay, as described above. Results are
presented as percentage of untreated control cells. Data are expressed
as means SEM of three independent experiments. The analysis was
accomplished using GraphPad Prism 5 Software applying One-way
ANOVA followed by Dunnett’s multiple comparison posttest. Levels
of significance: * p < 0.05; ** p < 0.01; *** p < 0.001.
Western Blotting. Equal amounts of protein (10−20 μg) per lane
were used for SDS−PAGE. All samples were separated using 4−12%
Criterion XT Precast Bis−Tris Gels (Biorad, Hercules, CA, USA).
Proteins were transferred to nitrocellulose membranes, and the quality
of protein measurement, electrophoresis, and transfer were checked
by Ponceau S staining. Membranes were blocked with 5% skim milk
in TBS-T (20 mM Tris buffer pH 7.5, 0.5 M NaCl, 0.1% Tween 20)
for 1 h at room temperature and incubated overnight at 4 °C with the
primary antibody diluted in 5% BSA in TBS/0.05% Tween 20. HRP-
conjugated rabbit antiactin (#5125, 1/20 000), anti-Nrf2 (#sc-13032,
1/500), antiphospho ERK (#9101, 1/1000), antitotal ERK (#9102,
1/1000), antiphospho p38 (#9212, 1/1000), and antitotal p38
(#9212,1/1000) from Cell Signaling (Danvers, MA, USA) were used
as the primary antibody. Subsequently, blots were washed in TBS/
Ferroptosis in HT22 Cells. Cells (3 × 103 cells per well) were
seeded into sterile 96-well plates and incubated overnight. The next
day, the medium was exchanged with fresh medium, and 300 nM
RSL3 was added with vehicle (DMSO) to induce oxidative stress, or
K
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0.05% Tween 20 and incubated for 1 h at room temperature in
horseradish peroxidase goat antirabbit or antimouse (Biorad) diluted
1/5000 in 5% skim milk in TBS-T. After additional washing, protein
bands were detected by chemiluminescence using the Super Signal
West Pico Substrate (Pierce). Autoradiographs were scanned using a
Biorad GS800 scanner. Band density was measured using ImageJ.
Each Western blot was repeated at least three times with independent
protein samples.
washed with 500 μL of ice-cold methanol and sonicated for 4 s, and
after 10 min incubation rotating at 4 °C, the suspension was
centrifuged at 6500 g for 4 min at 4 °C. The supernatant was
removed, and the washing step was repeated once. For solubilization,
1 mL of 1.2% SDS in PBS was added to the pellet after the second
washing step, and it was sonicated for 4 s and boiled for 5 min when
the sample was transferred into a 15 mL falcon tube and diluted with
5 mL PBS to a final SDS concentration of 0.2%. For affinity
purification, streptavidin magnetic beads (GE Healthcare) were used
according to the manufacturer’s protocol with an extended incubation
time of 2 h for proteome incubation. After elution in 35 μL of 2.5x
sample buffer, a methanol/chloroform extraction was rendered to
prepare the samples for MS analysis. The samples were diluted in 140
μL of methanol and 35 μL of chloroform and vortexed. Milli-Q (105
μL) was added, and the mixture was vortexed again before
centrifugation at 15 000 rpm for 2 min at 4 °C. The upper layer
was removed, and 105 μL of methanol was added and vortexed. After
another centrifugation step, the supernatant was removed, and the
pellet was left to dry.
MS Analysis. Gel Electrophoresis. Precipitated proteins were
dissolved in NuPAGE LDS sample buffer (Life Technologies),
reduced with 50 mM DTT at 70 °C for 10 min, and alkylated with
120 mM Iodoacetamide at room temperature for 20 min. The
separation was performed on NuPAGE Novex 4−12% Bis−Tris gels
(Life Technologies) with MOPS buffer according to the manufac-
turer’s instructions. Gels were washed three times for 5 min with
water and stained for 1 h with Simply Blue Safe Stain (Life
Technologies). After washing with water for 1 h, each gel lane was cut
into 14 slices.
In-Gel Digestion. The excised gel bands were destained with 30%
acetonitrile in 0.1 M NH4HCO3 (pH 8), shrunk with 100%
acetonitrile, and dried in a vacuum concentrator (Concentrator
5301, Eppendorf, Germany). Digests were performed with 0.1 μg of
trypsin per gel band overnight at 37 °C in 0.1 M NH4HCO3 (pH 8).
After removing the supernatant, peptides were extracted from the gel
slices with 5% formic acid, and extracted peptides were pooled with
the supernatant.
NanoLC−MS/MS Analysis. NanoLC−MS/MS analyses were
performed on an Orbitrap Fusion (Thermo Scientific) equipped
with a PicoView Ion Source (New Objective) and coupled to an
EASY-nLC 1000 (Thermo Scientific). Peptides were loaded on
capillary columns (PicoFrit, 30 cm × 150 μm ID, New Objective)
self-packed with ReproSil-Pur 120 C18-AQ (1.9 μm) (Dr. Maisch)
and separated with a 30 min linear gradient from 3 to 30% acetonitrile
and 0.1% formic acid and a flow rate of 500 nL/min.
Both MS and MS/MS scans were acquired in the Orbitrap analyzer
with a resolution of 60 000 for MS scans and 7500 for MS/MS scans.
HCD fragmentation with 35% normalized collision energy was
applied. A Top Speed data-dependent MS/MS method with a fixed
cycle time of 3 s was used. Dynamic exclusion was applied with a
repeat count of 1 and an exclusion duration of 30 s; singly charged
precursors were excluded from selection. The minimum signal
threshold for precursor selection was set to 50 000. Predictive AGC
was used with an AGC target value of 2e5 for MS scans and 5e4 for
MS/MS scans. EASY-IC was used for internal calibration.
MS Data Analysis. Raw MS data files were analyzed with
MaxQuant version 1.6.2.2.61 The database search was performed
with Andromeda, which is integrated into the utilized version of
MaxQuant. The search was performed against the UniProt mouse
database. Additionally, a database containing common contaminants
was used. The search was performed with tryptic cleavage specificity
with 3 allowed miscleavages. Protein identification was under the
control of the false-discovery rate (FDR; < 1% FDR on protein and
PSM level). In addition to MaxQuant default settings, the search was
performed against the following variable modifications: Protein N-
terminal acetylation, Gln to pyro-Glu formation (N-term. Gln), and
oxidation (Met). Carbamidomethyl (Cys) was set as a fixed
modification. Further data analysis was performed using R scripts
developed in-house. LFQ intensities were used, and missing LFQ
intensities in the control samples were imputed with values close to
CuAAC with Cy3-Azide. Cells (5 × 105 cells per 60 mm dish) were
grown overnight, and then the compound or DMSO was added at the
indicated concentrations and incubated for 4 h. The CuAAC reaction
was performed in a final volume of 120 μL. Therefore, 100 μg of
protein were adjusted to the appropriate volume with PBS, and 20
μM Cy3-azide was added followed by freshly prepared tris(2-
carboxyethyl)phosphine hydrochloride (TCEP) (1 mM final
concentration), tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methylamine
(TBTA) (0.1 mM final concentration), and copper sulfate (1 mM
final concentration). The mixture was vortexed and incubated rotating
at room temperature for 1 h after which the reaction was quenched by
the addition of 120 μL of 5X sample buffer. The samples were
submitted to SDS−PAGE, transferred to a nitrocellulose membrane,
and examined according to the Western blot protocol described above
with anti-Cy3 (sc-166894, 1/1000) as a primary antibody.
CuAAC in Fixed Cells for Fluorescence Microscopy. Cells (0.4 ×
103 HT22 cells) stably expressing a mitochondria-targeted GFP
(mito-GFP) were grown overnight on 24-well plates with coverslips.
The next day, compounds were added at the indicated concentrations
and incubated for 30 min at 37 °C. After we washed the cells three
times for 5 min with warm PBS, the cells were fixed with freshly
prepared 4% paraformaldehyde (PFA) in PBS at 37 °C for 20 min
and permeabilized with 0.5% Triton X-100 in PBS at room
temperature for 15 min. The CuAAC reaction mixture was prepared
in an Eppendorf tube by mixing Cy3-azide (20 μM final
concentration), freshly prepared tris(2-carboxyethyl)phosphine hy-
drochloride (TCEP) (1 mM final concentration), tris[(1-benzyl-1H-
1,2,3-triazol-4-yl)methylamine (TBTA) (0.1 mM final concentration),
and copper sulfate (1 mM final concentration) in PBS. The CuAAC
reaction mixture was added to the permeabilized cells and incubated
for 1 h at room temperature in the dark. Three additional PBS
washing steps were performed before placing the coverslips on slides
with mounting media. Images were acquired using a Zeiss LSM 880
Rear Port Laser Scanning Confocal and Airyscan FAST Microscope.
GFP and Cy3 were excited sequentially using 488 and 561 laser lines,
respectively, and collected using the corresponding filters and the
Airyscan detector. The final analysis of the images was performed
using ImageJ FIJI software.
CuAAC with HT22 Cells and Affinity Purification of Target
Proteins. The procedure was based on Weerapana et al.60 and
Brodziak-Jarosz et al.21 with modifications. HT22 cells were grown in
10 cm dishes to 90% confluency. Compound 1 (200 μM) or DMSO
in fresh medium was added and incubated for 2 h. The cells were
washed with ice-cold PBS, scraped into 2 mL of ice-cold PBS, and
transferred to Eppendorf tubes. After centrifugation at 5000 rpm for
one min, the cells were suspended in 500 μL of lysis buffer (TBS with
1% NP-40, pH 7.4) with protease inhibitor cocktail and left shaking
for 20 min at 4 °C. Lysates were centrifuged at 14 000 rpm for 20 min
at 4 °C to pellet cellular debris. The supernatant was transferred to
fresh Eppendorf tubes, and the protein concentration was determined
by the bicinchoninic acid method (Pierce).
The CuAAC reaction was conducted in an Eppendorf tube in a
final volume of 500 μL. Protein (2000 μg) was adjusted to the needed
volume with PBS and 150 μM Biotin-PEG3-azide (Sigma-Aldrich,
Munich, Germany); 1 mM freshly prepared tris(2-carboxyethyl)-
phosphine hydrochloride, 0.1 mM tris[(1-benzyl-1H-1,2,3-triazol-4-
yl)methylamine, and 1 mM copper sulfate were added. The reaction
mixture was vortexed, and then the reaction proceeded for 3 h at
room temperature with gentle shaking at which time a precipitate
formed. After centrifugation at 6500 g for 4 min at 4 °C, the
supernatant was collected and immediately submitted to affinity
pulldown following the procedure described below. The pellet was
L
https://dx.doi.org/10.1021/acschemneuro.0c00589
ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
the baseline. Data imputation was performed with values from a
standard normal distribution with a mean of the 5% quantile of the
combined log10-transformed LFQ intensities and a standard
deviation of 0.1. For the identification of significantly enriched
proteins, mean log2 transformed protein ratios were calculated from
the two replicate experiments, and boxplot outliers were identified in
intensity bins of at least 300 proteins. Log2 transformed protein ratios
of the sample versus control with values outside a 1.5x (significance 1)
or 3x (significance 2) interquartile range (IQR), respectively, were
considered as significantly enriched in the individual replicates.
Summed significance values were used to identify best target
candidates (significance 2 in both replicates = significance 4).
GoTerms were added using Perseus.62
Academic Exchange Service (DAAD) with funds of the
Federal Ministry of Education and Research (BmBF) under
project number 57508635 is gratefully acknowledged. P.M. was
supported by grants from NIH (RO1 AG046153, RF1
AG054714, and RF1 AG061296).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thankfully acknowledge support by Dr. Antonio Currais
(Cellular Biology Laboratory, Salk Institute for Biological
Studies) for providing HT22-mitoGFP cells.
Transfection. HT22 cells were plated at 5 × 105 cells per dish in 60
mm tissue culture dishes and grown overnight. The cells were then
transfected with 166 pmol siRNA (ANT-1 no. sc-42354; SERCA no.
sc-36485, both from Santa Cruz Biotechnology) or control siRNA
(no. 1027280; Quiagen) using RNAi max (Invitrogen) according to
the manufacturer’s instructions. After growth overnight, the cells were
subcultured for analysis, as described above.
ABBREVIATIONS
■
7CT, 7-O-cinnamoyltaxifolin; GSH, glutathione; IAA, iodo-
acetic acid
REFERENCES
■
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acschemneuro.0c00589.
■
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LC−MS traces and NMR analysis of target compounds
1 and 2 and full list of identified proteins by MS analysis
(PDF)
Protein ID and related information (XLSX)
AUTHOR INFORMATION
Corresponding Authors
Michael Decker − Pharmaceutical and Medicinal Chemistry,
Institute of Pharmacy and Food Chemistry, Julius Maximilian
■
University of Wurzburg, Am Hubland 97074, Wurzburg,
̈ ̈
Germany; orcid.org/0000-0002-6773-6245; Phone: +49
931 3189676; Email: michael.decker@uni-wuerzburg.de
Pamela Maher − The Salk Institute for Biological Studies, La
Jolla 92037, California, United States of America; Phone: +1
858 453 4110 1932; Email: pmaher@salk.edu
Authors
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Sandra Gunesch − Pharmaceutical and Medicinal Chemistry,
Institute of Pharmacy and Food Chemistry, Julius Maximilian
University of Wurzburg, Am Hubland 97074, Wurzburg,
̈ ̈
Germany
David Soriano-Castell − The Salk Institute for Biological
Studies, La Jolla 92037, California, United States of America
Stephanie Lamer − Rudolf-Virchow-ZentrumCenter for
Integrative and Translational Bioimaging, Julius Maximilian
University of Wurzburg, 97080 Wurzburg, Germany
̈ ̈
Andreas Schlosser − Rudolf-Virchow-ZentrumCenter for
Integrative and Translational Bioimaging, Julius Maximilian
University of Wurzburg, 97080 Wurzburg, Germany
̈ ̈
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The manuscript was written through contributions of all
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Financial support by the Bavaria California Technology Center
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