Natural-Product-Inspired Aminoepoxybenzoquinones Kill Members of the Gram-Negative Pathogen Salmonella by Attenuating Cellular Stress Response

Article abstract of DOI:10.1002/anie.201607338

Gram-negative bacteria represent a challenging task for antibacterial drug discovery owing to their impermeable cell membrane and restricted uptake of small molecules. We herein describe the synthesis of natural-product-derived epoxycyclohexenones and explore their antibiotic activity against several pathogenic bacteria. A compound with activity against Salmonella Typhimurium was identified, and the target enzymes were unraveled by quantitative chemical proteomics. Importantly, two protein hits were linked to bacterial stress response, and corresponding assays revealed an elevated susceptibility to reactive oxygen species upon compound treatment. The consolidated inhibition of these targets provides a rationale for antibacterial activity and highlights epoxycyclohexenones as natural product scaffolds with suitable properties for killing Gram-negative Salmonella.

Full text of DOI:10.1002/anie.201607338

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Communications
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International Edition: DOI: 10.1002/anie.201607338
German Edition: DOI: 10.1002/ange.201607338
Drug Discovery Very Important Paper
Natural-Product-Inspired Aminoepoxybenzoquinones Kill Members of
the Gram-Negative Pathogen Salmonella by Attenuating Cellular
Stress Response
Franziska A. Mandl+, Volker C. Kirsch+, Ilke Ugur, Elena Kunold, Jan Vomacka,
Christian Fetzer, Sabine Schneider, Klaus Richter, Thilo M. Fuchs, Iris Antes,* and
Stephan A. Sieber*
Abstract: Gram-negative bacteria represent a challenging task
for antibacterial drug discovery owing to their impermeable
cell membrane and restricted uptake of small molecules. We
herein describe the synthesis of natural-product-derived epox-
ycyclohexenones and explore their antibiotic activity against
several pathogenic bacteria. A compound with activity against
Salmonella Typhimurium was identified, and the target
enzymes were unraveled by quantitative chemical proteomics.
Importantly, two protein hits were linked to bacterial stress
response, and corresponding assays revealed an elevated
susceptibility to reactive oxygen species upon compound
treatment. The consolidated inhibition of these targets provides
a rationale for antibacterial activity and highlights epoxycy-
clohexenones as natural product scaffolds with suitable
properties for killing Gram-negative Salmonella.
therapeutics in clinical use. However, the majority of natural-
product antimicrobial drugs only address a limited scope of
cellular targets, comprising cell wall, protein, and DNA
biosynthesis. Thus, to get around resistance within these
targets, searching for less explored natural product scaffolds
and in-depth studies on their mechanism of action could
provide additional and thus far unexploited cellular pathways
suitable for drug development. Aminoepoxycyclohexenones
(AECs) represent a prime example of such a privileged
scaffold present in numerous natural products. They comprise
the
subclass
of
aminoepoxyhydroxycyclohexenones
(AEHCs) with the amino functional group at either the
2’- (ortho, type I) or 5’-position (meta, type II), including LL-
C10037a (1), asukamycin A1 (2), epoxyquinomycin C (3),
and cetoniacytone A (4; Figure 1A). Moreover, structural
diversity is increased by the biosynthesis of related amino-
epoxybenzoquinones (AEBQs, here referred to as type III)
such as epoxyquinomycin B (5) and G-7063-2 (6; Figure 1A).
Interestingly, AEHCs as well as related AEBQs exhibit pre-
validated activity against Gram-positive and even Gram-
negative bacteria.[4] The presence of both an intact epoxide
moiety and the Michael acceptor system is crucial for their
antibacterial bioactivity, suggesting that alkylation of enzyme
active sites could be a substantial part of the thus far
uncharacterized cellular mechanism.[5] Herein, we exploited
the antibiotic activity and cellular mechanism of structurally
diverse aminoepoxycyclohexenones (I–III) by chemical, bio-
chemical, and proteomic methods. Two compounds were
identified that exhibit low micromolar activity against
S. Typhimurium by addressing enzymes controlling oxidative
stress. Moreover, the structure and mechanism of one target
protein with unknown function were explored, giving unpre-
cedented insight into the aminoepoxycyclohexenone mode of
action.
T
he increasing spread of multiresistant bacteria represents
a fundamental threat to human health. While a few antibiotics
of last resort are still effective against resistant Gram-positive
bacteria, the most pressing problem stems from untreatable
clinical Gram-negative isolates.[1] Here, the largely imperme-
able cell membrane impairs small-molecule uptake, which
represents a major challenge for drug discovery.[2] For
example, infections caused by resistant non-typhoidal Salmo-
nella serotypes are of particular concern.[3] In the past, natural
products such as b-lactams and aminoglycosides have been
a rich source for antibiotic discovery, providing effective
[*] Dr. F. A. Mandl,[+] V. C. Kirsch,[+] E. Kunold, J. Vomacka, C. Fetzer,
Dr. S. Schneider, Dr. K. Richter, Prof. Dr. S. A. Sieber
Center for Integrated Protein Science
Department Chemie, Technische Universitꢀt Mꢁnchen
Lichtenbergstrasse 4, 85747 Garching (Germany)
E-mail: stephan.sieber@tum.de
Dr. I. Ugur, Prof. Dr. I. Antes
To determine the bioactivities and cellular targets of
aminoepoxycyclohexenones I–III, we synthesized a suite of
molecules equipped with aliphatic and aromatic amide side
chains and functionalized them with an alkyne tag (Fig-
ure 1B). The alkyne tag served as a benign handle for target
identification by Huisgen/Sharpless/Meldal cycloaddition
(click chemistry, CC) to rhodamine or biotin azides.[6]
Furthermore, corresponding benzoquinone probes lacking
the crucial epoxide moiety were prepared as controls for
cellular and proteomic studies. The syntheses of the probes
were based on established procedures[7] and initiated by
oxidative dearomatization of Boc-protected 2,5-dimethoxy-
Center for Integrated Protein Science
Department fꢁr Biowissenschaften, Technische Universitꢀt Mꢁnchen
Erlenmeyer-Forum 8, 85354 Freising (Germany)
E-mail: antes@tum.de
Prof. Dr. T. M. Fuchs
Lehrstuhl fꢁr Mikrobielle ꢂkologie
Zentralinstitut fꢁr Ernꢀhrungs- und Lebensmittelforschung
Technische Universitꢀt Mꢁnchen
Weihenstephaner Berg 3, 85354 Freising (Germany)
[+] These authors contributed equally to this work.
Supporting information for this article can be found under:
http://dx.doi.org/10.1002/anie.201607338.
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compounds were tested for the inhibition
of bacterial growth against a panel of 16
different strains, including major pathogens
such as S. aureus, multiresistant S. aureus
(MRSA), and S. Typhimurium (Table S2).
Interestingly, type III AEBQs FM205
and FM243 as well as type I AEHC FM255
inhibited the growth of Gram-positive
strains, including MRSA, with minimal
inhibitory concentrations (MICs) of
10 mm. Contrary, analogues of these com-
pounds with an acetal group (12a–12h,
Scheme 1) did not show growth inhibition
(up to 300 mm), suggesting that the carbonyl
or reduced hydroxy moieties are essential
for the antibiotic activity. Strikingly,
AEBQs FM233 and FM239 inhibited the
growth of S. Typhimurium LT2 and TA100
and S. enteritidis with MICs of about 30 mm
while the corresponding controls lacking
the epoxide (Figure 1B and Scheme S1)
did not affect viability. Satisfyingly, FM233
displayed only low toxicity (< 15% apop-
totic cells) in apoptosis assays against A549
cells at concentrations up to 150 mm (Fig-
Figure 1. A) Representative aminoepoxycyclohexenone natural products: LL-C10037a (1),
asukamycin A1 (2), epoxyquinomycin C (3), and cetoniacytone A (4) belong to the AEHC
subclass (type I/II). Epoxyquinomycin B (5) and G-7063-2 (6) are members of the AEBQ
subclass (type III).[4,8] B) Structures of diverse type I–III aminoepoxycyclohexenone ABPP
probes and their corresponding epoxide-lacking benzoquinone controls.
ure S1). However, as the metabolic activity was impaired in
human cells (Figure S2 and Table S3), we utilized a more
complex and representative model of toxicity evaluation
based on a whole organism. The nematode Caenorhabditis e-
legans[9] tolerated high doses of FM233 as well as the inactive
control FM375 (highest tested concentration: 500 mm) over
a period of five days without any signs of toxicity or
developmental defects. Moreover, normal production of L1
larvae and eggs was observed whereas doxorubicin (100 mm),
a toxic control, led to growth arrest (Figure S3). Contrary to
extensive investigations of AEHC and AEBQ targets in
human cells, the bacterial mode of action of this compound
class has remained elusive.[10] Thus we applied our probe
molecules in activity-based protein profiling (ABPP)[11]
experiments and incubated several Gram-positive and
Gram-negative strains with probe molecules in situ. The
cells were lyzed, the probe-tagged proteins were clicked to
rhodamine azide and separated by SDS-PAGE, and labeled
proteins were then visualized by fluorescence scanning (Fig-
ure 2A). Whereas all S. aureus strains exhibited largely
comparable labeling patterns of discrete cytosolic protein
bands (Figure S4), a closer inspection of the Salmonella
specific targets revealed striking differences between the
antibiotic AEBQs (FM239 and FM233) and their correspond-
ing inactive controls lacking the epoxide moiety (FM257 and
FM375; Figure S5). For example, FM233 labeled several
distinct proteins (e.g., a prominent band at 30 kDa) that are
absent in the FM375 treated sample, suggesting that the
epoxide addresses targets essential for bacterial viability
(Figure 2A,B). In contrast, these probes did not reveal
pronounced labeling in S. aureus and exhibited no MIC,
suggesting a species-specific mode of action (Figure S6).
Owing to the challenging discovery of druggable targets
especially in Gram-negative strains, we selected antibiotic
Scheme 1. Synthesis of type I and II AEHCs and type III AEBQs.[7]
Reagents and conditions: a) 1. Boc2O, THF, 708C, 16 h, 99%; 2.
iodobenzene dipivalinate, MeOH, 08C!RT, 16 h, 84%; 3. Boc2O,
THF, 708C, 16 h, 68%; b) 1. H2O2, NaOH, THF, RT, 16 h, 53%; 2.
trifluoroacetic acid, CH2Cl2, RT, 2 h, 96%; c) synthesized acids or acid
chlorides 11a–11e, lithium tert-butoxide, THF, ꢀ108C!RT, 4–16 h,
30–73%; d) BF3·OEt3, CH2Cl2, ꢀ208C!RT, 14 h, 68–93%; e) NaBH-
(OAc)3, MeOH, 08C!RT, 2 h, 53%; f) 1. LiBHEt3, THF, ꢀ788C!RT,
3 h; 2. montmorillonite K10, CH2Cl2, RT, 16 h, 70% over two steps.
aniline (7) with iodobenzene dipivalinate (a; Scheme 1),
followed by epoxidation using aqueous hydrogen peroxide
solution and subsequent deprotection with trifluoroacetic
acid (b). The Michael acceptor–epoxide key fragment 9 was
further coupled to various alkyne-tagged acids or acid
chlorides (see the Supporting Information, Section S9.1) by
amide bond formation (c). Next, the acetal of the partially
protected quinones (12a–12h) was removed with boron
trifluoride diethyl etherate leading to type III AEBQs;
subsequent reduction gave access to type II AEHCs. Upon
inverting these two steps (d, e), type I AEHCs were generated
(f). Benzoquinone analogues lacking the epoxide moiety
(control, Figure 1B) served as reference compounds for
phenotypic studies and labeling experiments (Scheme S1
and Table S1). Prior to proteomic target identification, all
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Figure 2. A) Fluorescence SDS gel of S. Typhimurium LT2 labeled in situ with the active probe FM233 and the inactive control FM375.
B) Structures of the probes used for labeling. C) Gel-free quantitative proteomics studies based on dimethyl labeling. D) Volcano plot of the
enrichment analysis. Blue dots represent proteins that were significantly enriched at an FDR of 0.01 (Student’s t-test, Benjamini–Hochberg
truncation). E) Target validation by concentration-dependent labeling of recombinant TrxA. DT=heat-denatured TrxA labeled with 10 mm FM233.
F) Inhibition of TrxA by FM233 monitored in a turbidimetric assay of insulin disulfide reduction. Shown are two independent experiments, each
performed in technical triplicate; error bars represent the standard deviation of the mean.
FM233 as a promising starting point for a detailed mecha-
nism-of-action analysis in S. Typhimurium LT2.
tase protecting the cell from thiol-specific stress caused by
quinone electrophiles,[12] was discovered as well. When this
experiment was carried out with the insoluble fraction, no
significant hits were discovered. These results suggest that the
molecule is cell-permeable and largely addresses cytosolic
proteins (Figure S8). Furthermore, the characteristic cytosolic
30 kDa band could be assigned to be SCRP-27A by gel-based
MS studies (Figure S9). Whereas GrxB and TrxA are
associated with bacterial redox pathways, little is known
about SCRP-27A.[13] To validate probe binding and to
investigate the mechanism of inhibition, all targets were
overexpressed, purified, and analyzed in various assay
systems. Labeling studies with recombinant SCRP-27A,
TrxA, and GrxB proteins and corresponding expression
strains confirmed FM233 binding whereas, in line with the
proteome data, the FM375 control probe was largely inactive
(Figure 2E and Figures S10–S14).
SCRP-27A turned out to be most challenging as it is
a protein of unknown function. It belongs to the DJ-1 protein
superfamily[14] with a predicted Cys-His-Glu triad character-
istic of glutamine amidotransferases, hydrolases, and pro-
teases.[15] To gain insight into the mechanism, we first
crystallized recombinant SCRP-27A and determined its
X-ray structure to 1.75 ꢁ resolution. (Figure 3A).[21] Interest-
For quantitative proteomic studies, cells were incubated
with either active FM233, inactive FM375, or a DMSO
control, lyzed, separated into soluble and insoluble fractions,
and clicked to biotin azide. Afterwards, labeled proteins were
enriched on avidin and digested by trypsin, and finally, the
peptides were modified with either heavy or light isotopes by
dimethyl labeling (DL, Figure 2C). Both samples were
pooled and analyzed by liquid chromatography (LC) in
combination with high-resolution mass spectrometry (MS).
Proteins that were specifically bound by the probe revealed
a high enrichment (criteria: log2 ꢁ 2 and statistically signifi-
cant; Studentꢀs t-test, Benjamini–Hochberg FDR of 0.01)
compared to those that are background binders and also
present in the DMSO control (Figure S7). To focus only on
target proteins that are associated with the antibiotic mode of
action, we performed additional gel-free studies in compar-
ison with the inactive control FM375 (Figure 2D) and
identified three significantly enriched targets in the soluble
fraction: glutaredoxin 2 (GrxB), thioredoxin-1 (TrxA), and
sigma cross-reacting protein 27A (SCRP-27A). Although not
matching the significance criteria, it is worth noting that
FMN-dependent azoreductase R (AzoR), a quinone reduc-
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Figure 3. A) Crystal structure of S. Typhimurium SCRP-27A determined with a resolution of 1.75 ꢃ.[21] The dimeric biomolecule (shown as a ribbon
in green and blue) is overlaid with its semi-transparent surface and completed by the crystallographic symmetry. Enlarged image of the predicted
active site: Cys135 is displayed as a stick model, with the corresponding FoꢀFc difference electron density contoured at 2s (gray mesh). Cys135
adopts two alternative conformations and is most likely oxidized to sulfenic acid.[18] B) Concentration-dependent labeling of FM233 in SCRP-27A
and the SCRP-27A-C135A mutant (left) as well as in S. Typhimurium LT2 and LT2-DyhbL (right) confirmed the binding site of FM233. C) ROS
assay with active FM233 and inactive FM375 at a concentration of 100 mm compared to the untreated control (no ROS). ns=not significant; *:
p<0.05; **: p<0.01.
ingly, an exposed Cys135 residue located in a distinct pocket
was observed, which is highly conserved among DJ-1 family
members.[16a] To analyze the role of this residue for probe
binding and catalysis, a point mutation was introduced by site-
directed mutagenesis (Cys135Ala, C135A). Fluorescence
SDS-PAGE analysis as well as mass spectrometry on the
full-length proteins revealed, contrary to the wild type (wt)
protein, a lack of FM233 probe binding, suggesting an
important role of Cys135 for SCRP-27A function (Figure 3B,
left; see also Figures S15 and S16).
To narrow down its catalytic mechanism, we tested the
enzymatic activity in various assays (see the Supporting
Information for details). Interestingly, only the esterase assay
with para-nitrophenyl acetate revealed slight turnover, which
was abolished with the mutant and inhibited by FM233 in
a concentration-dependent manner (Figure S17). To then test
the relevance of this putative esterase for bacterial viability,
we constructed a non-polar deletion mutant of gene yhbL
(STM3327) in S. Typhimurium LT2. Labeling of the 30 kDa
protein was not observed in this mutant (Figure 3B, right),
validating target binding and demonstrating that yhbL indeed
encodes SCRP-27A. However, growth assays with FM233 did
not alter the MIC, indicating that this protein is not involved
in the antibacterial mechanism of action.
tivating oxidatively damaged proteins, and strains lacking
thioredoxin and glutaredoxin oxidoreductases show impaired
growth.[16] These mechanisms suggest that our compounds
could mimic endogenous quinones and act as suicide inhib-
itors of TrxA and GrxB. We confirmed this notion by
inhibition assays with both enzymes. TrxA as well as GrxB
were inhibited in a concentration-dependent manner solely
by FM233 and, in line with the bioactivity data, not by the
inactive control FM375 (Figures 2E, S18, and S19).
Although we were not successful in determining the
binding site by direct MS/MS sequencing owing to the poor
ionization ability of the AEBQ moiety, MS analysis on the
intact full-length protein confirmed irreversible modification
of both enzymes (Figure S20). Moreover, FM233 binding to
GrxB and TrxA was further elucidated by molecular docking
followed by molecular dynamics simulations and QM/MM
calculations.[17] The surface representation of GrxB shows
that FM233 is buried in a deep pocket (Figure 4A) and
stabilized by several hydrogen bonds (Figure 4B). In contrast,
probe FM375, being devoid of the epoxide moiety, lacked the
crucial contact to Lys129, resulting in 11 kcalmolꢀ1 weaker
interaction energy (Figure 4D and Table S4). A similar
difference of 8 kcalmolꢀ1 was observed for the binding of
the active and inactive probes to TrxA (Figure 4F,H and
Table S4). Here, the binding pocket is more surface-exposed
with less stabilization, resulting in a 26 to 30 kcalmolꢀ1
weaker interaction compared to GrxB (Figure 4E and
GrxB and TrxA are well characterized enzymes that
catalyze redox reactions of cysteine residues with crucial roles
in related stress pathways.[13] They are important for reac-
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Figure 4. Conformation of the QM/MM-minimized structures of FM233 within the receptors: GrxB (A: surface, B: ribbon) and TrxA (E: surface, F:
ribbon). The covalently bound complexes of FM233 to Cys12 and Cys32 within GrxB and TrxA are shown in (C) and (G), respectively. The QM/
MM-minimized structures of the inactive control FM375 within GrxB and TrxA are shown in (D) and (H). Only significant atoms are shown for
clarity, all distances are given in ꢃ.
Table S4). Based on the QM/MM-minimized structures, we
modeled a possible reaction between FM233 and the redox-
active cysteine residues Cys12 and Cys32 of GrxB and TrxA,
respectively. Both nucleophiles readily react with the electro-
philic epoxide in an exothermal reaction (Figure 4C,G and
Table S4). MD simulations performed for several docked
complexes revealed fluctuations of the ligand positions within
the binding site. This indicates that the partially surface-
exposed location of the active sites enables diffusion of the
ligands into the solvent. However, in the case of FM233, the
covalent-bond formation, which is highly exothermic, keeps
the probe within the protein, leading to stable inhibition.
With this mechanistic validation, we finally investigated
the impact of FM233 on oxidative stress in a cellular reactive
oxygen species (ROS) assay (Figure 3C).[19] Interestingly,
under ROS conditions, only active FM233, but not inactive
FM375, induced a significant reduction in bacterial viability
compared to the untreated control. The antibiotic activity of
FM233 thus stemmed to a large extent from the down-
regulation of the enzymatic stress response and the corre-
sponding sensitization of the cells to stress. In fact, quinones
are known to trigger ROS production through the formation
of semiquinone radicals. Moreover, quinone-mediated thiol
S-alkylation results in the aggregation and depletion of thiol-
containing proteins that are important for cellular homeo-
stasis.[20]
stress response significantly impaired bacterial viability.
Targeting GrxB and TrxA with natural-product-inspired
quinone mimics thus represents an intriguing approach for
the discovery of novel antibacterial compounds.
Acknowledgments
This work was funded by the Deutsche Forschungsgemein-
schaft, SFB749, CIPSM, FOR1406, and Fonds der chemi-
schen Industrie. We thank Prof. Michael Groll for access to
the crystallization facility and Fabian Koczian for help with
the FACS assay. Moreover, we are grateful to Astrid Kçnig,
Katja Bꢂuml, and Mona Wolff for support.
Keywords: antibiotic resistance · covalent inhibitors ·
Gram-negative bacteria · stress response ·
quantitative proteomics
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Received: July 29, 2016
Published online: && &&, &&&&
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Communications
Drug Discovery
F. A. Mandl, V. C. Kirsch, I. Ugur,
E. Kunold, J. Vomacka, C. Fetzer,
S. Schneider, K. Richter, T. M. Fuchs,
I. Antes,* S. A. Sieber*
&&&& — &&&&
Natural-Product-Inspired
Aminoepoxybenzoquinones Kill
Members of the Gram-Negative
Pathogen Salmonella by Attenuating
Cellular Stress Response
Aminoepoxycyclohexenones inhibit the
growth of Gram-negative Salmonella in
the low micromolar range. The bacterial
targets of these antibiotics were analyzed
by quantitative ABPP, which revealed
a link to bacterial stress response that
was validated by various assays. Amino-
epoxybenzoquinones were thus validated
as natural product scaffolds with suitable
properties for the growth inhibition of
Salmonella.
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