Sulfone-Based Probes Unraveled Dihydrolipoamide S-Succinyltransferase as an Unprecedented Target in Phytopathogens

Article abstract of DOI:10.1021/acs.jafc.9b02059

Target validation of current drugs remains the major challenge for target-based drug discovery, especially for agrochemical discovery. The bactericide 0 represents a novel lead structure and has shown potent efficacy against those diseases that are extremely difficult to control, such as rice bacterial leaf blight. However, no detailed target analysis of this bactericide has been reported. Here, we developed a panel of 0-derived probes 1?6, in which a conservative modification (alkyne tag) was introduced to keep the antibacterial activity of 0 and provide functionality for target identification via click chemistry. With these cell-permeable probes, we were able to discover dihydrolipoamide S-succinyltransferase (DLST) as an unprecedented target in living cells. The probes showed good preference for DLST, especially probe 1, which demonstrated distinct selectivity and reactivity. Also, we reported 0 as the first covalent DLST inhibitor, which has been used to confirm the involvement of DLST in the regulation of energy production.

Full text of DOI:10.1021/acs.jafc.9b02059

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Agricultural and Environmental Chemistry
Sulfone-based probes unraveled dihydrolipoamide S-
succinyltransferase as an unprecedented target in phytopathogens
Biao Chen, Qingsu Long, Yong-Liang Zhao, Yuanyuan Wu, Shasha Ge, Peiyi
Wang, Cai-Guang Yang, Yonggui Robin Chi, Baoan Song, and Song Yang
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b02059 • Publication Date (Web): 31 May 2019
Downloaded from http://pubs.acs.org on June 1, 2019
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Sulfone-based probes unraveled dihydrolipoamide S-succinyltransferase as an
unprecedented target in phytopathogens
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Biao Chen,† Qingsu Long,† Yongliang Zhao,† Yuanyuan Wu,† Shasha Ge,† Peiyi
Wang,† Cai-Guang Yang,§ Yonggui Chi,† Baoan Song,†,* and Song Yang†,‡,*
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†Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key
Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of
Education, Guizhou University, Huaxi District, Guiyang 550025, China.
§Laboratory of Chemical Biology, State Key Laboratory of Drug Research, Shanghai
Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
‡College of Pharmacy, East China University of Science & Technology, Shanghai
200237, China.
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*Corresponding author
*E-mail: jhzx.msm@gmail.com (S.Y.); basong@gzu.edu.cn (B.S.).
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Abstract
Target validation of current drugs remains the major challenge for target-based
drug discovery, especially for the agrochemical discovery. The bactericide 0
represents a novel lead structure and has showed potent efficacy against those
diseases that are extremely difficult to control, such as rice bacterial leaf blight.
However, no detailed target analysis of this bactericide has been reported. Here we
developed a panel of 0-derived probes 1–6, in which a conservative modification
(alkyne tag) was introduced to keep the antibacterial activity of 0 and provide
functionality for target identification via click chemistry. With these cell-permeable
probes, we were able to discover dihydrolipoamide S-succinyltransferase (DLST) as
an unprecedented target in living cells. The probes showed a good preference for
DLST, especially probe 1, which demonstrated distinct selectivity and reactivity. Also,
we reported 0 as the first covalent DLST inhibitor, which has been used to confirm
the involvement of DLST in the regulation of energy production.
Keywords
Bactericide 0; Target; DLST; Covalent inhibitor; Energy production
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1. Introduction
Since the late 19th century, crop protection chemistry has become a high-tech
science in supporting the sustainable production of food from the available farmland.1
Advancements in the discovery of highly effective agrochemicals will be required to
tackle the challenge of increasing food production. Generally, the discovery of a
commercialized agrochemical will take about 10 years and a cost of approximately
U.S. $286 million and a screening of above 150,000 compounds.2 An important step
that accelerates the agrochemical discovery process is to enlarge the number of
druggable targets as well as to deeply understand the molecular and cellular
mechanism of action of current agrochemicals. With a known target, multiple
techniques such as crystallography, computational modeling, biochemistry and
binding kinetics can be used to analyze the target interaction with drugs, enabling
efficient target-based drug design.3 However, target validation of current drugs
remains the major challenge for target-based drug discovery, especially for the
agrochemical discovery. The definite mechanism of most agrochemicals are unclear,
which results in so few targets that can be used for target-based molecular design.
Bacterial plant pathogens, especially the top 10,4 severely infect a number of
economically important crops and seriously reduce the crop yield. The bacterial
outbreaks are generally difficult to control due to lack of effective bactericides and the
increasing resistance problem, thereby emphasizing an urgent demand for the
discovery of new antibacterial agents. The sulfone reagents represent promising
biologically active privileged structures that can react covalently with the active sites
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of certain proteins.5-7 Among them, the drug candidate 0 (jiahuangxianjunzuo, in
registration stage ) and its analogs have been reported to show potent efficacy against
those diseases8-12 that are extremely difficult to control, such as Xanthomonas oryzae
pv. oryzae (Xoo) and Xanthomonas axonopodis pv. citri (Xac). However, no detailed
target analysis of this bactericide has been conducted so far in living systems.
Recently, an unbiased chemical proteomic strategy termed “activity-based
protein profiling” (ABPP)13-15 has been used as a powerful method for the target
deconvolution of bioactive molecules. ABPP used activity-site directed probes to
profile the function state of enzymes directly in native biological systems, which has
been developed for multiple enzyme classes, such as cysteine proteases, serine
hydrolases, phosphatases and kinases.16 In order to investigate the interactions
between 0 and its protein targets in bacteria, we designed a series of
alkyne-functionalized probes 1–6 (Figure 1A) and systematically evaluated their
reactivity and selectivity directly in living bacterial cells. As expected, minor change
in a common structure resulted in a selective labeling event of ∼45 kDa protein. All
probes containing an alkyne handle were used for the modification with a reporter
group (e.g., biotin) via the 1,3-dipolar Huisgen cycloaddition (click chemistry, CC)
after cell penetration and lysis.17,18 Labeled proteins are separated by SDS gels,
visualized by streptavidin blot, and subsequently identified by mass spectrometry
(Figure 1B).19
2. Materials and methods
2.1 Instruments and Chemicals
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1H and 13C NMR spectral analysis were performed on a JEOL-ECX 500 NMR
spectrometer using CDCl3 or DMSO-d6 as the solvent and TMS as an internal
standard. High resolution mass spectra (HRMS) was conducted on an LTQ Orbitrap
(Thermo Scientific). Imaging analysis was conducted on the the ChemiDoc XRS+
System (Bio-Rad). All reagents from Chinese Chemical Reagent Company were
reagent-grade and used without further purification.
2.2 General Synthetic Procedures for the Intermediates 1−6
The intermediates 1−4 were prepared according to previously reported methods.8
Thioether derivatives 4 were prepared by esterification, hydrazidation, cyclization and
thioetherification reactions. To a solution of 4 (0.03 mol) in 50 mL CCl4 was added
N-bromosuccinimide (0.036 mol) and a catalytic amount of azobisisobutyronitrile
(AIBN, 0.003 mol). The mixture was refluxed for 48 h and then the CCl4 was
evaporated to obtain the crude product, which was further washed with saturated
aqueous NaHCO3 and dried over Na2SO4. The organic layer was evaporated and the
residue was purified by silica gel column chromatography to obtain the intermediates
5a−5f with yields of 43−77%. To a solution of intermediate 5 (5 mmol) and propargyl
alcohol (5.1 mmol) in 20 mL dried DMF, 60% sodium hydride (6 mmol) was added.
The reaction was stirred for 2 h under the ice bath. The mixture was quenched by
adding saturated NH4Cl (aq) and extracted by ethyl acetate. The organic layer was
washed with saturated NH4Cl for two times and dried over Na2SO4, filtered and the
solvent was removed under reduced pressure. The crude product was purified by
silica gel column chromatography to obtain the intermediates 6a−6f with yields of
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43−66%.
2.3 General Synthetic Procedures for the probes 1−6
To a stirred solution of intermediate 6 (5 mmol) in 15 mL CH2Cl2 was added
m-CPBA (15 mmol). The reaction was stirred for 2 h and the mixture was filtered.
After evaporation, the obtained crude product was purified by silica gel column
chromatography with ethyl acetate/petroleum ether (v/v = 1:6) to give the pure probes
1–6 with yields of 40−70%. The representative data for probe 1 are presented below.
2-(methylsulfonyl)-5-(4-fluoro-3-((prop-2-yn-1-yloxy)methyl)phenyl)-1,3,4-oxa
diazole (Probe 1). Yield 70%; white solid; 1H NMR (500 MHz, CDCl3) δ 8.26 (dd, J
= 6.6, 2.2 Hz, 1H, Ph-6-H), 8.10 (m, 1H, Ph-2-H), 7.25 (t, J = 8.9 Hz, 1H, Ph-5-H),
4.74 (s, 2H, Ph-CH2O-), 4.30 (d, J = 2.4 Hz, 2H, -OCH2-C≡CH), 3.53 (s, 3H,
13
-SO2CH3), 2.52 (t, J = 2.4 Hz, 1H, -C≡CH); C NMR (125 MHz, CDCl3) δ 165.8,
164.4, 162.4, 162.2, 129.8, 129.5, 127.1, 118.5, 116.9, 78.9, 75.5, 64.5, 58.2, 43.0;
HRMS (ESI) calculated for C13H11FN2O4S [M+H]+ m/z 311.0496, found 311.0493.
2.4 General Synthetic Procedures for negative probe (NP)
Sodium hydroxide (120 mg, 3 mmol) dissolved in 5 mL distilled water was added to a
solution of probe 1 (465 mg, 1.5 mmol) in 5 mL THF. The reaction was stirred at RT
for 2 h. The mixture was acidified with dilute HCl to pH 4−5 and extracted with 20
mL CH2Cl2 for two times. The organic layer was evaporated and the crude product
was purified by silica gel column chromatography with ethyl acetate/petroleum ether
(v/v = 1:6) to give the pure NP.
5-(4-fluoro-3-((prop-2-yn-1-yloxy)methyl)phenyl)-1,3,4-oxadiazol-2-ol
(NP).
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Yield 85%; white solid; H NMR (500 MHz, DMSO-D6) δ 12.60 (s, 1H, -OH), 7.84
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(d, J = 6.8 Hz, 1H, Ph-6-H), 7.79 (m, 1H, Ph-2-H), 7.39 (t, J = 8.8 Hz, 1H, Ph-5-H),
4.64 (s, 2H, Ph-CH2O-), 4.26 (d, J = 2.4 Hz, 2H, -OCH2-C≡CH), 3.51 (t, J = 2.4 Hz,
1H, -C≡CH); 13C NMR (125 MHz, CDCl3) δ 163.6, 161.5, 154.7, 154.6, 128.0, 127.5,
126.3, 120.2, 116.4, 79.0, 75.3, 64.6, 58.0; HRMS (ESI) calculated for C12H9N2O3F
[M−H]– m/z 247.0513, found 247.0523.
2.5 In Vitro Antibacterial Bioassay
In this study, the antibacterial activities of target probes against Xoo and Xac were
screened by a turbidimeter test. About 40 μL of liquid nutrient broth (1 g of yeast
powder, 3 g of beef extract, 10 g of glucose, 5 g of peptone, and 1 L of distilled water,
pH 7.0−7.2) containing Xoo or Xac was added to 5 mL of nutrient broth (NB) media
containing the test compounds as experimental groups and DMSO as a blank control.
The inoculated test tubes were incubated at 28 ± 1 °C with a constant shake at 180
rpm until the bacteria reached logarithmic growth phase. The growth of the culture
was monitored on a microplate reader by measuring the optical density at 600 nm
(OD600). The in vitro inhibition rates I (%) are calculated by the formula described
below, where C represents the corrected absorbance value (OD600) of the DMSO NB
media and T represents the corrected absorbance value (OD600) of the compounds
treated NB media.
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Inhibition rate I (%) = ( C−T) / C × 100
The half-maximal inhibitory concentration (IC50) was determined from the equation
via software SPSS 17.0. Each experiment was repeated three times.
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2.6 Cell culture
Xanthomonas oryzae pv. oryzae (Xoo) strain PXO99A and Xanthomonas axonopodis
pv. citri (Xac) strain Xac 29−1 cells were cultured in liquid nutrient broth medium
(3.0 g beef extract, 5.0 g peptone, 1.0 g yeast powder, 10.0 g glucose, added into 1 L
distilled water, pH 7.0−7.2) at 28 °С for 12 h. Cells were cultivated in a constant
temperature shaker with a speed of 180 rpm.
2.7 Preparation of proteomes for in vitro experiments
Xoo or Xac cells were cultured in 1 L liquid nutrient broth medium at 28 °С until the
OD600 reached 0.6 and then harvested by centrifugation at 6000 rpm. The bacterial
cell pellet was washed with PBS three times, resuspended in 40 mL PBS (pH 7.2)
buffer and lysed by sonication with a VCX150 (Sonics) instrument under ice cooling.
The cell mixture was separated by centrifugation at 12000 rpm for 45 min and the
protein concentration of supernatant was determined by the Bradford assay, which
was further adjusted to 1 mg/mL by dilution with PBS and stored at −80 °C until use.
2.8 In vitro Labeling of Bacterial Proteomes
Proteome samples (44 µL of 1 mg/mL protein in PBS) were treated with 1 µL desired
concentrations of probe 1 or 2 for 2 h at 25 °C. For competition experiments with
compound 0, proteomes (43 µL) were first incubated with various concentrations of 0
(1 µL) for 2 h at 25 °C, followed by addition of probe 1 (5 µM) or 2 (50 µM) for
another 2 h. After incubation, 1 µL biotin-azide reagent (200 µM) was added,
followed by 1 µL freshly prepared sodium ascorbate solution (5 mM). Samples were
gently vortexed, and the cycloaddition was initiated by the addition of 3 µL
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CuSO4/BTTAA mixture. A 50 mM BTTAA solution was preincubated with 50 mM
CuSO4 solution (2:1 v/v) before use. The reaction mixtures were then incubated at 37
°C for 2 h in the dark. After click chemistry, 50 µL 2× SDS loading buffer was added
into the mixture to stop the reaction, which was further heated at 95 °C for 10 min. 10
µg protein per lane was loaded and separated by SDS-PAGE.
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2.9 In situ labeling of living cells
Xoo or Xac cells were grown to OD600=0.6 in liquid nutrient broth medium at 28 °С.
The growth media was removed by centrifugation at 6000 rpm, followed by washing
with PBS for three times. The cell pellet was resuspended in 990 µL PBS and treated
with various concentrations of probes (10 µL) or equal DMSO control at 25 °C for 2 h.
In the case of competitive ABPP experiments, cells were resuspended in 980 µL PBS
and preincubated with various concentrations of 0 (10 µL) at 25 °C for 2 h, and then
treated with probe 1 (5 µM) or 2 (50 µM) at 25 °C for another 2 h. After incubation
with probes, the supernatant was separated by centrifugation and excessive probes
were further removed by washing the cell pellet with PBS for three times. The cell
pellet was lysed by sonication on ice in 100 µL PBS, followed by centrifugation at
12000 rpm for 30 min. Protein concentration of supernatant was determined by
Bradford assay and diluted to 1 mg/mL in PBS. The follow-up steps were performed
as described above.
2.10 Streptavidin blot
Proteins on the SDS-gel were transferred onto a PVDF membrane at 4 °C for 60 min.
The membrane was further blocked in 5% nonfat dried milk solution for 2 h at room
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temperature. After washing with PBST (PBS with 0.05% Tween 20), the membrane
was incubated with streptavidin-horseradish peroxidase (Sangon Biotech) (with
1:1000 dilution in 5% nonfat dried milk PBST solution) for 2 h at 4 °C, and detected
by ECL western blotting detection reagents (Bio-Rad). The PVDF membranes were
scanned using the ChemiDoc XRS+ System (Bio-Rad).
2.11 Biotin-streptavidin enrichment experiments
After the in situ labeling experiments as described above, the cell pellet was
resuspended with PBS and lysed by sonication on ice. The supernatant was separated
by centrifugation and the protein concentration was measured by Bradford assay,
which was further adjusted to the same concentration. To a 950 µL supernatant, 10 µL
biotin-azide (200 µM), 10 µL sodium ascorbate solution (5 mM) and 30 µL premixed
CuSO4/BTTAA solution were added, followed by incubation at 37 °C for 2 h. The
reaction was then quenched by addition of 4 mL pre-chilled acetone. After incubation
at –80 °C overnight, the mixture was subsequently centrifuged at 12000 rpm for 15
min to obtain precipitated proteins. The supernatant was removed and precipitate was
washed with pre-chilled MeOH thrice. The pellet was air dried and resuspended in 1
mL 0.2% SDS by sonication. Subsequently, the supernatant was incubated with 1 mL
prewashed streptavidin beads (Promega) for 2 h. After enrichment, beads were
washed with 1% SDS/PBS, 6 M urea and PBS. 50 µL 2×SDS loading buffer was
added to the beads, which was further heated at 95 °C for 10 min to release the target
proteins from the beads. The supernatant was collected by centrifugation and
subjected to a preparative SDS-gel. The enriched bands were visualized by coomassie
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blue staining.
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2.12 In-gel digestion analysis and target identification
The protein band on the SDS-gel was excised and cut into 1 mm2 pieces using a razor
blade. The gel pieces were washed with ddH20, 100 mM ammonium bicarbonate and
dehydrated with 100% CH3CN, which were further reduced by 10 mM DTT for 45
min at 37 °C with gentle shaking. Then the gel pieces were alkylated by 55 mM
iodoacetamide for 1 hour at 37 °C in the dark. For trypsin digestion, 0.5 µg trypsin
(Promega) in 100 µL 25 mM ammonium bicarbonate was added and the reaction was
incubated at 37 °C overnight. The tryptic peptides were further separated by
centrifugation and analyzed using a Nano-Liquid Chromatogram coupled with Triple
TOF 5600 MS (AB SCIEX). The mass spectrometry data were searched using the
MaxQuant software (v.1.5.2.8) against the corresponding Xanthomonas proteome of
the uniprot database. For protein modifications, N-terminal acetylation and oxidation
on methionine were chosen as variable modification and carbamidomethylation on
cysteine as fixed modification. Mass tolerances of the precursor and fragment ions
were set to 6 and 20 ppm. The false discovery rates for peptide and protein
identification were set to 1%.
2.13 Western blot
After the enrichment experiment by streptavidin beads, proteins on the SDS-gel were
transferred onto a PVDF membrane, which was further blocked in 5% nonfat dried
milk solution for 2 h. After washing with PBST, the membrane was incubated with
primary rabbit polyclonal anti-DLST antibody (Wuhan GeneCreate Biological
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Engineering Co., Ltd., 1:2000 dilution in 5% milk in PBST) for 2 h at 4 °C, followed
by PBST washing. Subsequently, goat anti-rabbit IgG HRP-conjugated secondary
antibody (Bio-Rad, 1:2,000 dilution in 5% nonfat dried milk PBST solution) was add
and the membrane was incubated at 4 °C for 2 h. Finally, the membrane was washed
with PBST and signals were detected by ECL western blotting detection reagents
(Bio-Rad) using ChemiDoc XRS+ System (Bio-Rad).
2.14 Cloning and recombinant expression of DLST (uniprot: A0A0K0GL90)
The DLST gene (sucB) with NdeI/XhoI restriction enzyme sites was cloned into the
pet-28a (+) plasmid vector by ligation independent cloning using the following
primers:
DLST-pET-28a (+) Forward: cgcggcagccatatggccaccgaagttaaagttcc
DLST-pET-28a (+) Reverse: tggtggtgctcgagttacagaccaaacagcatac
DLST-pET-28a (+) construct was transformed into E. coli BL21 cells for expression
and selected on LB agar plates with a 50 µg/ml concentration of kanamycin. Cells
were incubated with kanamycin (50 µg/ml) at 37 °C until an OD600 of 0.6. Then
isopropyl β-D-thiogalactopyranoside (IPTG, 500 µM) was added to induce protein
expression. Bacteria were harvested and the cell pellet was then resuspended in lysis
buffer, followed by sonication (30% power, 30 min) on cie. The mixture was
separated by centrifugation and the supernatant was loaded on a pre-equilibrated 5 mL
HisTrapTM HP column (GE Healthcare). DLST was purified via a linear gradient
elution with buffer A (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 50 mM imidazole, 5%
glycerol, 1 mM DTT) and buffer B (50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 400
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mM imidazole, 5% glycerol, 1 mM DTT) using an Äkta purifier system (GE
Healthcare). Fractions containing DLST were pooled, concentrated and digestion by
thrombin for 4 h, followed by further purification with HisTrapTM column. Finally,
fractions containing DLST were concentrated and adjusted to a concentration of 1
mg/mL for in vitro labeling experiments and then stored at −80 °C.
2.15 In vitro labeling of recombinant DLST
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50 ng recombinant DLST (in 44 μL PBS) was treated with desired concentration of
probe 1 (1 μL) and the reaction was incubated at 25 °C for 2 h. In competition
experiment, 50 ng recombinant DLST was pretreated with different concentrations of
0 at 25 °C for 2 h, followed by addition of 5 μM probe 1 for another 2 h. The followed
steps were described as above.
2.16 ATP content detection
Then 5 ml of each cell culture (OD600=0.2) were incubated with desired concentration
of 0 at 28 °C for 2 h with the speed of 180 rpm. After incubation, the cells were
harvested by centrifugation and washed with PBS. The pellet was resuspended with
lysis buffer from the ATP detection kit and lysed by sonication (30% power) on ice.
The supernatant was separated by centrifugation and ATP content was determined by
the luciferin-luciferase bioluminescence assay kit following the kit literature
(Beyotime, China).
3. Results and discussion
3.1 Antibacterial activity test of probes 1−6
To unravel the biological targets of 0, we introduced a short alkyne tag to its benzene
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ring to synthesize a series of sulfone probes (Scheme 1). Also, we designed a negative
probe for control experiments, which was synthesized by probe 1 under an intense
base condition (Scheme 2). To test whether the structural modifications influence the
antibacterial activity of all probes, we determined their individual half-maximal
inhibitory concentration (IC50) against Xoo and Xac (Table 1) by a turbidimeter test,
as described previously.8 As expected, all probes showed good antibacterial activity
against Xoo and Xac, suggesting that the addition of the clickable alkyne tag does not
interfere with drug antibacterial activity. Notably, the antibacterial activity of probe 1
(IC50=3.58 μM) against Xoo was similar to 0 (IC50=3.51 μM), and the antibacterial
activity of probe 2 (IC50=7.06 M) against Xac was slightly better than 0 (IC50=8.59
μM). Probes 1 and 2 perfectly keep the antibacterial activity of 0, which is a
prerequisite for ABPP target discovery. Meanwhile, the NP showed a dramatic drop
in potency (IC50>500 μM) both in Xoo and Xac compared with 0, thereby implying
that the sulfonyl group may be critical for its antibacterial activity.
3.2 Target analysis in Xoo and Xac by probes 1 and 2
We next tested whether these probes are cell permeable and can label enzymes in
living pathogens. In this study, 5 μM probes 1–6 and 50 μM probes 1–4 were
incubated with Xoo and Xac cells for 2 h, respectively. The subsequent cell lysis and
CC with biotin-azide were followed by SDS-PAGE analysis. Analysis of the labeled
proteins by streptavidin blot detection showed a major band at ∼45 kDa (Figure S1)
both in Xoo and Xac, thereby indicating that these probes can enter the cells. All
probes exhibited similar target preferences, thereby indicating that protein labeling is
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primarily driven by the scaffold rather than the attached modifications. In accordance
with their activity, probe 1 showed a higher reactivity with p45 than probes 2–6 in
Xoo cells, and probe 2 outbalanced others in the Xac cells. Therefore, we used probes
1 and 2 to conduct all subsequent studies as a representative example.
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To evaluate the reactivity of the probes, probe 1 was used to treat Xoo cellular
lysates and living cells and probe 2 for Xac labeling experiments with different
concentrations. Both in vitro and in situ studies of Xoo have showed that the labeling
intensity of p45 increased over the concentration of probe 1, which indicated a
dose-dependent manner with p45 (Figure 2A). An intense labeling of this band could
be observed as low as 2.5 μM, thereby indicating a high-affinity binding of p45 with
probe 1. Moreover, probe 1 demonstrated a distinct selectivity with p45 in Xoo cells.
As shown in Figure 2A, labeling of Xoo lysates resulted in a much higher background
and relative weaker labeling intensity compared with Xoo cells at the same
concentration of probe 1, thereby emphasizing the significance of in situ labeling as a
precondition for specific target identification. Consistent with probe 1, probe 2 also
showed good target selectivity for p45 at low concentration in Xac labeling
experiments. Both in vitro and in situ studies of Xac have revealed a dose-dependent
manner with p45 and even in a high concentration of 100 μM, p45 was identified as
an undoubted major target (Figure 2B).
To test whether the probes specifically interact with p45, we conducted in vitro
and in situ competitive experiments with the pretreatment of 0. By treating Xoo cells
with 0 at the indicated concentrations for 2 h before the addition of 5 μM of probe 1,
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we measured an approximate IC50 value of 50 μM for 0 labeling of p45 (Figure 2C).
Preincubation with 100 μM 0 resulted in a total loss of labeling signals, thereby
indicating that 0 and the corresponding probe compete for the same binding sites.
Consistent with the in situ labeling, we also found that pretreatment with 0 in Xoo
cellular lysates abolished this labeling event with a similar potency to that detected in
living cells (Figure S2A). By contrast, p45 again appeared to be a specific target both
in Xac cells and lysates because its labeling signal was gradually reduced in the
presence of an excess of 0 (Figure 2D, Figure S2B). Taken together, p45 is the
specific and main target of 0 both in Xoo and Xac.
To visualize the rate of the labeling reaction, Xoo and Xac cells were incubated
with sulfone probes for various incubation times. The cells were lysed by sonication,
separated by centrifugation at 12000 rpm and protein concentration was measured by
the Bradford assay. After CC reaction with biotin-azide and SDS-PAGE analysis, the
labeled proteins were detected by streptavidin blot. As a result, we found a detectable
signal appeared within 5 min, indicating that the sulfone probes can be used for the
rapid detection of p45 in living cells. In addition, the labeling intensity of p45
increased over time, which emphasizes that the probe binds to p45 in a
time-dependent manner (Figures 3A).
3.3 Reactive mode of sulfone probes with p45
As described above, the sulfone probes 1-6 showed good antibacterial activity against
Xoo and Xac, while the NP probe almost showed no antibacterial activity. We
speculated that the sulfonyl group is critical for the labeling ability of the probes. To
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test this hypothesis, 10 μM probe 1 and NP were used to treat Xoo cells. Furthermore,
50 μM probe 2 and NP were used for Xac cells, followed by CC reaction and
streptavidin blot. Interestingly, the probe 1 and 2 strongly labeled p45 and no labeling
event was detected by NP even at the concentration of 50 μM (Figure 3B), indicating
that the oxadiazole ring was covalently attacked by the nucleophilic residues of
protein targets and sulfonyl group served as a leaving group. This result was in
accordance with the reactivity of sulfone reagents as reported previously, thereby
emphasizing the crucial role of sulfonyl group for labeling protein targets.5-7 When the
sulfonyl group is substituted, the probe cannot react with the nucleophilic residue of
target proteins and the corresponding antibacterial activity was significantly
decreased.
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3.4 Target identification by MS analysis and validation by western bolt
After these initial experiments, we next aimed to identify the p45 protein by a
chemical proteomics approach. We treated Xoo and Xac cells with 100 μM probe 1
and 200 μM probe 2 for 2 h, respectively, followed by lysis and CC reaction. The
target proteins were affinity purified by streptavidin beads, separated by SDS-PAGE
and stained by coomassie blue (Coo). As a result, the probes 1 and 2 strongly purified
the p45 both in Xoo and Xac cells (Figure 3C and 3D). The p45 band was then diced
and subjected to in-gel trypsin digestion.20,21 The resulting peptides were analyzed by
LC–MS/MS, and dihydrolipoamide S-succinyltransferase (DLST) was identified as
the likely target of sulfone probes (Table 2, Table S1). As expected, the intensity of
p45 in probe 1 treated group is much higher than DMSO control. The predicted
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molecular weight of DLST is 42.3 kDa, which is in accordance with the major
labeling band detected in all labeling experiments.
To further validate the MS results, we implemented target enrichment by a
pull-down experiment that was then applied to western blot analysis22 using the DLST
antibody. For anti-DLST western blot, the enriched target protein was prepared in the
identical way as biotin-streptavidin enrichment experiments as described above. After
SDS-PAGE, proteins were transferred onto a PVDF membrane, blocked with 5%
non-fat dried milk solution and incubated with anti-DLST antibody, followed by the
incubation with goat anti-rabbit IgG HRP-conjugated secondary antibody. The signals
were detected by ECL western blotting detection reagents. As a result, an intense
labeling band appeared in the probe-treated sample but not in the DMSO control,
indicating the purified p45 by probes 1 and 2 was DLST (Figure 3E and 3F). These
results have confirmed DLST as the target of probe 1 in Xoo and probe 2 in Xac.
3.5 In vitro interaction of probe 1 with recombinant DLST
For additional validation experiments, recombinantly expressed DLST (from Xoo)
was labeled by probe 1 in a dose-dependent manner, and no labeling event was
detected by NP (Figure 4A). The labeling of DLST was markedly decreased with a
preincubation of excess 0, thereby suggesting a specific interaction of the DLST with
probe 1 (Figure 4B). For sensitivity detection experiment, a series of concentrations of
recombinant DLST was incubated with probe 1 for 2 h. After CC reaction and
streptavidin blot, we showed that probe 1 is highly sensitive for DLST with a
detection limit of 3.1 nM (Figure 4C). Furthermore, the labeling of DLST was
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completely blocked by preincubation with phenylmethylsulfonyl fluoride (PMSF)
(Figure S3), which is a known active-site inhibitor of serine proteases,23 thereby
indicating that probe 1 may covalently bind to the serine residues. Among these
results, we have unambiguously confirmed that DLST is the target of probe 1 in cells.
3.6 Function study of DLST by 0
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DLST is the structural and catalytic core of the 2-oxoglutarate dehydrogenase
complex (KGDHC),24 which catalyzes the oxidative decarboxylation of
2-oxoglutarate to succinyl-CoA and acts as a key regulatory enzyme of energy
production in the tricarboxylic acid (TCA) cycle.25 To date, efficacious and selective
DLST inhibitors are unavailable and the functional studies of DLST were mostly
conducted by genetic approaches.26-28 To determine whether the inhibition of DLST
will alter the energy production in cells, we treated Xoo cells with various
concentrations of 0 and determined the ATP content by luciferin-luciferase
bioluminescence assay. As expected, the ATP content was decreased with the
increase of 0 and resulted in more than 40% decrease at the concentration of 10 IC50
as compared with the DMSO control (Figure 4D). When DLST was inhibited, the
energy production was interrupted, thereby indicating that the compound 0 may serve
as an effective tool for the function study of DLST in bacteria.
In summary, we have developed a series of cell-permeable probes for target
identification of bactericide 0 in living cells. Through chemical proteomic strategies,
we validated DLST as an unprecedented target, which was also the first reported
target of this novel structure. As a central enzyme in the TCA cycle, targeting this
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energy regulator may, therefore, represent an attractive strategy for antibacterial agent
discovery. To date, the function of DLST remains largely unknown and was studied
mostly by genetic approaches due to lack of effective inhibitors. For the first time, we
reported the covalent DLST inhibitor, the drug candidate 0, which may serve as a
useful tool for the function study of DLST in cells. Taken together, we anticipate
target deconvolution of this sulfone-based antibacterial reagent may offer a guidance
for the discovery of novel agrochemicals.
Abbreviations Used
ABPP, activity-based protein profiling; Xoo, Xanthomonas oryzae pv. oryzae; Xac,
Xanthomonas axonopodis pv. citri; CC, click chemistry; AIBN, azobisisobutyronitrile;
m-CPBA, 3-Chloroperbenzoic acid; DLST, dihydrolipoamide S-succinyltransferase;
BTTAA, 2-(4-((bis((1-tert-butyl-1H-1,2,3-triazol-4-yl)methyl)amino)methyl)-1H-1,2,
3-triazol-1-yl)acetic acid
Acknowledgment
We acknowledge financial support by the Key Technologies R&D Program
(2014BAD23B01), National Natural Science Foundation of China (21877021,
21662009), the Research Project of Chinese Ministry of Education (213033A,
20135201110005).
Supporting Information
Supplementary data including Figure S1–S3, Table S1, Synthesis and characterization
data for other target compounds, 1H NMR, 13CNMR, and HRMS spectra, in the online
version, at https://pubs.acs.org/journal/jafcau.
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Conflict of interest
The authors declare no conflict of interest.
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Figure captions
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Scheme 1. Synthetic route of 0-derived probes.
Scheme 2. Synthetic route of negative probe.
Figure 1. Design of 0-derived probes. (A) Chemical structures of 0 and its clickable
analogs. (B) Detection and identification of probe-labeled proteome by the ABPP
strategy.
Figure 2. In vitro and in situ labeling of bacterial pathogens by the 0-derived probes.
(A) Dose response of in vitro and in situ labeling revealed the potential engagement of
probes 1 and 2 to an unknown target of ∼45 kDa (p45) both in Xoo and Xac (B). Coo,
Coomassie blue staining shows protein loading. (C) Dose-dependent blockade of
labeling of p45 by pretreatment with 0 in Xoo and Xac cells (D). A–D: gel analysis by
streptavidin blot.
Figure 3. Kinetic analysis, covalent reaction and target identification of DLST in Xoo
and Xac. (A) Time-course experiments revealed a time-dependent manner with p45.
(B) In situ labeling of Xoo and Xac by probes 1–2 and NP indicated a crucial role of
sulfonyl group for the probes’ labeling ability. A–B: gel analysis by streptavidin blot.
(C) Streptavidin pull-down experiment by probe 1 enriched p45 in Xoo cells. (D)
Streptavidin pull-down experiment by probe 2 enriched p45 in Xac cells. C–D: gel
analysis by coomassie blue staining. Immunoblot analysis using an antibody
confirmed the protein pulled down by probe 1 (E) and probe 2 (F) was DLST. E–F:
gel analysis by western blot.
Figure 4. Labeling of recombinant DLST and its function study by 0. (A)
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Dose-dependent labeling of recombinant DLST with probe 1. (B) Increasing
concentrations of 0 prevented labeling of recombinant DLST with probe 1. (C)
Labeling of different concentrations of recombinant DLST indicated a high sensitivity
with probe 1. A–C: gel analysis by streptavidin blot. (D) Equant Xoo cells were
treated with the concentrations of 0–10 IC50 of 0, resulting in a dramatic decrease of
ATP content. Error bars represent s.d. in three independent replicates.
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Table 1. Antibacterial Activities of 0 and 0-derived probes against Xoo and Xac in
vitro.
Xoo
Xac
compound
regression equation
y = 7.64x + 5.53
y = 10.40x + 4.51
y = 7.53x + 1.90
y = 28.44x – 0.92
y = 27.40x – 12.27
y = 4.19x + 4.33
y = 4.80x + 3.05
r
IC50 (μM)
3.51 ± 0.04
3.58 ± 0.03
7.95 ± 0.09
5.51 ± 0.03
13.94 ± 0.03
3.99 ± 0.08
6.80 ± 0.16
>500
regression equation
y = 4.39x + 3.61
y = 3.75x + 2.24
y = 2.55x + 4.08
y = 1.93x + 3.73
y = 2.34x + 3.62
r
IC50 (μM)
8.59 ± 0.16
17.53 ± 0.10
7.06 ± 0.25
15.60 ± 0.14
12.63 ± 0.33
0
0.98
0.99
0.98
0.99
0.99
0.99
0.98
0.99
0.97
0.99
0.97
0.98
Probe 1
Probe 2
Probe 3
Probe 4
Probe 5
Probe 6
NP
>500
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