Discovery of human TyrRS inhibitors by structure-based virtual screening, structural optimization, and bioassays

Article abstract of DOI:10.1039/c9ra00458k

The human tyrosyl transfer-RNA (tRNA) synthetase (TyrRS), which is well known for its essential aminoacylation function in protein synthesis, has been shown to translocate to the nucleus and protect against DNA damage caused by external stimuli. Small molecules that can fit into the active site pocket of TyrRS are thought to affect the nuclear role. The exploitation of TyrRS inhibitors has attracted attention recently. In this investigation, we adopted a structure-based virtual screening strategy and subsequent structure-activity relationship analysis to discover new TyrRS inhibitors, and identified a potent compound 5,7-dihydroxy-6,8-bis((3-hydroxyphenyl)thio)-2-phenyl-4H-chromen-4-one (compound 11, K_i = 8.8 μM). In intact HeLa cells, this compound showed a protective effect against DNA damage. Compound 11 is a good lead compound for the further development of drugs against disorders caused by DNA damage.

Full text of DOI:10.1039/c9ra00458k

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PAPER
Discovery of human TyrRS inhibitors by structure-
based virtual screening, structural optimization,
and bioassays†
Cite this: RSC Adv., 2019, 9, 9323
Shenzhen Huang, ‡a Xiang Wang,‡bc Guifeng Lin,a Jie Cheng,a Xiuli Chen,a
a
Weining Sun,d Rong Xiang,b Yamei Yu,a Linli Lid and Shengyong Yang
*
The human tyrosyl transfer-RNA (tRNA) synthetase (TyrRS), which is well known for its essential
aminoacylation function in protein synthesis, has been shown to translocate to the nucleus and protect
against DNA damage caused by external stimuli. Small molecules that can fit into the active site pocket
of TyrRS are thought to affect the nuclear role. The exploitation of TyrRS inhibitors has attracted
attention recently. In this investigation, we adopted a structure-based virtual screening strategy and
subsequent structure–activity relationship analysis to discover new TyrRS inhibitors, and identified
Received 18th January 2019
Accepted 18th March 2019
a
potent compound 5,7-dihydroxy-6,8-bis((3-hydroxyphenyl)thio)-2-phenyl-4H-chromen-4-one
(compound 11, Ki ¼ 8.8 mM). In intact HeLa cells, this compound showed a protective effect against DNA
damage. Compound 11 is a good lead compound for the further development of drugs against disorders
caused by DNA damage.
DOI: 10.1039/c9ra00458k
rsc.li/rsc-advances
nucleus. The nuclear-localized TyrRS activates transcription
factor E2F1 to upregulate the expression of DNA damage repair
genes such as BRCA1 and RAD51.9 In particular, RSV can also
Introduction
Resveratrol (3,4,5-trihydroxystilbene, RSV), a natural product, is
an important component of red wine. RSV has long been
thought to impart benets in lifespan expectancy,1,2 cancer
chemopreventive activity,3 cardioprotective,4 and neuro-
protective effects,5 by launching a stress response.6,7 However,
because it has low activity and the target of its action is not
clear;8 RSV is only marketed as a health care product. Human
tRNA synthetase is a potent PARP1-activating effector target for
RSV.
Recently, Sajish et al. demonstrated that RSV could interact
with human tyrosyl transfer-RNA synthetase (TyrRS) and hence
play its benecial role.7 TyrRS was originally known for its
essential aminoacylation function in protein synthesis. Recent
studies have found a new function for TyrRS in DNA damage
protection.7,9,10 External stimuli, such as oxidative stress, can
induce TyrRS to rapidly translocate from the cytosol to the
trigger the translocation of TyrRS to the nucleus by directly
binding to the active site of TyrRS, leading to the activation of
NAD+-dependent auto-poly-ADP ribosylation of poly(ADP
ribose) polymerase 1 (PARP-1). PARP-1 is a major stress
response and DNA repair factor that has a considerable impact
on longevity.11 PARP-1 activated by TyrRS leads to activation of
a series of protective genes, including the tumor suppressor
gene p53, FOXO3A, or SIRT6.7 Therefore, TyrRS is an attractive
target for developing therapeutics for diseases of the nervous
system diseases and for DNA damage protection.
To date, RSV is the only small molecule compound with an
inhibition constant (Ki) value in the micromolar range that has
been identied as an inhibitor of TyrRS.7 However, RSV has also
been identied as a modulator against many targets.12–15
Therefore, discovery of novel potent and selective TyrRS inhib-
itors, with scaffolds different from RSV, is desirable.
Here, we report the discovery of a new series of TyrRS
inhibitors containing the scaffold 5,7-dihydroxy-2-phenyl-6,8-
bis(phenylthio)-4H-chromen-4-one. These compounds were
identied by a high throughput virtual screening and subse-
quent structure–activity relationship (SAR) analysis. The most
active compound 5,7-dihydroxy-6,8-bis((3-hydroxyphenyl)thio)-
2-phenyl-4H-chromen-4-one (11) was further investigated in
binding mode analysis with TyrRS and bio-functional
studies.7,9,10,16,17 Compound 11 is a promising lead compound
for the further development of drugs for DNA damage related
diseases.
aState Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan
University/Collaborative Innovation Center of Biotherapy, Chengdu, Sichuan 610041,
China. E-mail: yangsy@scu.edu.cn
bDepartment of Clinical Medicine, School of Medicine, Nankai University, Tianjin
300071, China
cDepartment of Chemistry, Fudan University, Shanghai 200433, China
dKey Laboratory of Drug Targeting and Drug Delivery System of Ministry of Education,
West China School of Pharmacy, Sichuan University, Chengdu, Sichuan 610041, China
† Electronic supplementary information (ESI) available: General procedure for the
synthesis of compounds (1–31), Fig S1–S7, and Table S1. See DOI:
10.1039/c9ra00458k
‡ These authors contributed equally to this work.
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manner with Ki values of 187.9, 69.1, and 58.6 mM, respec-
tively. The positive control compound RSV showed weak
potency (Ki ¼ 107.7 mM) (Fig. 1C and S1†). Because the hit
compounds AH-357/03514064 and SKLB2002 have similar
scaffolds, and compound SKLB2002 was the most active
compound and contained the new scaffold 5,7-dihydroxy-2-
phenyl-4H-chromen-4-one, further structural optimization and
SAR analysis were carried out on this compound.
Results and discussion
Structure-based virtual screening of novel hit compounds
against TyrRS
The virtual screening process is schematically shown in Fig. 1A.
The chemical databases included the commercial chemical
library Specs and an in-house database. In the rst step,
compounds were ltered by Lipinski's rules and for pan-assay
interference compounds (PAINS) in Pipeline Pilot 8.5, which
is used for ltering pan-assay interference compounds18,19 and
has been used with great success in drug discovery.18,20 Then
molecular docking was carried out with the remaining
compounds. Here, the GOLD program was used because it
provides good docking performance.21 All the calculations were
carried out on the platform of Accelrys Discovery Studio 3.1
(Accelrys Inc., San Diego, CA, USA). The crystal structure of
TyrRS in complex with RSV (PDB entry 4Q93) was used for the
docking studies,7 and RSV was used as a reference molecule
Chemistry
All of the target compounds were prepared from the commer-
cially available 5,7-dihydroxy-2-phenyl-4H-chromen-4-one. The
general synthetic routes for compounds 1 (SKLB2002), 2–12,
and 14–31 shown in Scheme 1. In route 1, intermediate 1a was
prepared by a similar synthetic method as described in the
literature.23 Target compounds 1–12 were then obtained by
a conventional coupling reaction of various benzenethiols and
intermediate 1a in DMF in yields of 40–70%. In route 2, inter-
mediate 13a was obtained by a displacement reaction with
iodomethane and commercially available 5,7-dihydroxy-2-
phenyl-4H-chromen-4-one. Intermediate 13b was then obtained
by a classic electrophilic substitution reaction between NIS and
the previously synthesized intermediate 13a in quantitative
yield. Target compounds 14–22 were synthesized from various
benzenethiols and intermediate 13b in DMF. Finally, the target
compounds 23–31 were obtained by demethylation of
compounds 14–22 with boron tribromide.24
˚
with a root-mean-square deviation (RMSD) of 0.57 A (Fig. S2†).
Next, the consensus scoring strategy was used to score the
compounds that may target TyrRS. Here, the scoring functions,
GoldScore incorporated in the GOLD program and ID-Score
developed by our laboratory,22 were selected for the consensus
scoring strategy because they have provide good performance
for scoring.18,22 Finally, 30 compounds were selected from the
top ranked compounds by the consensus scoring and visual
inspection.
SAR analysis and optimization of hit compounds
TyrRS enzyme inhibition assays
The inhibitory effects of the synthesized compounds on human
TyrRS are displayed in Tables 1 and 2. As can be seen from Table
1, the introduction of methyl, methoxyl, and tert-butyl groups,
that is compounds 3, 6, 8, and 9, which substantially dimin-
ished activity. The series of derivatives (1, 2, 5, 7, 11, and 12)
revealed that the presence of halogen or hydroxyl groups in the
The 30 in silico-screened compounds were tested for inhibitory
activity against TyrRS using the ATP–pyrophosphate exchange
assay (Fig. 1B). From the enzyme inhibition assay, the hit
compounds
AK-087/41343686,
AH-357/03514064,
and
SKLB2002 were identied, they showed an enzyme activity <
60% against TyrRS at the concentration of 100 mM and inhibited
TyrRS activity in biochemical assays in a dose-dependent
Fig. 1 (A) Schematic diagram of the underlying workflow of the TyrRS
drug discovery. (B) Enzyme activity of the 30 candidate molecules
based on virtual screening at 100 mM. The red columnar bar represents Scheme 1 Reagents and conditions: (a) HNO3, I2, CH3COOH, RT. (b
the activity of the reference compound RSV. (C) The structures of the and e) Pd2(dba)3, X-phos, Cs2CO3, DMF, N2, 110 ꢀC. (c) CH3I, K2CO3,
hit compounds and RSV.
DCM, RT. (d) NIS, CF3COOH, 80 ꢀC. (f) BBr3, DCM, RT.
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Table 1 Chemical structures and bioactivities of SKLB2002 derivatives
with R1 varied
potency compared with the benzenethiol-substituted
compounds. Compound 11, Ki ¼ 8.8 mM, was the most active
compound and was the most promising inhibitor in the present
series of derivatives.
Differential scanning uorimetry (DSF) and differential
scanning calorimetry (DSC) assays
DSF is a good platform for screening protein stability conditions
and has the advantages of being fast and inexpensive. Because
of the small amount and low concentration of protein required,
DSF can be used to identify low-molecular-weight ligands. A
DSF assay was employed to understand the relationship
between TyrRS inhibitor binding and protein construct
stability25,26 (Fig. 3A and B). The impact of the binding of RSV
and compound 11 on the TyrRS was explored by determining
the changes in the melting temperature (Tm) of the protein in
the absence and the presence of ligand. The shi in the Tm as
dened by the relative midpoint of the denaturation prole
assay indicated that the thermal stability was dependent on the
ligand concentration. As shown in Fig. 3A, the presence of
compound 11 increased the Tm value of the native TyrRS
protein, showing that this class of inhibitor retained the ability
to bind and stabilize the protein construct. Similarly, a signi-
cant increase in the Tm value was also observed for RSV,
a specic binding ligand for this protein (Fig. 3B).
Cpd
R1
Enzyme activity@100 mM (%)
1
2
3
4
5
6
7
8
m-Br
o-Br
45.63
31.76
88.86
53.87
47.76
76.17
19.24
75.93
85.29
24.48
4.57
o-OCH3
m-OCH3
p-F
p-CH3
m-F
o-CH3
p-(CH3)3
4-Pyridinea
m-OH
o-OH
9
10
11
12
29.92
a
The pyridine ring was use to replace the benzenoid structure.
skeletal structure of these molecules was desirable; compound
7 and, in particular, compound 11, displayed an inhibition rate
larger than 80% at a concentration of 100 mM. Compounds 7
and 11 strongly inhibited TyrRS with Ki values of 10.6 and 8.8
mM, respectively, which were considerably higher than that
shown by the positive control RSV (Ki ¼ 107.7 mM, Fig. 2 and
S1†).
As can be seen from Table 2, the R2 and R3 groups were also
optimized. However, deletion of the benzenethiol-substituted
unit at the 18-position of in the skeletal structure of these
molecules was not ideal. Compounds 14–31 showed weaker
DSC, a thermal analysis method, has become an efficient
experimental technique for determination of the thermody-
namic properties of biological macromolecules such as
proteins.27–29 A DSC assay was also performed to determine the
Tm values for TyrRS. In the DSC assay, the Tm values of the TyrRS
protein in the absence and presence of ligands were determined
by the midpoint of the rise in each curve30,31 (Fig. 3C and D). The
results obtained were comparable with the DSF method and
showed the same trends. As shown in Fig. 3C and D, the pres-
ence of RSV or compound 11 shied the native TyrRS protein Tm
ꢀ
value by more than 1.84 or 1.30 C higher, respectively.
Table 2 Chemical structures and bioactivities of SKLB2002 derivatives with R2 and R3 varied
Cpd
R2
Enzyme activity@100 mM (%)
Cpd
R2
Enzyme activity@100 mM (%)
14
15
16
17
18
19
20
21
22
m-Br
m-F
p-F
m-OCH3
o-F
o-Br
o-OCH3
p-OCH3
m-Cl
86.42
68.51
76.37
71.84
73.76
69.55
79.91
73.88
77.86
23
24
25
26
27
28
29
30
31
m-Br
m-F
p-F
m-OH
o-F
o-Br
o-OH
p-OH
m-Cl
91.4
75.12
56.25
93.16
87.04
72.44
81.88
78.55
87.07
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sulfanyl)phenol group at the 6, 8-position of 5,7-dihydroxy-2-
phenyl-4H-chromen-4-one is orientated with the H atom of the
OH group and the S atom hydrogen-bonded to the Asp173 or
Trp40 residues in the active site of TyrRS. The O atom in the 7-
hydroxy branch of compound 11 formed another hydrogen-
bonding interaction with the Gln170 residue. Compound 11
also formed hydrophobic interactions with residues Tyr39,
Tyr52, Leu72, and Phe183 in the active site of TyrRS.
Fig. 2 Enzyme activity data for TyrRS with compounds 7 and 11. (A and
B) Determination of the Ki values of compounds 7 and 11, respectively.
Protection against DNA damage
DNA damage can be induced by various stimuli, including UV
radiation from the environment, oxygen produced by endoge-
nous reactive oxygen species, and chemicals for disease treat-
ment. DNA damage is considered a major source of gene
mutations and a cause of many diseases, for example, neuro-
degenerative diseases, cardiovascular disease, immune
dysfunction, and cancer.32,33 Even though there is a DNA
damage response in cells to ght against DNA damage caused
by endogenous or environmental stimulus, it is not sufficient
for cells to eliminate all DNA damage. Thus, chemicals that can
help cells ght against DNA damage are desirable. Serum star-
vation (SS, oxidative stress) and RSV have been shown to affect
TyrRS nuclear localization.7,9 This observation prompted us to
investigate whether compound 11 can regulate TyrRS nuclear
localization in HeLa cells. By immunoblotting of the cyto-
plasmic and nuclear fractions from cells, we found that treat-
ment with compound 11 dramatically promoted the nuclear
localization of TyrRS in a concentration-dependent manner in
HeLa cells, without the inuence of TyrRS in the cytoplasm
(Fig. 5).
Having demonstrated that nuclear localization of TyrRS is
promoted by compound 11, we investigated the potential for
TyrRS inhibitors to protect cells from DNA damage. We pre-
treated HeLa cells with compound 11 for 24 h before inducing
DNA damage in cells by treatment with 30 mM cisplatin for 24 h.
To visualize the DNA damage level in cells, phosphorylated
histone H2A.X at S139 (g-H2A.X), used as a biomarker for DNA
double-strand breaks, was detected by immunoblotting.
Without cisplatin treatment, the level of g-H2A.X was below the
detection limit (Fig. 6). However, with cisplatin treatment, g-
H2A.X could be detected. We found that treatment with
compound 11 dramatically decreased the level of g-H2A.X in
HeLa cells, suggesting that nuclear TyrRS had a protective effect
on DNA damage. Compound 7 had a similar effect as shown in
Fig. S7.†
Fig. 3 DSF and DSC were used to test the stabilization of TyrRS by RSV
and compound 11. (A) The Tm value of compound 11. (B) The Tm value
of RSV. Also shown are graphs of the unfolding transition of 2 mM TyrRS
in the presence of 50 mM RSV or compound 11, respectively. (C and D)
Representative DSC melting profiles for TyrRS with RSV or compound
11; DTm of RSV and compound 11 obtained from DSC.
Binding-mode analysis
Fig. 4 shows a possible binding mode of compound 11 with
TyrRS. For comparison, RSV was also shown in the active site of
TyrRS (PDB entry 4Q93). Obviously compound 11 occupies the
same binding site but a larger area than RSV does. The 3-(l1-
Experimental
Ligand preparation
Compound libraries, including In-house and Specs databases,
were used for virtual screening in this investigation. The total
number of compounds is approximately 220 000. All the
compounds were used for docking, which were prepared with
‘Prepare Ligands’ module in Discovery Studio (DS) 3.1 (Accelrys
Inc., San Diego, CA, USA). Parameter values for “Change Ioni-
zation, Generate Tautomers, and Generate Isomers” were set to
Fig. 4 Binding modes of compound 11 and RSV with TyrRS (PDB code
4Q93). Compound 11 is shown as green sticks, and RSV is displayed as
marine sticks.
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hydrogen atoms to the protein, and assigning force eld (here
the CHARMm forceeld was adopted). For GOLD, the “pre-
dened generic algorithm (GA)” setting of ‘automatic’ was
employed and others were set to default.
General procedure for the synthesis of compounds 1–31
All commercially available starting materials, solvents, and
reagents were obtained from commercial suppliers and used
without further purication unless otherwise indicated. Reac-
tions were monitored by thin layer chromatography (TLC) on
Merck silica gel 60 F-254 thin layer plates. 1H NMR spectra was
recorded on a Bruker AV-400 spectrometer at 400 MHz. Spin
multiplicities are described as s (singlet), br. s (broad singlet), t
(triplet), q (quartet), and m (multiplet). Chemical shis (d) are
listed in parts per million (ppm) relative to tetramethylsilane
(TMS) as an internal standard. Mass spectral (MS) data were
acquired on a Waters Q-TOF Premier mass spectrometer
(Micromass, Manchester, U.K.). The 1H NMR, 13C NMR, and MS
of compounds 1–31 are listed in the ESI.†
Purication of human TyrRS protein
Human TyrRS was puried as previously described.7,36,37 The
plasmid of mini-TyrRS (amino acids 1–341) as a gi from Paul
Schimmel. Briey, this plasmid was transformed into E. coli
Arctic Express (DE3) cells. The cells were cultured at 37 C in
fresh LB liquid medium (5 g of NaCl, 10 g of bactotrypton, and
Fig. 5 Compound 11 promoted TyrRS nuclear localization in HeLa
cells. Hela cells were treated with compound 11 (1.1, 3.3, 10, or 30 mM)
or DMSO for 8 h. Cells cultured in serum-free media for 8 h were
under serum starvation. The cytoplasmic and nuclear fractions from
ꢀ
cells were subjected to immunoblot assays. Quantification of immu- 10 g of yeast extract per litre). Isopropyl-b-D-1-thiogalactopyr-
noblots is shown below.
anoside (IPTG, 0.5 mM) was used to induce expression when
attenuance D600 nm was 1, and the culture was grown for 20 h at
16 ꢀC. Cells were collected by centrifugation at 4000 rpm minꢁ1
for 15 min at 4 ꢀC and then re-suspended in lysis buffer (50 mM
Hepes-Na, pH 7.5, 5 mM imidazole pH 7.5, 400 mM NaCl, 1ꢂ
PMSF, and 2 mM BME). Cells were lysed using a cell disrupter.
Aer centrifugation at 14 000 rpm minꢁ1 for 25 min at 4 ꢀC, the
supernatant was loaded onto a nickel-affinity chromatography
column (GE-Healthcare) pre-equilibrated with lysis buffer. The
Fig. 6 Nuclear TyrRS promoted by compound 11 protected cells from
protein was eluted with a linear gradient of imidazole (0–300
DNA damage in HeLa cells. HeLa cells were pre-treated with
mM). The desired fractions were pooled, concentrated and
compound 11 (30, 10, 3.3, 1.1, or 0.37 mM), RSV (1 mM), or DMSO for
subjected to gel-ltration (Superdex 200; GE-Healthcare) chro-
matography and the protein peak corresponding to homoge-
nous protein in buffer (20 mM Hepes-Na, pH 7.5, 50 mM NaCl,
and 2 mM BME) was collected (see Fig. S3†). The quality of the
protein purication was validated by SDS-PAGE analysis (see
Fig. S4†).
24 h. To induce DNA damage, cells were treated with cisplatin (30 mM)
or DMSO in the presence of the test compounds or DMSO for 24 h.
Then, whole cell lysates were subjected to immunoblot assays.
false. Other parameters were set to their default values. The
compounds were retrieved according to previous reported.18,34,35
Selected compounds were purchased in milligram quantities
from chemical vendors or synthesis by our laboratory. Purity of
compounds was $95%, as declared by the chemical vendors.
In vitro enzymatic inhibition assays
In vitro enzymatic inhibition assays were performed using an
ATP–pyrophosphate exchange assay as previously described7
and the results provided by Reaction Biology Corp, Malvern,
United States. Briey, tyrosyl adenylate synthesis was measured
using the tyrosine-dependent ATP–pyrophosphate (PPi)
Structure-based virtual screening of novel hit compounds
against TyrRS
In this investigation, the docking method GOLD was involved in exchange assay. A mixture containing 100 mM HEPES-Na (pH
the platform of DS 3.1. Before molecular docking, the receptor 7.5), 20 mM KCl, 2 mM ATP, 1 mM NaPPi, 2 mM DTT, 100 mM L-
protein was prepared by the DS 3.1 soware package with tyrosine (T3754-50G, Sigma), 0.02% Brij35 (w/v), 10 mM MgCl2,
standard preparation procedures (protein preparation and approximately 0.01 mCi mlꢁ1 Na[32P]PPi (NEX019001MC,
protocol), which include removing water molecules, adding PerkinElmer) was added to 12.5 nM puried TyrRS, pre-
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incubated with 0–300 mM of test compounds or 0–1000 mM of Then whole cell lysates were extracted with radio-
positive control RSV (ref. 7) at 4 ꢀC for 30 min. The ATP–PPi immunoprecipitation assay (RIPA) lysis buffer (Beyotime
exchange reaction mixture was incubated at room temperature Biotechnology) that contained phenylmethanesulfonyl uoride
for 2 h, and aliquots were removed at specied time intervals (PMSF, Sigma) and protease inhibitor cocktail (Sigma) and
and quenched in a mixture containing 40 mM NaPPi, 1.4% (w/v) subjected to immunoblot assays. Anti-Histone H2A.XS139ph
HClO4, 0.4% (w/v) HCl, and 8% (w/v) of activated charcoal. Aer (phospho Ser139) antibodies (GeneTex, GTX127340, 1 : 1000
thorough mixing, the charcoal was ltered and washed with dilution) were used to detect DNA damage levels in cells. Anti-b-
a solution of 7% HClO4 (w/v) and 200 mM NaPPi using Spin-X actin antibodies (Proteintech, 60008-1-Ig, 1 : 1000 dilution)
Centrifuge Filters (Corning) containing 0.45 mm pore-size were used to probe b-actin as a control.
cellulose acetate lters. Aer drying, the charcoal was
punched into scintillation vials and the radioactivity of the ATP
bound to the charcoal mixture was measured by scintillation
Cellular localization of TyrRS
HeLa cells were treated with compound 7 (30, 10, 3.3, and 1.1
mM), compound 11 (30, 10, 3.3, and 1.1 mM), RSV (1 mM), or
DMSO for 8 h. Cells cultured in serum-free media for 8 h were
under serum starvation. The cytoplasmic and nuclear fractions
were separated by NE-PER® nuclear and cytoplasmic extraction
reagents (Thermo Scientic, 78835) as per the manufacturer's
instructions. Both fractions were then subjected to immunoblot
assays. Anti-tyrosyl tRNA synthetase antibodies (Abcam,
ab150429, 1 : 1000 dilution) were used to probe TyrRS in the
nucleus and cytoplasm. Anti-GAPDH antibodies (Proteintech,
60004-1-Ig, 1 : 1000 dilution) and anti-Lamin B1 (Proteintech,
1298.7-1-AP, 1 : 1000 dilution) antibodies were used to probe
GAPDH and Lamin B1 as controls for the cytoplasmic and
nuclear fractions, respectively.
counting. The results of the L-tyrosine Km values and enzyme
titration can be found in Fig. S5 and S6.† The Ki value was
calculated by the equation Ki ¼ IC50/(1+[S]/Km).
Differential scanning calorimetry (DSC) assay
Human TyrRS (1 mg mLꢁ1) was diluted in buffer (20 mM Hepes-
Na, pH 7.4, and 400 mM NaCl) and melted using a Microcal VP
Capillary DSC (GE Healthcare Life Sciences). Data were
collected at 20–110 ꢀC, with a 200 ꢀC hꢁ1 scan rate and 10 s
ltering period. Data were analyzed in origin soware. TyrRS
was incubated with 200 mM RSV or compound 11 for at least
0.5 h before melting.
Differential scanning uorimetry (DSF) assay
DSF experiments were performed on a RT-PCR detection system
(BIO-RAD CFX96) according to the known protocol.25 SYPRO
orange (5000ꢂ, Invitrogen) was monitored using the lters of
FRET at the wavelength of 492 nm for excitation and of ROX at
the wavelength of 610 nm for emission. Each reaction solution
containing 2 mM protein in buffer (20 mM Hepes-Na, pH 7.4,
and 100 mM NaCl), 5 ꢂ SYPRO orange, and compound (10 mL)
was heated from 25 to 95ꢁꢀ1C. Fluorescence intensities were
Immunoblot assays
Proteins from cells were resolved by 12% or 10% sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
and transferred to polyvinylidene uoride (PVDF) membranes
(Millipore). The membranes were blocked with TBS-T blocking
buffer (5% nonfat milk in TBS-T) for 1–4 h at RT and then
ꢀ
probed with primary antibodies overnight at 4 C with gentle
shaking. Aer three washes with TBS-T, the membranes were
incubated with horseradish peroxidase-conjugated goat anti-
mouse or anti-rabbit antibody (Proteintech, 1 : 5000 dilution)
for 1 h at 37 ꢀC with gentle shaking. Aer extensive washing
with TBS-T, the membranes were preserved in TBS, and the
immunoblots were visualized by ECL (Abbkine). Immunoblots
were quantied using Adobe photoshop soware.
recorded every 1 ꢀC min
and plotted as a function of
temperature. The inection point of the transition curve (Tm)
was calculated by tting the Boltzmann equation to the
sigmoidal curve in GraphPad Prism 5.0. Each condition was
tested in triplicate.
Cell cultures
HeLa cells were maintained in Dulbecco's modied Eagle's
medium which was supplemented with 10% (v/v) fetal bovine
serum. To mimic oxidative stress in cells, serum starvation
under which condition cells were cultured in serum-free media
was used. Cells were grown at 37 ꢀC and 5% CO2 in a humidied
incubator.
Conclusions
A structure-based docking strategy was adopted to screen for
novel TyrRS inhibitors. Aer two campaigns including virtual
screening and bioassays, hit compounds with the novel scaffold
5,7-dihydroxy-2-phenyl-4H-chromen-4-one were selected for
structural optimization and SAR analysis. The most active
compound against TyrRS was compound 11 with a Ki value of
Detection of DNA damage in cells
To determine the protection of TyrRS inhibitors against DNA 8.8 mM. The results of orthogonal assays, including DSC and
damage, HeLa cells pre-seeded in 6-well plate were pre-treated DSF assays, revealed that these compounds bind to the active
with compound 7 (30, 10, 3.3, 1.1, or 0.37 mM), compound 11 site of TyrRS. Furthermore, compound 11 could protect against
(30, 10, 3.3, 1.1, or 0.37 mM), RSV (1 mM), or DMSO for 24 h. To the DNA damage caused by cisplatin in intact HeLa cells.
induce DNA damage, cells were treated with cisplatin (30 mM) or Therefore, these compounds represent useful probes for further
DMSO in the presence of test compounds or DMSO for 24 h. research into the biological function of TyrRS.
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Conflicts of interest
There are no conicts to declare.
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Acknowledgements
This work was supported by the National Natural Science
Foundation of China (81573349, 81773633, and 21772130),
National
Science
and
Technology
Major
Project
and
(2018ZX09711002-014-002,
2018ZX09711002-011-019,
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2018ZX09711003-003-006), and 1.3.5 project for disciplines of
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